BACKGROUND Hepatic metastases are common and difficult to treat after colorectal cancer(CRC)surgery.The predictive value of carcinoembryonic antigen(CEA),cancer antigen(CA)125 and CA19-9 combined tests for liver metas...BACKGROUND Hepatic metastases are common and difficult to treat after colorectal cancer(CRC)surgery.The predictive value of carcinoembryonic antigen(CEA),cancer antigen(CA)125 and CA19-9 combined tests for liver metastasis is unclear.AIM To evaluate predictive value of combined tests for CEA,CA125,and CA19-9 levels in patients with liver metastases of CRC.METHODS The retrospective study included patients with CRC alone(50 cases)and patients with CRC combined with liver metastases(50 cases)who were hospitalized between January 2021 and January 2023.Serum CEA,CA125 and CA19-9 levels were compared between the two groups,and binary logistic regression was used to analyze the predictive value of the combination of these tumor markers in liver metastasis.In addition,we performed receiver operating characteristic(ROC)curve analysis to assess its diagnostic accuracy.RESULTS The results showed that the serum CEA,CA125 and CA19-9 levels in the CRC with liver metastasis group were significantly higher than those in the CRC alone group.Specifically,the average serum CEA level in the CRC with liver metastasis group was 162.03±810.01 ng/mL,while that in the CRC alone group was 5.71±9.76 ng/mL;the average serum CA125 levels were 43.47±83.52 U/mL respectively.and 13.5±19.68 U/mL;the average serum CA19-9 levels were 184.46±473.13 U/mL and 26.55±43.96 U/mL respectively.In addition,binary logistic regression analysis showed that CA125 was significant in predicting CRC liver metastasis(P<0.05).ROC curve analysis results showed that the areas under the ROC curves of CEA,CA125 and CA19-9 were 0.607,0.692 and 0.586.CONCLUSION These results suggest that combined detection of these tumor markers may help early detection and intervention of CRC liver metastasis,thereby improving patient prognosis.展开更多
Objective To investigate the role and molecular mechanism of exosomal miR-224-5p in colorectal cancer(CRC).Methods The miR-224-5p expression in CRC patient tissues and cell-derived exosomes was measured by laser captu...Objective To investigate the role and molecular mechanism of exosomal miR-224-5p in colorectal cancer(CRC).Methods The miR-224-5p expression in CRC patient tissues and cell-derived exosomes was measured by laser capture microdissection and qRT-PCR,respectively.Dual-luciferase reporter gene assay was used to determine the target gene of miR-224-5p.The protein expressions of p53 and unc-51 like kinase 2(ULK2)in CRC cells were detected by western blot.Flow cytometry was used to detect cell cycle and apoptosis.Cell proliferation was measured by CCK8 and EdU assay.Results The miR-224-5p expression was upregulated in CRC tissues and increased progressively with the rise of CRC stage.CRC cells secreted extracellular miR-224-5p mainly in an exosome-dependent manner,and then miR-224-5p could be transferred to surrounding tumor cells to regulate cell proliferation in the form of autocrine or paracrine.Moreover,ULK2 was characterized as a direct target of miR-224-5p and was downregulated in CRC tissues.Interestingly,ULK2 inhibited CRC cell proliferation in a p53-dependent manner.Furthermore,exosome-derived miR-224-5p partially reversed the proliferation regulation of ULK2 on CRC cells.Conclusion Our findings demonstrate that exosome-transmitted miR-224-5p promotes p53-dependent cell proliferation by targeting ULK2 in CRC,which may offer promising targets for CRC prevention and therapy.展开更多
BACKGROUND Gastric cancer(GC)is a prevalent malignant tumor of the gastrointestinal system.ZNF710 is a transcription factor(TF),and zinc finger protein 710(ZNF710)-AS1-201 is an immune-related long noncoding RNA(lncRN...BACKGROUND Gastric cancer(GC)is a prevalent malignant tumor of the gastrointestinal system.ZNF710 is a transcription factor(TF),and zinc finger protein 710(ZNF710)-AS1-201 is an immune-related long noncoding RNA(lncRNA)that is upregulated in GC cells.AIM To assess the correlation between ZNF710-AS1-201 and immune microenvir-onment features and to investigate the roles of ZNF710-AS1-201 in the invasion and metastasis processes of GC cells.METHODS We obtained data from The Cancer Genome Atlas and Wujin Hospital.We assessed cell growth,migration,invasion,and programmed cell death using cell counting kit-8,EdU,scratch,Transwell,and flow cytometry assays.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to identify the potential downstream targets of ZNF710-AS1-201.RESULTS In GC tissues with low ZNF710-AS1-201 expression,immunoassays detected significant infiltration of various antitumor immune cells,such as memory CD8 T cells and activated CD4 T cells.In the low-expression group,the half-maximal inhibitory concentrations(IC_(50)s)of 5-fluorouracil,cisplatin,gemcitabine,and trametinib were lower,whereas the IC_(50)s of dasatinib and vorinostat were higher.The malignant degree of GC was higher and the stage was later in the high-expression group.Additionally,patients with high expression of ZNF710-AS1-201 had lower overall survival and disease-free survival rates.In vitro,the overexpression of ZNF710-AS1-201 greatly enhanced growth,metastasis,and infiltration while suppressing cell death in HGC-27 cells.In contrast,the reduced expression of ZNF710-AS1-201 greatly hindered cell growth,enhanced apoptosis,and suppressed the metastasis and invasion of MKN-45 cells.The expression changes in ZNF710 were significant,but the corresponding changes in isocitrate dehydrogenase-2,Semaphorin 4B,ARHGAP10,RGMB,hsa-miR-93-5p,and ZNF710-AS1-202 were not consistent or statistically significant after overexpression or knockdown of ZNF710-AS1-201,as determined by qRT-PCR.CONCLUSION Immune-related lncRNA ZNF710-AS1-201 facilitates the metastasis and invasion of GC cells.It appears that ZNF710-AS1-201 and ZNF710 have potential as effective targets for therapeutic intervention in GC.Nevertheless,it is still necessary to determine the specific targets of the ZNF710 TF.展开更多
BACKGROUND Gastric cancer(GC)is a common malignant tumor,long non-coding RNA and microRNA(miRNA)are important regulators that affect tumor proliferation,metastasis and chemotherapy resistance,and thus participate in t...BACKGROUND Gastric cancer(GC)is a common malignant tumor,long non-coding RNA and microRNA(miRNA)are important regulators that affect tumor proliferation,metastasis and chemotherapy resistance,and thus participate in tumor progression.CASC19 is a new bio-marker which can promote tumor invasion and metastasis.However,the mechanism by which CASC19 affects the progression of GC through miRNA is not clear.AIM To explore the role of the CASC19/miR-491-5p/HMGA2 regulatory axis in GC.METHODS To explore the expression and prognosis of CASC19 in GC through clinical samples,and investigate the effects of inhibiting CASC19 on the proliferation,migration,invasion and other functions of GC cells through cell counting Kit-8(CCK-8),ethynyldeoxyuridine,Wound healing assay,Transwell,Western blot and flow cytometry experiments.The effect of miR-491-5p and HMGA2 in GC were also proved.The regulatory relationship between CASC19 and miR-491-5p,miR-491-5p and HMGA2 were validated through Dual-luciferase reporter gene assay and reverse transcription PCR.Then CCK-8,Transwell,Wound healing assay,flow cytometry and animal experiments verify the role of CASC19/miR-491-5p/HMGA2 regulatory axis.RESULTS The expression level of CASC19 is related to the T stage,N stage,and tumor size of patients.Knockdown of the expression of CASC19 can inhibit the ability of proliferation,migration,invasion and EMT conversion of GC cells,and knocking down the expression of CASC19 can promote the apoptosis of GC cells.Increasing the expression of miR-491-5p can inhibit the proliferation of GC cells,miR-491-5p mimics can inhibit EMT conversion,and promote the apoptosis of GC cells,while decreasing the expression of miR-491-5p can promote the proliferation and EMT conversion and inhibit the apoptosis of GC cells.The expression of HMGA2 in GC tissues is higher than that in adjacent tissues.At the same time,the expression level of HMGA2 is related to the N and T stages of the patients.Reducing the level of HMGA2 can promote cell apoptosis and inhibit the proliferation of GC cells.Cell experiments and animal experiments have proved that CASC19 can regulates the expression of HMGA2 through miR-491-5p,thereby affecting the biological functions of GC.CONCLUSION CASC19 regulates the expression of HMGA2 through miR-491-5p to affect the development of GC.This axis may serve as a potential biomarker and therapeutic target of GC.展开更多
