Exosome-Transmitted miR-224-5p Promotes Colorectal Cancer Cell Proliferation via Targeting ULK2 in p53-Dependent Manner

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.

PbGIF1 promoting cell-proliferation in pear fruit is transcriptionally activated by Pb RR1

As a cell proliferation regulator involved in wide biological processes in plants,GRF-INTERACTING FACTOR(GIF)controls different tissues development.However,whether GIF participates in fruit development remains unclear.According to transcriptome data,we identified PbGIF1was highly expressed during fruit development in cytokinins induced parthenocarpy pear.In the present study,the biofunction of PbGIF1 was initially verified.Overexpression of PbGIF1 promoted fruit size of transgenic tomato.The size of flesh fruit was not affected by cell expansion but the cell proliferation was promoted by overexpressing Pb GIF1.The accelerated cell proliferation process was also observed in PbGIF1-overexpressed transgenic pear fruit calli.The transcriptional regulation of cytokinins on PbGIF1 was further confirmed by exogenous CPPU treatments in pear fruitlets.To investigate the underlying mechanism,the cytokinins-responded factor,PbRR1,was further focused on.The results of Yeast-one-hybrid assay suggested that PbRR1 can bind to the promoter sequence of PbGIF1.The transcriptional activation of PbRR1 on PbGIF1 was also confirmed by Dual-Luciferase assays.Taken together,the results showed that cytokinins control pear fruit development via the transcriptional activation of PbGIF1 by PbRR1.

High-frequency repetitive transcranial magnetic stimulation promotes neural stem cell proliferation after ischemic stroke

Prolife ration of neural stem cells is crucial for promoting neuronal regeneration and repairing cerebral infarction damage.Transcranial magnetic stimulation(TMS)has recently emerged as a tool for inducing endogenous neural stem cell regeneration,but its underlying mechanisms remain unclea r In this study,we found that repetitive TMS effectively promotes the proliferation of oxygen-glucose deprived neural stem cells.Additionally,repetitive TMS reduced the volume of cerebral infa rction in a rat model of ischemic stro ke caused by middle cerebral artery occlusion,im p roved rat cognitive function,and promoted the proliferation of neural stem cells in the ischemic penumbra.RNA-sequencing found that repetitive TMS activated the Wnt signaling pathway in the ischemic penumbra of rats with cerebral ischemia.Furthermore,PCR analysis revealed that repetitive TMS promoted AKT phosphorylation,leading to an increase in mRNA levels of cell cycle-related proteins such as Cdk2 and Cdk4.This effect was also associated with activation of the glycogen synthase kinase 3β/β-catenin signaling pathway,which ultimately promotes the prolife ration of neural stem cells.Subsequently,we validated the effect of repetitive TMS on AKT phosphorylation.We found that repetitive TMS promoted Ca2+influx into neural stem cells by activating the P2 calcium channel/calmodulin pathway,thereby promoting AKT phosphorylation and activating the glycogen synthase kinase 3β/β-catenin pathway.These findings indicate that repetitive TMS can promote the proliferation of endogenous neural stem cells through a Ca2+influx-dependent phosphorylated AKT/glycogen synthase kinase 3β/β-catenin signaling pathway.This study has produced pioneering res ults on the intrinsic mechanism of repetitive TMS to promote neural function recove ry after ischemic stro ke.These results provide a stro ng scientific foundation for the clinical application of repetitive TMS.Moreover,repetitive TMS treatment may not only be an efficient and potential approach to support neurogenesis for further therapeutic applications,but also provide an effective platform for the expansion of neural stem cells.

TM9SF1 is implicated in promoting the proliferation and invasion of bladder cancer cells

Zhuo et al looked into the part of transmembrane 9 superfamily member 1(TM9SF1)in bladder cancer(BC),and evaluated if it can be used as a therapeutic target.They created a permanent BC cell line and tested the effects of TM9SF1 overexpression and suppression on BC cell growth,movement,invasion,and cell cycle advancement.Their results show that TM9SF1 can boost the growth,movement,and invasion of BC cells and their access into the G2/M stage of the cell cycle.This research gives a novel direction and concept for targeted therapy of BC.