BACKGROUND Gastric cancer is a kind of malignant tumor which is prevalent all over the world.Although some progress has been made in the treatment of gastric cancer,its prognosis is still not optimistic,so it is of gr...BACKGROUND Gastric cancer is a kind of malignant tumor which is prevalent all over the world.Although some progress has been made in the treatment of gastric cancer,its prognosis is still not optimistic,so it is of great significance to find reliable prog-nostic indicators to guide the treatment and management of patients with gastric cancer.AIM To explore the relationship between serum levels of five biomarkers[carcinoem-bryonic antigen(CEA),carbohydrate antigen(CA)19-9,CA72-4,CA24-2,and ferritin]and prognosis in patients with gastric cancer.METHODS This study included 200 patients with gastric adenocarcinoma,and conducted an in-depth analysis of their baseline characteristics,relationship between tumor markers and staging,and prognosis.The study found that CA19-9 has a signi-ficant correlation with tumor stage,the average levels of CA24-2,CEA,CA72-4 and ferritin were slightly increased disregarding the stage of tumor.Survival analysis showed that increases in CEA,CA19-9,CA24-2,and ferritin were all associated with shortened overall survival of patients.Further multivariate ana-lysis revealed that elevated serum CA72-4 levels were an inde-pendent adverse prognostic factor.RESULTS This study reveals that there is a significant correlation between the expression levels of serum tumor markers CEA,CA19-9,CA72-4,CA24-2 and ferritin in patients with gastric cancer and prognosis,and can be used as important indicators for prognostic evaluation of gastric cancer.In particular,markers that appear abnormally elevated initially may help identify gastric cancer patients with poor prognosis.CONCLUSION Serum CEA and CA19-9 play an important role in the prognosis assessment of gastric cancer,and are effective tools to guide clinical practice and optimize individualized treatment strategies for gastric cancer patients.展开更多
BACKGROUND Heterogeneous ribonucleoprotein A1(hnRNPA1)has been reported to enhance the Warburg effect and promote colon cancer(CC)cell proliferation,but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in ...BACKGROUND Heterogeneous ribonucleoprotein A1(hnRNPA1)has been reported to enhance the Warburg effect and promote colon cancer(CC)cell proliferation,but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated.AIM To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway.METHODS Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b.The relationship between the expression values and the clinicopathological features of the patients was investigated.Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction,while differences in protein expression were analyzed using western blot.Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2’-deoxyuridine assays,and cell cycle and apoptosis were detected using flow cytometric assays.The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay.The Warburg effect was evaluated by glucose uptake and lactic acid production assays.RESULTS The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls(P<0.05).Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC,including stage I,II-III,and IV.Furthermore,the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification.HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway,thereby promoting proliferation of HCT116 and SW620 cells.However,the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b,effectively blocking the Warburg effect.CONCLUSION These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.展开更多
BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC...BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.展开更多
BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is ...BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the molecular mechanism of 18β-glycyrrhetinic acid(18β-GRA)regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.METHODS CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by a wound healing assay,the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated,and the cell autophagy level was determined by MDC staining.TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention,and then the protein-protein interaction was predicted using STRING(https://string-db.org/).MicroRNAs(miRNAs)transcriptome analysis was used to detect the miRNA differential expression profile,and use miRBase(https://www.mirbase/)and TargetScan(https://www.targetscan.org/)to predict the miRNA and complementary binding sites.Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells,and western blot was used to detect the expression of autophagy related proteins.Finally,the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.RESULTS 18β-GRA could inhibit GC cells viability,promote cell apoptosis,block cell cycle,reduce cell wound healing ability,and inhibit the GC cells growth in vivo.MDC staining results showed that 18β-GRA could promote autophagy in GC cells.By TMT proteomic analysis and miRNAs transcriptome analysis,it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells.Subsequently,we verified that TGM2 is the target of miR-345-5p,and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2.Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced,and LC3II,ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA.Overexpression of miR-345-5p not only inhibited the expression of TGM2,but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.CONCLUSION 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.展开更多
BACKGROUND microRNA-627-5p(miR-627-5p)dysregulation has been observed in several cancer types,such as hepatocellular carcinoma,oral squamous cell carcinoma,glioblastoma multiforme,and gastric cancer.The biological fun...BACKGROUND microRNA-627-5p(miR-627-5p)dysregulation has been observed in several cancer types,such as hepatocellular carcinoma,oral squamous cell carcinoma,glioblastoma multiforme,and gastric cancer.The biological function of miR-627-5p in colorectal cancer(CRC)growth and metastasis is yet unclear.AIM To investigate the effects of miR-627-5p on the malignant biological properties of colorectal malignant tumour cells by targeting Wnt2.METHODS The levels of miR-627-5p in colorectal tumour tissues were assessed in Gene Expression Omnibus datasets.In order to identify Wnt2 transcript expression in CRC tissues,quantitative real-time polymerase chain reaction(qRT-PCR)analysis was used.Luciferase reporter tests were used to explore whether miR-627-5p might potentially target Wnt2.Wnt2 transcript and protein levels were detected in CRC cells with high miR-627-5p expression.To learn more about how miR-627-5p affects CRC development,migration,apoptosis,and invasion,functional experiments were conducted.Cotransfection with the