Oncogenic BRAF^(V600E) induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

Activating V600E in v-Raf murine sarcoma viral oncogene homolog B(BRAF)is a common driver mutation in cancers of multiple tissue origins,including melanoma and glioma.BRAF^(V600E) has also been implicated in neurodegeneration.The present study aims to characterize BRAF^(V600E) during cell death and proliferation of three major cell types of the central nervous system:neurons,astrocytes,and microglia.Multiple primary cultures(primary cortical mixed culture)and cell lines of glial cells(BV2)and neurons(SH-SY5Y)were employed.BRAF^(V600E) and BRAF^(WT) expression was mediated by lentivirus or retrovirus.Blockage of downstream effectors(extracellular signal-regulated kinase 1/2 and JNK1/2)were achieved by siRNA.In astrocytes and microglia,BRAF^(V600E) induces cell proliferation,and the proliferative effect in microglia is mediated by activated extracellular signal-regulated kinase,but not c-Jun N-terminal kinase.Conditioned medium from BRAF^(V600E)-expressing microglia induced neuronal death.In neuronal cells,BRAF^(V600E) directly induces neuronal death,through c-Jun N-terminal kinase but not extracellular signal-regulated kinase.We further show that BRAF-related genes are enriched in pathways in patients with Parkinson’s disease.Our study identifies distinct consequences mediated by distinct downstream effectors in dividing glial cells and in neurons following the same BRAF mutational activation and a causal link between BRAF-activated microglia and neuronal cell death that does not require physical proximity.It provides insight into a possibly important role of BRAF in neurodegeneration as a result of either dysregulated BRAF in neurons or its impact on glial cells.

TATA-box-binding protein-associated factor 15 is a novel biomarker that promotes cell proliferation and migration in gastrointestinal stromal tumor

BACKGROUND Gastrointestinal stromal tumor(GIST)is a common neoplasm with high rates of recurrence and metastasis,and its therapeutic efficacy is still not ideal.There is an unmet need to find new molecular therapeutic targets for GIST.TATA-boxbinding protein-associated factor 15(TAF15)contributes to the progress of various tumors,while the role and molecular mechanism of TAF15 in GIST progression are still unknown.AIM To explore new molecular therapeutic targets for GIST and understand the biological role and underlying mechanisms of TAF15 in GIST progression.METHODS Proteomic analysis was performed to explore the differentially expressed proteins in GIST.Western blotting and immunohistochemical analysis were used to verify the expression level of TAF15 in GIST tissues and cell lines.Cell counting kit-8,colony formation,wound-healing and transwell assay were executed to detect the ability of TAF15 on cell proliferation,migration and invasion.A xenograft mouse model was applied to explore the role of TAF15 in the progression of GIST.Western blotting was used to detect the phosphorylation level and total level of RAF1,MEK and ERK1/2.RESULTS A total of 1669 proteins were identified as differentially expressed proteins with 762 upregulated and 907 downregulated in GIST.TAF15 was selected for the further study because of its important role in cell proliferation and migration.TAF15 was significantly over expressed in GIST tissues and cell lines.Overexpression of TAF15 was associated with larger tumor size and higher risk stage of GIST.TAF15 knockdown significantly inhibited the cell proliferation and migration of GIST in vitro and suppressed tumor growth in vivo.Moreover,the inhibition of TAF15 expression significantly decreased the phosphorylation level of RAF1,MEK and ERK1/2 in GIST cells and xenograft tissues,while the total RAF1,MEK and ERK1/2 had no significant change.CONCLUSION TAF15 is over expressed in GIST tissues and cell lines.Overexpression of TAF15 was associated with a poor prognosis of GIST patients.TAF15 promotes cell proliferation and migration in GIST via the activation of the RAF1/MEK/ERK signaling pathway.Thus,TAF15 is expected to be a novel latent molecular biomarker or therapeutic target of GIST.