overexpression vector of Wnt2 and miR-627-5p mimics was utilized to verify whether overexpression of Wnt2 could cancel the impact of miR-627-5p in CRC.Western blot and qRT-PCR were conducted to investigate the effects of miR-627-5p on the Wnt/β-catenin signalling pathway.RESULTS miR-627-5p was notably decreased in colorectal tumour tissues,while the gene level of Wnt2 was notably upregulated.A dual luciferase reporter assay revealed that miR-627-5p specifically targets the 3’-untranslated regions of Wnt2 and miR-627-5p upregulation markedly reduced the protein and gene expression of Wnt2 in CRC cells.In vitro gain-of-function assays displayed that miR-627-5p overexpression decreased CRC cells’capabilities to invade,move,and remain viable while increasing apoptosis.Wnt2 overexpression could reverse the suppressive functions of miR-627-5p.Moreover,upregulation of miR-627-5p suppressed the transcript and protein levels of the downstream target factors in the canonical Wnt/β-catenin signalling,such as c-myc,CD44,β-catenin,and cyclinD1.CONCLUSION miR-627-5p acts as a critical inhibitory factor in CRC,possibly by directly targeting Wnt2 and negatively modulating the Wnt/β-catenin signalling,revealing that miR-627-5p could be a possible treatment target for CRC.展开更多
AIM: To assess the diagnostic accuracy of multidetectorrow computed tomography(MDCT) as compared with conventional magnetic resonance imaging(MRI), in identifying mesorectal fascia(MRF) invasion in rectal cancer patie...AIM: To assess the diagnostic accuracy of multidetectorrow computed tomography(MDCT) as compared with conventional magnetic resonance imaging(MRI), in identifying mesorectal fascia(MRF) invasion in rectal cancer patients.METHODS: Ninety-one patients with biopsy proven rectal adenocarcinoma referred for thoracic and abdominal CT staging were enrolled in this study. The contrast-enhanced MDCT scans were performed on a 256 row scanner(ICT, Philips) with the following acquisition parameters: tube voltage 120 KV, tube current 150-300 m As. Imaging data were reviewed as axial and as multiplanar reconstructions(MPRs) images along the rectal tumor axis. MRI study, performed on 1.5 T with dedicated phased array multicoil, included multiplanar T2 and axial T1 sequences and diffusion weighted images(DWI). Axial and MPR CT images independently were compared to MRI and MRF involvement was determined. Diagnostic accuracy of both modalities was compared and statistically analyzed.RESULTS: According to MRI, the MRF was involved in 51 patients and not involved in 40 patients. DWI allowed to recognize the tumor as a focal mass with high signal intensity on high b-value images, compared with the signal of the normal adjacent rectal wall or with the lower tissue signal intensity background. The number of patients correctly staged by the native axial CT images was 71 out of 91(41 with involved MRF; 30 with not involved MRF), while by using the MPR 80 patients were correctly staged(45 with involved MRF; 35 with not involved MRF). Local tumor staging suggested by MDCT agreed with those of MRI, obtaining for CT axial images sensitivity and specificity of 80.4% and 75%, positive predictive value(PPV) 80.4%, negative predictive value(NPV) 75% and accuracy 78%; while performing MPR the sensitivity and specificity increased to 88% and 87.5%, PPV was 90%, NPV 85.36% and accuracy 88%. MPR images showed higher diagnostic accuracy, in terms of MRF involvement, than native axial images, as compared to the reference magnetic resonance images. The difference in accuracy was statistically significant(P = 0.02). CONCLUSION: New generation CT scanner, using high resolution MPR images, represents a reliable diagnostic tool in assessment of loco-regional and whole body staging of advanced rectal cancer, especially in patients with MRI contraindications.展开更多
BACKGROUND As reported,multiple circular RNAs(circRNAs)interfere with colorectal cancer(CRC)progression.Here,circRNA_0001658(circ_0001658)is focused on studying how it works in CRC.AIM Clarify the expression pattern,b...BACKGROUND As reported,multiple circular RNAs(circRNAs)interfere with colorectal cancer(CRC)progression.Here,circRNA_0001658(circ_0001658)is focused on studying how it works in CRC.AIM Clarify the expression pattern,biological function,and underlying mechanism of circ_0001658 of CRC tumorigenesis.METHODS In CRC-related chip data retrieved using the database named Gene Expression Omnibus,different expressions of circRNAs between CRC and normal tissue samples were identified.Quantitative Real-time PCR and Western blot ensured the analysis on circ_0001658,microRNA-590-5P(miR-590-5p),and methyltransferase-like 3(METTL3)mRNA expressions in tissues and cells.Cell counting kit-8 and flow cytometry were used to detect cell proliferation,apoptosis and migration.The targeting relations between circ_0001658,miR-590-5p,and METTL3 mRNA 3'-untranslated region were under the verification of bioinformatics prediction and dual luciferase-based reporter gene assays.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were employed on the downstream targets of miR-590-5p using the Database for Annotation,Visualization and Integrated Discovery database.RESULTS Circ_0001658 and METTL3 mRNA was elevated in CRC tissues and cells,whereas miR-590-5p was decreased.Circ_0001658 overexpression promoted the proliferation of HT29 cells,inhibited apoptosis,and accelerated the cell cycle.In SW480 cells,knocking down circ_0001658 had the opposite effect.Circ_0001658 could specifically bind to miR-590-5p and negatively modulate its expressions;METTL3 is a miR-590-5p target that can be positively regulated by circ 0001658.Circ 0001658 was inversely associated with miR-590-5p expression while positively with METTL3 expressions.CONCLUSION Circ_0001658 regulates the miR-590-5p/METTL 3-axis to increase CRC cell growth and decrease apoptosis.展开更多
Objective:To investigate the role of miR-129-5p in regulating HMGB1 expression in pancreatic cancer cell apoptosis.Methods:The untreated pancreatic cancer SW1990 cells were used as the control group.Mimics-NC(empty ve...Objective:To investigate the role of miR-129-5p in regulating HMGB1 expression in pancreatic cancer cell apoptosis.Methods:The untreated pancreatic cancer SW1990 cells were used as the control group.Mimics-NC(empty vector),miR-129-5p mimics,inhibitor-NC(empty vector)and miR-129-5p inhibitor were transfected into SW1990 cells by liposome transfection method as the mimics-NC group,miR-129-5p overexpression group(miR-129-5p mimics group),inhibitor-NC group and miR-129-5p low expression group(miR-129-5p inhibitor group).The binding site of miR-129-5p and HMGB1 was predicted by online target gene prediction website Target genes,and the targeting relationship between miR-129-5p and HMGB1 was verified by dual luciferase gene report experiment.The expression of miR-129-5p in each group was detected by qRT-PCR,and the expression of HMGB1 protein and apoptosis-related proteins Caspase 3 and Bcl-2 by Western blot.Hoechst staining was used to observe the changes of apoptosis.Results:Compared with the mimics-NC group and control group,miR-129-5p mimics transfection significantly up-regulated miR-129-5p level(P<0.01),inhibited HMGB1(P<0.01)and Bcl-2(P<0.05)protein expression,pro-moted Caspase 3 protein expression(P<0.05),and promoted apoptosis;compared with the inhibitor-NC group and control group,miR-129-5p inhibitor transfection significantly down-regulated miR-129-5p level(P<0.05),promoted HMGB1 and Bcl-2 protein expression(all P<0.05),inhibited Caspase 3protein expression(P<0.01),and inhibited apoptosis.The results of dual luciferase reporter gene assay showed that miR-129-5p could inhibit the fluorescence activity of wildtype HMGB1 cells and target the expression of HMGB1.Conclusion:miR-129-5p promotes the apoptosis of pancreatic cancer SW1990 cells by targeting inhibition of HMGB1 expression.展开更多