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals,microRNAs(miRNAs)play key roles in multiple biological processes by regulating the expression of target genes.Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors;however,its function remains unclear.In this study,we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation(P<0.01),tyrosinase activity(P=0.001)and significantly reduced(P<0.001)melanin production.Functional prediction revealed that the 3′-untranslated region(UTR)of MAP3K8 has a putative miR-370-5p binding site,and the interaction between these two molecules was confirmed using luciferase reporter assays.In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits(P<0.01)MAP3K8 expression via direct targeting of its 3′UTR.Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition(P<0.01)of melanocyte proliferation and significant reduction(P<0.001)in melanin production,which is consistent with our observations for miR-370-5p.Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA(containing sites for the targeted binding to miR-370-5p)was significantly rescued(P≤0.001),which subsequently promoted significant increases in cell proliferation(P<0.001)and melanin production(P<0.01).Collectively,these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background:Activated hepatic stellate cells(HSCs)are closely involved in the initiation,perpetuation,and resolution of liver fibrosis.Pro-inflammatory cytokine levels are positively correlated with the transition from liver injury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis.Methods:In this study,we investigated the effect of the pro-inflammatory cytokine interleukin(IL)-1βon the proliferation and signaling pathways involved in fibrogenesis in LX-2 cells,an HSC cell line,using western blotting and cell proliferation assays.Results:IL-1βincreased the proliferation rate andα-smooth muscle actin(SMA)expression of LX-2 cells in a dose-dependent manner.Within 1 h after IL-1βtreatment,c-Jun N-terminal kinase(JNK),p38,and nuclear factor-κB(NF-κB)signaling was activated in LX-2 cells.Subsequently,protein kinase B(AKT)phosphorylation and an increase inα-SMA expression were observed in LX-2 cells.Each inhibitor of JNK,p38,or NF-κB decreased cell proliferation,AKT phosphorylation,andα-SMA expression in IL-1β-treated LX-2 cells.Conclusion:These results indicate that JNK,p38,and NF-κB signals converge at AKT phosphorylation,leading to LX-2 activation by IL-1β.Therefore,the AKT signaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.

YWHAH activates the HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect the proliferation of gastric cancer cells

Fos-related antigen 1(Fra-1)is a nuclear transcription factor that regulates cell growth,differentiation,and apoptosis.It is involved in the proliferation,invasion,apoptosis and epithelial mesenchymal transformation of malignant tumor cells.Fra-1 is highly expressed in gastric cancer(GC),affects the cycle distribution and apoptosis of GC cells,and participates in GC occurrence and development.However,the detailed mechanism of Fra-1 in GC is unclear,such as the identification of Fra-1-interacting proteins and their role in GC pathogenesis.In this study,we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta(YWHAH)as a Fra-1-interacting protein in GC cells using co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry.Experiments showed that YWHAH positively regulated Fra-1 mRNA and protein expression,and affected GC cell proliferation.Whole proteome analysis showed that Fra-1 affected the activity of the high mobility group AT-hook 1(HMGA1)/phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(AKT)/mechanistic target of rapamycin(mTOR)signaling pathway in GC cells.Western blotting and flow cytometry confirmed that YWHAH activated HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect GC cell proliferation.These results will help to discover new molecular targets for the early diagnosis,treatment,and prognosis prediction of GC.

Single-cell sequencing analysis reveals the molecular mechanism of promotion of SCAP proliferation upon AZD2858 treatment

The Wnt/β-catenin signaling pathway is the main target of tooth regeneration regulation.Treatment of cells with AZD2858 stimulates the Wnt/β-catenin signaling pathway,yet the function of this pathway in tooth regeneration remains unclear.Here,we found that AZD2858 promotes the accumulation ofβ-catenin in the nuclei of stem cells from the apical papilla(SCAPs)and enhances cell proliferation.Single-cell sequencing was performed on SCAPs treated with AZD2858.Eight clusters were identified,namely SCAPs-CNTNAP2,SCAPs-DTL,SCAPs-MYH11,SCAPs-MKI67,SCAPs-CXCL8,SCAPs-TPM2,SCAPs-IFIT2 and SCAPs-NEK10.The pseudo-time trajectory analysis showed that AZD2858 enhanced the evolution of SCAPs from SCAPs-TMP2 clusters to SCAPs-MYH11,SCAPs-CNTNAPs and SCAPs-NEK10 clusters via up-regulation of PRKCA,SMURF2,MAGI2,RBMS3,EXT1,CAMK2D,PLCB4,and PLCB1.These results demonstrate that AZD2858 enhances the proliferation of SCAPs-TPM2 cluster by activating the non-canonical Wnt/β-catenin signaling pathway.