BACKGROUND The molecular mechanisms of colorectal cancer development and progression are far from being elucidated.AIM To investigate the role of microRNA-363-3p(miR-363-3p)in the progression of colorectal cancer.METH...BACKGROUND The molecular mechanisms of colorectal cancer development and progression are far from being elucidated.AIM To investigate the role of microRNA-363-3p(miR-363-3p)in the progression of colorectal cancer.METHODS Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues.PITA 6 was utilized to predict the targets of miR-363-3p.Dual-luciferase reporter system was used to validate the target of miR-363-3p.Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells’clonogenic survival ability and migration ability,respectively.Cell proliferation was examined by cell counting kit-8 assay.Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1(IFITM1)in colorectal cancer tissues and adjacent tissues.The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients.A colorectal cancer cell line with a deficiency of IFITM1 was constructed,and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified.RESULTS MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues.IFITM1 was characterized as a direct target of miR-363-3p.Overexpression of miR-363-3p led to decreased clonogenic survival,proliferation,and migration of colorectal cancer cells,which could be reversed by forced IFITM1 expression.CONCLUSION MiR-363-3p can constrain clonogenic survival,proliferation,and migration of colorectal cancer cells via targeting IFITM1.展开更多
BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer...BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.展开更多
Objective: To study the relationship between the methylation status of multi-drug resistance protein (MRP) gene and the expression of its mRNA and protein in lung cancer cell lines. Methods: Human embryo lung cell...Objective: To study the relationship between the methylation status of multi-drug resistance protein (MRP) gene and the expression of its mRNA and protein in lung cancer cell lines. Methods: Human embryo lung cell line WI-38, lung adenocarcinoma cell line SPCA-1 and its drug-resistant cells induced by different concentrations of doxorubicin were treated with restriction endonuclease Eco47III. The methylation status of MRP was examined by PCR, and the expressions of its mRNA and protein were evaluated by in situ hybridization and immunohistochemistry. Results: MRP gene promoter region of WI-38 cells was in hypermethylation status, but the promoter region of MRP in SPCA-1 cells and their resistant derivatives induced by different concentrations of doxorubicin were in hypomethylation status. There were significant differences in the expression of MRP mRNA among WI-38 cell line, SPCA-1 cells and their drug-resistant derivatives induced by different concentration of doxorubicin. Consistently, MRP immunostaining presented similar significant differences. Conclusion: The promoter region of MRP in SPCA-1 lung adenocarcinoma cells was in hypomethylation status. The hypomethylation status of 5' regulatory region of MRP promoter is an important structural basis that can increase the activity of transcription and results in the development of drug resistance in lung cancer.展开更多
Objective To investigate miR-183-5p targeting to forkhead box protein O1(FOXO1)and its corresponding effect on the proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)of non-small cell lung canc...Objective To investigate miR-183-5p targeting to forkhead box protein O1(FOXO1)and its corresponding effect on the proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)of non-small cell lung cancer(NSCLC)cells.Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection.Several cell lines were cultured in vitro,including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549,SPCA-1,PC-9,and 95-D.miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR;the corresponding correlations in NSCLC tissues were analyzed using the Pearson test,and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed.The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase,RNA-binding protein immunoprecipitation(RIP)assay,and pull-down experiments.A549 cells were divided into control,anti-miR-NC,anti-miR-183-5p,miR-NC,miR-183-5p,miR-183-5p+pcDNA3.1,and miR-183-5p+pcDNA3.1-FOXO1 groups.Cell proliferation,invasion,migration,apoptosis,and cell cycle distribution were detected using an MTT assay,clone formation assay,Transwell assay,scratch test,and flow cytometry,respectively.The expression of EMT-related proteins in the cells was analyzed by western blotting.The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice.Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues,whereas FOXO1 mRNA expression was significantly down-regulated.There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues(P<0.05).Additionally,the expression of miR-183-5p was significantly correlated with tumor size,tumor differentiation,and tumor-node-metastasis stage in patients with NSCLC(P<0.05).miR-183-5p targeted and inhibited FOXO1 expression.Compared to the anti-miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group,whereas the protein expression of E-cadherin andα-catenin and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group(P<0.05).Compared to the miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells in the miR-183-5p group were significantly higher,whereas the E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower;furthermore,the frequency of colony formation and invasion were significantly higher in the miR-183-5p group(P<0.05).Compared with the miR-183-5p+pcDNA3.1 group,the OD value,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group,whereas E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group(P<0.05).Overall,silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression.Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation,migration,invasion,and EMT,of NSCLC cells by down-regulating FOXO1 expression.展开更多
Magnetic resonance (MR) imaging has been increasingly used in the evaluation of prostate cancer. As studies have suggested that the majority of cancers arise from the peripheral zone (PZ), MR imaging has focused on th...Magnetic resonance (MR) imaging has been increasingly used in the evaluation of prostate cancer. As studies have suggested that the majority of cancers arise from the peripheral zone (PZ), MR imaging has focused on the PZ of the prostate gland thus far. However, a considerable number of cancers (up to 30%) originate in the transition zone (TZ), substantially contributing to morbidity and mortality. Therefore, research is needed on the TZ of the prostate gland. Recently, MR imaging and advanced MR techniques have been gaining acceptance in evaluation of the TZ. In this article, the MR imaging features of TZ prostate cancers, the role of MR imaging in TZ cancer detection and staging, and recent advanced MR techniques will be discussed in light of the literature.展开更多