18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3

BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GRA)has a variety of pharmacological effects.The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC,in order to provide effective ideas for the clinical prevention and treatment of GC.AIM To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.METHODS Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs(miRNAs)in GC cells after 18β-GRA intervention.Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability,cell colony formation ability was detected by the clone formation assay,and flow cytometry was used to detect the cell cycle and apoptosis.A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells.Tumor tissue morphology was observed by hematoxylin and eosin staining,and microtubule-associated protein light chain 3(LC3)expression was detected by immunohistochemistry.TransmiR,STRING,and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3(STAT3)-related information.Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction(qPCR)and the expression levels of STAT3,phosphorylated STAT3(p-STAT3),and LC3 were detected by western blot analysis.The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system.AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label.LC3 was labeled and autophagy flow was observed under a confocal laser microscope.RESULTS The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells(P=4.51E-06).Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability,arrested the cell cycle in the G0/G1 phase,promoted cell apoptosis,and inhibited the growth of subcutaneous tumors in BALB/c nude mice(P<0.01).No obvious necrosis was observed in the tumor tissue in the negative control group(no drug intervention or lentivirus transfection)and vector group(the blank vector for lentivirus transfection),and more cells were loose and necrotic in the miR-328-3p group.Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3,and STAT3 was closely related to autophagy markers such as p62.After overexpressing miR-328-3p,the expression level of STAT3 mRNA was significantly decreased(P<0.01)and p-STAT3 was downregulated(P<0.05).The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT33’untranslated regions of the wild-type reporter vector group was significantly decreased(P<0.001).Overexpressed miR-328-3p combined with bafilomycin A1(Baf A1)was used to detect the expression of LC3 II.Compared with the vector group,the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated(P<0.05),and compared with the Baf A1 group,the expression level of LC3 II in the overexpressed miR-328-3p+Baf A1 group was upregulated(P<0.01).The expression of LC3 II was detected after intervention of 18β-GRA in GC cells,and the results were consistent with the results of miR-328-3p overexpression(P<0.05).Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis(P<0.001).qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention(P<0.05).Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention(P<0.05).CONCLUSION 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

Study on the mechanism of tRNA-ValAAC-5 in promoting the proliferation and migration of hepatocellular carcinoma cells

Objective:To demonstrate the role and mechanism of tRNA-ValAAC-5 expression in hepatocellular carcinoma(HCC)cells.Methods:The expression levels of tRNA-ValAAC-5 in HCC(Hep3B,HuH7,SNU398,Hep3G2)and human hepatocellular carcinoma(THLE2,THLE3)were detected by real-time PCR.HEP3B and Hep3G2 cells were respectively transfected with tRNA-ValAAC-5-inhibitor and tRNA-ValAAC-5-NC as the inhibitor group and the NC group.Then the ability of cell proliferation was detected by CCK-8 assay and the ability of invasion and metastasis was detected by Transwell assay.The protein expression levels of p21,Matrix metalloproteinase 2(MMP2)and Matrix metalloproteinase 9(MMP9)were determined by Western blot.Results:The relative expression of tRNA-ValAAC-5 in Hep3B,HuH7,SNU398 and Hep3G2 cells were significantly higher than THLE2 and THLE3 cells,the differences were statistically significant(P<0.05).After tRNA-ValAAC-5-inhibitor transfection,the expression of tRNA-ValAAC-5 in Hep3B and Hep3G2 cells were reduced than tRNA-ValAAC-NC group.Both of the differences were statistically significant(t=36.52,27.45,P<0.001),which indicated the transfection was successful.The proliferative ability of Hep3B and Hep3G2 cells transfected with tRNA-ValAAC-5-inhibitor after 24,48,72,96 h were inhibited effectively compared with tRNA-ValAAC-5-NC group.All of the differences were statistically significant in Hep3B(t=5.25,8.23,7.33,14.16,P<0.001)and Hep3G2(t=4.25,5.11,9.39,7.59,P<0.001)cells.The number of invasion and metastasis of Hep3B and Hep3G2 cells were reduced in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,there was significant difference(t=14.01,21.85,P<0.001).The protein expression levels of P21 were lower,MMP2 and MMP9 were higher in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,the differences were statistically significant in Hep3B(t=8.96,12.80,4.652,P<0.001)cells and Hep3G2(t=15.17,22.36,12.61,P<0.001)cells.Conclusion:tRNA-ValAAC-5 can effectively promote the proliferation,invasion and metastasis of HCC,and its possible mechanism is related to regulating the expression of p21,MMP2 and MMP9.