Background:Hepatocellular carcinoma(HCC),the most common type of primary liver cancer,is the fourth leading cause of cancer-related deaths worldwide.Previous evidence shows that the expression of circulating RNA ZFR(c...Background:Hepatocellular carcinoma(HCC),the most common type of primary liver cancer,is the fourth leading cause of cancer-related deaths worldwide.Previous evidence shows that the expression of circulating RNA ZFR(circZFR)is upregulated in HCC tissues.However,the molecular mechanism of circZFR in HCC is unclear.Methods:Quantitative reverse transcriptase polymerase chain reaction(qRT-PCR)was employed to detect the expression of circZFR,microRNA-624-3p(miR-624-3p)and WEE1 in HCC tissues and cells.RNase R assay and actinomycin D treatment assay were used to analyze the characteristics of circZFR.For functional analysis,the capacities of colony formation,cell proliferation,cell apoptosis,migration and invasion were assessed by colony formation assay,5-ethynyl-2-deoxyuridine(EdU)assay,flow cytometry assay and transwell assay.Western blot was used to examine the protein levels of WEE1 and epithelial-mesenchymal transition(EMT)-related proteins.The interactions between miR-624-3p and circZFR or WEE1 were validated by dual-luciferase reporter assay and RNA immunoprecipitation(RIP)assay.Xenograft models were established to determine the role of circZFR in vivo.Results:circZFR and WEE1 were upregulated,while miR-624-3p expression was reduced in HCC tissues and cells.circZFR could sponge miR-624-3p,and WEE1 was a downstream gene of miR-624-3p.Knockdown of circZFR significantly reduced the malignant behaviors of HCC and that co-transfection with miR624-3p inhibitor restored this change.Overexpression of WEE1 abolished the inhibitory effect of miR624-3p mimic on HCC cells.Mechanistically,circZFR acted as a competitive endogenous RNA(ceRNA)to regulate WEE1 expression by targeting miR-624-3p.Furthermore,in vivo studies have illustrated that circZFR knockdown inhibited tumor growth.Conclusions:circZFR knockdown reduced HCC cell proliferation,migration and invasion and promoted apoptosis by regulating the miR-624-3p/WEE1 axis,suggesting that the circZFR/miR-624-3p/WEE1 axis might be a potential target for HCC treatment.展开更多
文摘BACKGROUND Hepatic metastases are common and difficult to treat after colorectal cancer(CRC)surgery.The predictive value of carcinoembryonic antigen(CEA),cancer antigen(CA)125 and CA19-9 combined tests for liver metastasis is unclear.AIM To evaluate predictive value of combined tests for CEA,CA125,and CA19-9 levels in patients with liver metastases of CRC.METHODS The retrospective study included patients with CRC alone(50 cases)and patients with CRC combined with liver metastases(50 cases)who were hospitalized between January 2021 and January 2023.Serum CEA,CA125 and CA19-9 levels were compared between the two groups,and binary logistic regression was used to analyze the predictive value of the combination of these tumor markers in liver metastasis.In addition,we performed receiver operating characteristic(ROC)curve analysis to assess its diagnostic accuracy.RESULTS The results showed that the serum CEA,CA125 and CA19-9 levels in the CRC with liver metastasis group were significantly higher than those in the CRC alone group.Specifically,the average serum CEA level in the CRC with liver metastasis group was 162.03±810.01 ng/mL,while that in the CRC alone group was 5.71±9.76 ng/mL;the average serum CA125 levels were 43.47±83.52 U/mL respectively.and 13.5±19.68 U/mL;the average serum CA19-9 levels were 184.46±473.13 U/mL and 26.55±43.96 U/mL respectively.In addition,binary logistic regression analysis showed that CA125 was significant in predicting CRC liver metastasis(P<0.05).ROC curve analysis results showed that the areas under the ROC curves of CEA,CA125 and CA19-9 were 0.607,0.692 and 0.586.CONCLUSION These results suggest that combined detection of these tumor markers may help early detection and intervention of CRC liver metastasis,thereby improving patient prognosis.
基金supported by the National Natural Science Foundation of China[Grant Number:81972803]。
文摘Objective To investigate the role and molecular mechanism of exosomal miR-224-5p in colorectal cancer(CRC).Methods The miR-224-5p expression in CRC patient tissues and cell-derived exosomes was measured by laser capture microdissection and qRT-PCR,respectively.Dual-luciferase reporter gene assay was used to determine the target gene of miR-224-5p.The protein expressions of p53 and unc-51 like kinase 2(ULK2)in CRC cells were detected by western blot.Flow cytometry was used to detect cell cycle and apoptosis.Cell proliferation was measured by CCK8 and EdU assay.Results The miR-224-5p expression was upregulated in CRC tissues and increased progressively with the rise of CRC stage.CRC cells secreted extracellular miR-224-5p mainly in an exosome-dependent manner,and then miR-224-5p could be transferred to surrounding tumor cells to regulate cell proliferation in the form of autocrine or paracrine.Moreover,ULK2 was characterized as a direct target of miR-224-5p and was downregulated in CRC tissues.Interestingly,ULK2 inhibited CRC cell proliferation in a p53-dependent manner.Furthermore,exosome-derived miR-224-5p partially reversed the proliferation regulation of ULK2 on CRC cells.Conclusion Our findings demonstrate that exosome-transmitted miR-224-5p promotes p53-dependent cell proliferation by targeting ULK2 in CRC,which may offer promising targets for CRC prevention and therapy.
基金Changzhou Sci and Tech Program,No.CJ20220008Young Talent Development Plan of Changzhou Health Commission,No.CZQM2020118+2 种基金Changzhou High-Level Medical Talents Training Project,No.2022CZBJ105Cultivation Project of Changzhou Medical Center,Nanjing Medical University,No.CMCB202211Development Foundation of Affiliated Hospital of Xuzhou Medical University,No.XYFC202304,and No.XYFM202307。
文摘BACKGROUND Gastric cancer(GC)is a prevalent malignant tumor of the gastrointestinal system.ZNF710 is a transcription factor(TF),and zinc finger protein 710(ZNF710)-AS1-201 is an immune-related long noncoding RNA(lncRNA)that is upregulated in GC cells.AIM To assess the correlation between ZNF710-AS1-201 and immune microenvir-onment features and to investigate the roles of ZNF710-AS1-201 in the invasion and metastasis processes of GC cells.METHODS We obtained data from The Cancer Genome Atlas and Wujin Hospital.We assessed cell growth,migration,invasion,and programmed cell death using cell counting kit-8,EdU,scratch,Transwell,and flow cytometry assays.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to identify the potential downstream targets of ZNF710-AS1-201.RESULTS In GC tissues with low ZNF710-AS1-201 expression,immunoassays detected significant infiltration of various antitumor immune cells,such as memory CD8 T cells and activated CD4 T cells.In the low-expression group,the half-maximal inhibitory concentrations(IC_(50)s)of 5-fluorouracil,cisplatin,gemcitabine,and trametinib were lower,whereas the IC_(50)s of dasatinib and vorinostat were higher.The malignant degree of GC was higher and the stage was later in the high-expression group.Additionally,patients with high expression of ZNF710-AS1-201 had lower overall survival and disease-free survival rates.In vitro,the overexpression of ZNF710-AS1-201 greatly enhanced growth,metastasis,and infiltration while suppressing cell death in HGC-27 cells.In contrast,the reduced expression of ZNF710-AS1-201 greatly hindered cell growth,enhanced apoptosis,and suppressed the metastasis and invasion of MKN-45 cells.The expression changes in ZNF710 were significant,but the corresponding changes in isocitrate dehydrogenase-2,Semaphorin 4B,ARHGAP10,RGMB,hsa-miR-93-5p,and ZNF710-AS1-202 were not consistent or statistically significant after overexpression or knockdown of ZNF710-AS1-201,as determined by qRT-PCR.CONCLUSION Immune-related lncRNA ZNF710-AS1-201 facilitates the metastasis and invasion of GC cells.It appears that ZNF710-AS1-201 and ZNF710 have potential as effective targets for therapeutic intervention in GC.Nevertheless,it is still necessary to determine the specific targets of the ZNF710 TF.
基金Supported by Natural Science Foundation of Anhui Province,No.2108085QH337Research Fund of Anhui Medical University,No.2022xkj156+1 种基金Key Projects of Anhui Provincial Department of Education,No.2023AH053330Anhui Institute of Translational Medicine Research Fund,No.2022zhyx-C88.
文摘BACKGROUND Gastric cancer(GC)is a common malignant tumor,long non-coding RNA and microRNA(miRNA)are important regulators that affect tumor proliferation,metastasis and chemotherapy resistance,and thus participate in tumor progression.CASC19 is a new bio-marker which can promote tumor invasion and metastasis.However,the mechanism by which CASC19 affects the progression of GC through miRNA is not clear.AIM To explore the role of the CASC19/miR-491-5p/HMGA2 regulatory axis in GC.METHODS To explore the expression and prognosis of CASC19 in GC through clinical samples,and investigate the effects of inhibiting CASC19 on the proliferation,migration,invasion and other functions of GC cells through cell counting Kit-8(CCK-8),ethynyldeoxyuridine,Wound healing assay,Transwell,Western blot and flow cytometry experiments.The effect of miR-491-5p and HMGA2 in GC were also proved.The regulatory relationship between CASC19 and miR-491-5p,miR-491-5p and HMGA2 were validated through Dual-luciferase reporter gene assay and reverse transcription PCR.Then CCK-8,Transwell,Wound healing assay,flow cytometry and animal experiments verify the role of CASC19/miR-491-5p/HMGA2 regulatory axis.RESULTS The expression level of CASC19 is related to the T stage,N stage,and tumor size of patients.Knockdown of the expression of CASC19 can inhibit the ability of proliferation,migration,invasion and EMT conversion of GC cells,and knocking down the expression of CASC19 can promote the apoptosis of GC cells.Increasing the expression of miR-491-5p can inhibit the proliferation of GC cells,miR-491-5p mimics can inhibit EMT conversion,and promote the apoptosis of GC cells,while decreasing the expression of miR-491-5p can promote the proliferation and EMT conversion and inhibit the apoptosis of GC cells.The expression of HMGA2 in GC tissues is higher than that in adjacent tissues.At the same time,the expression level of HMGA2 is related to the N and T stages of the patients.Reducing the level of HMGA2 can promote cell apoptosis and inhibit the proliferation of GC cells.Cell experiments and animal experiments have proved that CASC19 can regulates the expression of HMGA2 through miR-491-5p,thereby affecting the biological functions of GC.CONCLUSION CASC19 regulates the expression of HMGA2 through miR-491-5p to affect the development of GC.This axis may serve as a potential biomarker and therapeutic target of GC.
文摘BACKGROUND Gastric cancer is a kind of malignant tumor which is prevalent all over the world.Although some progress has been made in the treatment of gastric cancer,its prognosis is still not optimistic,so it is of great significance to find reliable prog-nostic indicators to guide the treatment and management of patients with gastric cancer.AIM To explore the relationship between serum levels of five biomarkers[carcinoem-bryonic antigen(CEA),carbohydrate antigen(CA)19-9,CA72-4,CA24-2,and ferritin]and prognosis in patients with gastric cancer.METHODS This study included 200 patients with gastric adenocarcinoma,and conducted an in-depth analysis of their baseline characteristics,relationship between tumor markers and staging,and prognosis.The study found that CA19-9 has a signi-ficant correlation with tumor stage,the average levels of CA24-2,CEA,CA72-4 and ferritin were slightly increased disregarding the stage of tumor.Survival analysis showed that increases in CEA,CA19-9,CA24-2,and ferritin were all associated with shortened overall survival of patients.Further multivariate ana-lysis revealed that elevated serum CA72-4 levels were an inde-pendent adverse prognostic factor.RESULTS This study reveals that there is a significant correlation between the expression levels of serum tumor markers CEA,CA19-9,CA72-4,CA24-2 and ferritin in patients with gastric cancer and prognosis,and can be used as important indicators for prognostic evaluation of gastric cancer.In particular,markers that appear abnormally elevated initially may help identify gastric cancer patients with poor prognosis.CONCLUSION Serum CEA and CA19-9 play an important role in the prognosis assessment of gastric cancer,and are effective tools to guide clinical practice and optimize individualized treatment strategies for gastric cancer patients.
基金Supported by the National Natural Science Foundation of China,No.82160405Jiangxi Provincial Natural Science Foundation,No.20232BAB206131,No.20212ACB206016,and No.20224BAB206114+1 种基金Jiangxi Provincial Health Commission Project,No.202310887the Development Fund of Jiangxi Cancer Hospital,No.2021J10.
文摘BACKGROUND Heterogeneous ribonucleoprotein A1(hnRNPA1)has been reported to enhance the Warburg effect and promote colon cancer(CC)cell proliferation,but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated.AIM To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway.METHODS Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b.The relationship between the expression values and the clinicopathological features of the patients was investigated.Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction,while differences in protein expression were analyzed using western blot.Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2’-deoxyuridine assays,and cell cycle and apoptosis were detected using flow cytometric assays.The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay.The Warburg effect was evaluated by glucose uptake and lactic acid production assays.RESULTS The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls(P<0.05).Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC,including stage I,II-III,and IV.Furthermore,the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification.HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway,thereby promoting proliferation of HCT116 and SW620 cells.However,the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b,effectively blocking the Warburg effect.CONCLUSION These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.
文摘BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.
基金Supported by the Ningxia Natural Science Foundation,No.2022AAC03144.
文摘BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the molecular mechanism of 18β-glycyrrhetinic acid(18β-GRA)regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.METHODS CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by a wound healing assay,the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated,and the cell autophagy level was determined by MDC staining.TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention,and then the protein-protein interaction was predicted using STRING(https://string-db.org/).MicroRNAs(miRNAs)transcriptome analysis was used to detect the miRNA differential expression profile,and use miRBase(https://www.mirbase/)and TargetScan(https://www.targetscan.org/)to predict the miRNA and complementary binding sites.Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells,and western blot was used to detect the expression of autophagy related proteins.Finally,the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.RESULTS 18β-GRA could inhibit GC cells viability,promote cell apoptosis,block cell cycle,reduce cell wound healing ability,and inhibit the GC cells growth in vivo.MDC staining results showed that 18β-GRA could promote autophagy in GC cells.By TMT proteomic analysis and miRNAs transcriptome analysis,it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells.Subsequently,we verified that TGM2 is the target of miR-345-5p,and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2.Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced,and LC3II,ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA.Overexpression of miR-345-5p not only inhibited the expression of TGM2,but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.CONCLUSION 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.
基金Supported by the National Key Development Plan for Precision Medicine Research,No.2017YFC0910002.