Inhibitory Effects of Propolis Water Extract on the Proliferation of Cervical Cancer Cells

[Objectives]To investigate the effects of propolis extract on HeLa cells to provide theoretical guidance for facilitating clinical treatment.[Methods]The effects of propolis on inflammation,and proliferation were analyzed based on the levels of DNA transcriptional regulation and mRNA and protein expression levels.[Results]Propolis water extract(PWE)could inhibit the cell proliferation and production of DNA damage in a dosedependent manner.Moreover,the propolis water extract could significantly downregulate the expression of iNOS-Luc,PTGS-2-Luc,and IL-8-Luc,and that it was related to the expression of the NF-κB family protein.After the induction of HeLa cells by propolis,the expression of the cell cycle inhibitor gene p21 was increased,while that of the cell proliferation gene Ki67 was decreased.[Conclusions]Propolis water extract could significantly inhibit the cell proliferation and production of DNA damage,suggesting propolis as a potential candidate for the development of adjunctive therapy against cervical cancer.

Chemokine platelet factor 4 accelerates peripheral nerve regeneration by regulating Schwann cell activation and axon elongation

Schwann cells in peripheral nerves react to traumatic nerve injury by attempting to grow and regenerate.Howeve r,it is unclear what factors play a role in this process.In this study,we searched a GEO database and found that expression of platelet factor 4 was markedly up-regulated after sciatic nerve injury.Platelet factor is an important molecule in cell apoptosis,diffe rentiation,survival,and proliferation.Further,polymerase chain reaction and immunohistochemical staining confirmed the change in platelet factor 4 in the sciatic nerve at different time points after injury.Enzyme-linked immunosorbent assay confirmed that platelet factor 4 was secreted by Schwann cells.We also found that silencing platelet factor 4 decreased the proliferation and migration of primary cultured Schwann cells,while exogenously applied platelet factor 4 stimulated Schwann cell prolife ration and migration and neuronal axon growth.Furthermore,knocking out platelet factor 4 inhibited the prolife ration of Schwann cells in injured rat sciatic nerve.These findings suggest that Schwann cell-secreted platelet factor 4 may facilitate peripheral nerve repair and regeneration by regulating Schwann cell activation and axon growth.Thus,platelet factor 4 may be a potential therapeutic target for traumatic peripheral nerve injury.

Role of hsa_circ_0007482 in pterygium development: insights into proliferation, apoptosis, and clinical correlations

AIM:To investigate the impact of hsa_circ_0007482 on the proliferation and apoptosis of human pterygium fibroblasts(HPFs)and its correlation with the severity grades of pterygium.METHODS:Pterygium and normal conjunctival tissues were collected from the superior area of the same patient’s eye(n=33).The correlation between pterygium severity and hsa_circ_0007482 expression using quantitative reversetranscription polymerase chain reaction(RT-qPCR)were analyzed.Three distinct siRNA sequences targeting hsa_circ_0007482,along with a negative control sequence,were transfected into HPFs.Cell proliferation was assessed using the cell counting kit-8.Expression levels of Ki67,proliferating cell nuclear antigen(PCNA),Cyclin D1,Bax,B-cell lymphoma-2(Bcl-2),and Caspase-3 were measured via RT-qPCR.Immunofluorescence staining was employed to detect Ki67 and vimentin expressions.Apoptosis was evaluated using flow cytometry.RESULTS:Hsa_circ_0007482 expression was significantly higher in pterygium tissues compared to normal conjunctival tissues(P<0.001).Positive correlations were observed between hsa_circ_0007482 expression and pterygium severity,thickness,and vascular density.Knockdown of hsa_circ_0007482 inhibited cell proliferation,reducing the mRNA expression of Ki67,PCNA,and Cyclin D1 in HPFs.Hsa_circ_0007482 knockdown induced apoptosis,increasing mRNA expression levels of Bax and Caspase-3,while decreasing Bcl-2 expression in HPFs.Additionally,hsa_circ_0007482 knockdown attenuated vimentin expression in HPFs.CONCLUSION:The downregulation of hsa_circ_0007482 effectively hampers cell proliferation and triggers apoptosis in HPFs.There are discernible positive correlations detected between the expression of hsa_circ_0007482 and the severity of pterygium.