文摘BACKGROUND microRNA-627-5p(miR-627-5p)dysregulation has been observed in several cancer types,such as hepatocellular carcinoma,oral squamous cell carcinoma,glioblastoma multiforme,and gastric cancer.The biological function of miR-627-5p in colorectal cancer(CRC)growth and metastasis is yet unclear.AIM To investigate the effects of miR-627-5p on the malignant biological properties of colorectal malignant tumour cells by targeting Wnt2.METHODS The levels of miR-627-5p in colorectal tumour tissues were assessed in Gene Expression Omnibus datasets.In order to identify Wnt2 transcript expression in CRC tissues,quantitative real-time polymerase chain reaction(qRT-PCR)analysis was used.Luciferase reporter tests were used to explore whether miR-627-5p might potentially target Wnt2.Wnt2 transcript and protein levels were detected in CRC cells with high miR-627-5p expression.To learn more about how miR-627-5p affects CRC development,migration,apoptosis,and invasion,functional experiments were conducted.Cotransfection with the overexpression vector of Wnt2 and miR-627-5p mimics was utilized to verify whether overexpression of Wnt2 could cancel the impact of miR-627-5p in CRC.Western blot and qRT-PCR were conducted to investigate the effects of miR-627-5p on the Wnt/β-catenin signalling pathway.RESULTS miR-627-5p was notably decreased in colorectal tumour tissues,while the gene level of Wnt2 was notably upregulated.A dual luciferase reporter assay revealed that miR-627-5p specifically targets the 3’-untranslated regions of Wnt2 and miR-627-5p upregulation markedly reduced the protein and gene expression of Wnt2 in CRC cells.In vitro gain-of-function assays displayed that miR-627-5p overexpression decreased CRC cells’capabilities to invade,move,and remain viable while increasing apoptosis.Wnt2 overexpression could reverse the suppressive functions of miR-627-5p.Moreover,upregulation of miR-627-5p suppressed the transcript and protein levels of the downstream target factors in the canonical Wnt/β-catenin signalling,such as c-myc,CD44,β-catenin,and cyclinD1.CONCLUSION miR-627-5p acts as a critical inhibitory factor in CRC,possibly by directly targeting Wnt2 and negatively modulating the Wnt/β-catenin signalling,revealing that miR-627-5p could be a possible treatment target for CRC.
文摘AIM: To assess the diagnostic accuracy of multidetectorrow computed tomography(MDCT) as compared with conventional magnetic resonance imaging(MRI), in identifying mesorectal fascia(MRF) invasion in rectal cancer patients.METHODS: Ninety-one patients with biopsy proven rectal adenocarcinoma referred for thoracic and abdominal CT staging were enrolled in this study. The contrast-enhanced MDCT scans were performed on a 256 row scanner(ICT, Philips) with the following acquisition parameters: tube voltage 120 KV, tube current 150-300 m As. Imaging data were reviewed as axial and as multiplanar reconstructions(MPRs) images along the rectal tumor axis. MRI study, performed on 1.5 T with dedicated phased array multicoil, included multiplanar T2 and axial T1 sequences and diffusion weighted images(DWI). Axial and MPR CT images independently were compared to MRI and MRF involvement was determined. Diagnostic accuracy of both modalities was compared and statistically analyzed.RESULTS: According to MRI, the MRF was involved in 51 patients and not involved in 40 patients. DWI allowed to recognize the tumor as a focal mass with high signal intensity on high b-value images, compared with the signal of the normal adjacent rectal wall or with the lower tissue signal intensity background. The number of patients correctly staged by the native axial CT images was 71 out of 91(41 with involved MRF; 30 with not involved MRF), while by using the MPR 80 patients were correctly staged(45 with involved MRF; 35 with not involved MRF). Local tumor staging suggested by MDCT agreed with those of MRI, obtaining for CT axial images sensitivity and specificity of 80.4% and 75%, positive predictive value(PPV) 80.4%, negative predictive value(NPV) 75% and accuracy 78%; while performing MPR the sensitivity and specificity increased to 88% and 87.5%, PPV was 90%, NPV 85.36% and accuracy 88%. MPR images showed higher diagnostic accuracy, in terms of MRF involvement, than native axial images, as compared to the reference magnetic resonance images. The difference in accuracy was statistically significant(P = 0.02). CONCLUSION: New generation CT scanner, using high resolution MPR images, represents a reliable diagnostic tool in assessment of loco-regional and whole body staging of advanced rectal cancer, especially in patients with MRI contraindications.
文摘BACKGROUND As reported,multiple circular RNAs(circRNAs)interfere with colorectal cancer(CRC)progression.Here,circRNA_0001658(circ_0001658)is focused on studying how it works in CRC.AIM Clarify the expression pattern,biological function,and underlying mechanism of circ_0001658 of CRC tumorigenesis.METHODS In CRC-related chip data retrieved using the database named Gene Expression Omnibus,different expressions of circRNAs between CRC and normal tissue samples were identified.Quantitative Real-time PCR and Western blot ensured the analysis on circ_0001658,microRNA-590-5P(miR-590-5p),and methyltransferase-like 3(METTL3)mRNA expressions in tissues and cells.Cell counting kit-8 and flow cytometry were used to detect cell proliferation,apoptosis and migration.The targeting relations between circ_0001658,miR-590-5p,and METTL3 mRNA 3'-untranslated region were under the verification of bioinformatics prediction and dual luciferase-based reporter gene assays.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were employed on the downstream targets of miR-590-5p using the Database for Annotation,Visualization and Integrated Discovery database.RESULTS Circ_0001658 and METTL3 mRNA was elevated in CRC tissues and cells,whereas miR-590-5p was decreased.Circ_0001658 overexpression promoted the proliferation of HT29 cells,inhibited apoptosis,and accelerated the cell cycle.In SW480 cells,knocking down circ_0001658 had the opposite effect.Circ_0001658 could specifically bind to miR-590-5p and negatively modulate its expressions;METTL3 is a miR-590-5p target that can be positively regulated by circ 0001658.Circ 0001658 was inversely associated with miR-590-5p expression while positively with METTL3 expressions.CONCLUSION Circ_0001658 regulates the miR-590-5p/METTL 3-axis to increase CRC cell growth and decrease apoptosis.
基金National Natural Science Foundation of China (No.82260134)Natural Science Foundation of Guangxi (No.2023GXNSFAA026118)+1 种基金High-level Talents Research Project of the Affiliated Hospital of Youjiang Medical College for Nationalities (No.R202011702)Guangxi Graduate Education Innovation Project (No.YCSW2023506)。
文摘Objective:To investigate the role of miR-129-5p in regulating HMGB1 expression in pancreatic cancer cell apoptosis.Methods:The untreated pancreatic cancer SW1990 cells were used as the control group.Mimics-NC(empty vector),miR-129-5p mimics,inhibitor-NC(empty vector)and miR-129-5p inhibitor were transfected into SW1990 cells by liposome transfection method as the mimics-NC group,miR-129-5p overexpression group(miR-129-5p mimics group),inhibitor-NC group and miR-129-5p low expression group(miR-129-5p inhibitor group).The binding site of miR-129-5p and HMGB1 was predicted by online target gene prediction website Target genes,and the targeting relationship between miR-129-5p and HMGB1 was verified by dual luciferase gene report experiment.The expression of miR-129-5p in each group was detected by qRT-PCR,and the expression of HMGB1 protein and apoptosis-related proteins Caspase 3 and Bcl-2 by Western blot.Hoechst staining was used to observe the changes of apoptosis.Results:Compared with the mimics-NC group and control group,miR-129-5p mimics transfection significantly up-regulated miR-129-5p level(P<0.01),inhibited HMGB1(P<0.01)and Bcl-2(P<0.05)protein expression,pro-moted Caspase 3 protein expression(P<0.05),and promoted apoptosis;compared with the inhibitor-NC group and control group,miR-129-5p inhibitor transfection significantly down-regulated miR-129-5p level(P<0.05),promoted HMGB1 and Bcl-2 protein expression(all P<0.05),inhibited Caspase 3protein expression(P<0.01),and inhibited apoptosis.The results of dual luciferase reporter gene assay showed that miR-129-5p could inhibit the fluorescence activity of wildtype HMGB1 cells and target the expression of HMGB1.Conclusion:miR-129-5p promotes the apoptosis of pancreatic cancer SW1990 cells by targeting inhibition of HMGB1 expression.
文摘BACKGROUND The molecular mechanisms of colorectal cancer development and progression are far from being elucidated.AIM To investigate the role of microRNA-363-3p(miR-363-3p)in the progression of colorectal cancer.METHODS Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues.PITA 6 was utilized to predict the targets of miR-363-3p.Dual-luciferase reporter system was used to validate the target of miR-363-3p.Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells’clonogenic survival ability and migration ability,respectively.Cell proliferation was examined by cell counting kit-8 assay.Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1(IFITM1)in colorectal cancer tissues and adjacent tissues.The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients.A colorectal cancer cell line with a deficiency of IFITM1 was constructed,and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified.RESULTS MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues.IFITM1 was characterized as a direct target of miR-363-3p.Overexpression of miR-363-3p led to decreased clonogenic survival,proliferation,and migration of colorectal cancer cells,which could be reversed by forced IFITM1 expression.CONCLUSION MiR-363-3p can constrain clonogenic survival,proliferation,and migration of colorectal cancer cells via targeting IFITM1.
基金Supported by the National Natural Science Funds of China,No.81974448Guangdong Medical Research Foundation,No.B2019126Shenzhen Science and Technology Innovation Commission,No.JCYJ20210324135005013.
文摘BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.
基金This work was supported by grants from Shanghai Educational Committee Funds(No.99B18).
文摘Objective: To study the relationship between the methylation status of multi-drug resistance protein (MRP) gene and the expression of its mRNA and protein in lung cancer cell lines. Methods: Human embryo lung cell line WI-38, lung adenocarcinoma cell line SPCA-1 and its drug-resistant cells induced by different concentrations of doxorubicin were treated with restriction endonuclease Eco47III. The methylation status of MRP was examined by PCR, and the expressions of its mRNA and protein were evaluated by in situ hybridization and immunohistochemistry. Results: MRP gene promoter region of WI-38 cells was in hypermethylation status, but the promoter region of MRP in SPCA-1 cells and their resistant derivatives induced by different concentrations of doxorubicin were in hypomethylation status. There were significant differences in the expression of MRP mRNA among WI-38 cell line, SPCA-1 cells and their drug-resistant derivatives induced by different concentration of doxorubicin. Consistently, MRP immunostaining presented similar significant differences. Conclusion: The promoter region of MRP in SPCA-1 lung adenocarcinoma cells was in hypomethylation status. The hypomethylation status of 5' regulatory region of MRP promoter is an important structural basis that can increase the activity of transcription and results in the development of drug resistance in lung cancer.
文摘Objective To investigate miR-183-5p targeting to forkhead box protein O1(FOXO1)and its corresponding effect on the proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)of non-small cell lung cancer(NSCLC)cells.Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection.Several cell lines were cultured in vitro,including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549,SPCA-1,PC-9,and 95-D.miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR;the corresponding correlations in NSCLC tissues were analyzed using the Pearson test,and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed.The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase,RNA-binding protein immunoprecipitation(RIP)assay,and pull-down experiments.A549 cells were divided into control,anti-miR-NC,anti-miR-183-5p,miR-NC,miR-183-5p,miR-183-5p+pcDNA3.1,and miR-183-5p+pcDNA3.1-FOXO1 groups.Cell proliferation,invasion,migration,apoptosis,and cell cycle distribution were detected using an MTT assay,clone formation assay,Transwell assay,scratch test,and flow cytometry,respectively.The expression of EMT-related proteins in the cells was analyzed by western blotting.The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice.Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues,whereas FOXO1 mRNA expression was significantly down-regulated.There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues(P<0.05).Additionally,the expression of miR-183-5p was significantly correlated with tumor size,tumor differentiation,and tumor-node-metastasis stage in patients with NSCLC(P<0.05).miR-183-5p targeted and inhibited FOXO1 expression.Compared to the anti-miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group,whereas the protein expression of E-cadherin andα-catenin and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group(P<0.05).Compared to the miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells in the miR-183-5p group were significantly higher,whereas the E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower;furthermore,the frequency of colony formation and invasion were significantly higher in the miR-183-5p group(P<0.05).Compared with the miR-183-5p+pcDNA3.1 group,the OD value,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group,whereas E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group(P<0.05).Overall,silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression.Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation,migration,invasion,and EMT,of NSCLC cells by down-regulating FOXO1 expression.
文摘Magnetic resonance (MR) imaging has been increasingly used in the evaluation of prostate cancer. As studies have suggested that the majority of cancers arise from the peripheral zone (PZ), MR imaging has focused on the PZ of the prostate gland thus far. However, a considerable number of cancers (up to 30%) originate in the transition zone (TZ), substantially contributing to morbidity and mortality. Therefore, research is needed on the TZ of the prostate gland. Recently, MR imaging and advanced MR techniques have been gaining acceptance in evaluation of the TZ. In this article, the MR imaging features of TZ prostate cancers, the role of MR imaging in TZ cancer detection and staging, and recent advanced MR techniques will be discussed in light of the literature.
文摘Background:Hepatocellular carcinoma(HCC),the most common type of primary liver cancer,is the fourth leading cause of cancer-related deaths worldwide.Previous evidence shows that the expression of circulating RNA ZFR(circZFR)is upregulated in HCC tissues.However,the molecular mechanism of circZFR in HCC is unclear.Methods:Quantitative reverse transcriptase polymerase chain reaction(qRT-PCR)was employed to detect the expression of circZFR,microRNA-624-3p(miR-624-3p)and WEE1 in HCC tissues and cells.RNase R assay and actinomycin D treatment assay were used to analyze the characteristics of circZFR.For functional analysis,the capacities of colony formation,cell proliferation,cell apoptosis,migration and invasion were assessed by colony formation assay,5-ethynyl-2-deoxyuridine(EdU)assay,flow cytometry assay and transwell assay.Western blot was used to examine the protein levels of WEE1 and epithelial-mesenchymal transition(EMT)-related proteins.The interactions between miR-624-3p and circZFR or WEE1 were validated by dual-luciferase reporter assay and RNA immunoprecipitation(RIP)assay.Xenograft models were established to determine the role of circZFR in vivo.Results:circZFR and WEE1 were upregulated,while miR-624-3p expression was reduced in HCC tissues and cells.circZFR could sponge miR-624-3p,and WEE1 was a downstream gene of miR-624-3p.Knockdown of circZFR significantly reduced the malignant behaviors of HCC and that co-transfection with miR624-3p inhibitor restored this change.Overexpression of WEE1 abolished the inhibitory effect of miR624-3p mimic on HCC cells.Mechanistically,circZFR acted as a competitive endogenous RNA(ceRNA)to regulate WEE1 expression by targeting miR-624-3p.Furthermore,in vivo studies have illustrated that circZFR knockdown inhibited tumor growth.Conclusions:circZFR knockdown reduced HCC cell proliferation,migration and invasion and promoted apoptosis by regulating the miR-624-3p/WEE1 axis,suggesting that the circZFR/miR-624-3p/WEE1 axis might be a potential target for HCC treatment.