SENP3 Promotes Mantle Cell Lymphoma Development through Regulating Wnt10a Expression

Objective SUMO-specific protease 3(SENP3),a member of the SUMO-specific protease family,reverses the SUMOylation of SUMO-2/3 conjugates.Dysregulation of SENP3 has been proven to be involved in the development of various tumors.However,its role in mantle cell lymphoma(MCL),a highly aggressive lymphoma,remains unclear.This study was aimed to elucidate the effect of SENP3 in MCL.Methods The expression of SENP3 in MCL cells and tissue samples was detected by RT-qPCR,Western blotting or immunohistochemistry.MCL cells with stable SENP3 knockdown were constructed using short hairpin RNAs.Cell proliferation was assessed by CCK-8 assay,and cell apoptosis was determined by flow cytometry.mRNA sequencing(mRNA-seq)was used to investigate the underlying mechanism of SENP3 knockdown on MCL development.A xenograft nude mouse model was established to evaluate the effect of SENP3 on MCL growth in vivo.Results SENP3 was upregulated in MCL patient samples and cells.Knockdown of SENP3 in MCL cells inhibited cell proliferation and promoted cell apoptosis.Meanwhile,the canonical Wnt signaling pathway and the expression of Wnt10a were suppressed after SENP3 knockdown.Furthermore,the growth of MCL cells in vivo was significantly inhibited after SENP3 knockdown in a xenograft nude mouse model.Conclusion SENP3 participants in the development of MCL and may serve as a therapeutic target for MCL.

Knockdown of circular RNA (CircRNA)_001896 inhibits cervical cancer proliferation and stemness in vivo and in vitro

Objective:Previous studies indicated that aberrant circular RNA(circRNA)expression affects gene expression regulatory networks,leading to the aberrant activation of tumor pathways and promoting tumor cell growth.However,the expression,clinical significance,and effects on cell propagation,invasion,and dissemination of circRNA_001896 in cervical cancer(CC)tissues remain unclear.Methods:The Gene Expression Omnibus(GEO)datasets(GSE113696 and GSE102686)were used to examine differential circRNA expression in CC and adjacent tissues.The expression of circRNA_001896 was detected in 72 CC patients usingfluorescence quantitative PCR.Correlation analysis with clinical pathological features was performed through COX multivariate and univariate analysis.The effect of circRNA_001896 downregulation on CC cell propagation was examined using the cell counting kit-8(CCK-8)test,clonogenic,3D sphere formation,and in vivo tumorigenesis assays.Results:Intersection of the GSE113696 and GSE102686 datasets revealed an increased expression of four circRNAs,including circRNA_001896,in CC tissues.Fluorescence quantitative PCR confirmed circRNA_001896 as a circular RNA.High expression of circRNA_001896 was considerably associated with lymph node metastasis,International Federation of Gynecologists and Obstetricians(FIGO)stage,tumor diameter,and survival period in CC patients.Proportional hazards model(COX)univariate and multivariate analyses revealed that circRNA_001896 expressions are a distinct risk factor affecting CC patients’prognosis.Cellular functional experiments showed that downregulating circRNA_001896 substantially suppressed CC cell growth,colony formation,and 3D sphere-forming ability.In vivo,tumorigenesis analysis in nude mice demonstrated that downregulating circRNA_001896 remarkably reduced the in vivo proliferation capacity of CC cells.Conclusion:CircRNA_001896 is highly expressed in CC tissues and is substantially related to lymph node metastasis,FIGO stage,tumor size,and survival period in patients.Moreover,downregulating circRNA_001896 significantly inhibits both in vivo and in vitro propagation of CC cells.Therefore,circRNA_001896 might be used as a biomarker for targeted therapy in cervical cancer.

Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine

BACKGROUND Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages.In humans,their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs.Studies suggested that mesenchymal stem cells(MSCs),necessary for repair and regeneration via transplantation,require doses ranging from 10 to 400 million cells.Furthermore,the limited expansion of MSCs restricts their therapeutic application.AIM To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols.METHODS Human umbilical cord(hUC)tissue derived MSCs were obtained and re-cultured.These cultured cells were subjected to the following evaluation pro-cedures:Immunophenotyping,immunocytochemical staining,trilineage differentiation,population doubling time and number,gene expression markers for proliferation,cell cycle progression,senescence-associatedβ-galactosidase assay,human telomerase reverse transcriptase(hTERT)expression,mycoplasma,cytomegalovirus and endotoxin detection.RESULTS Analysis of pluripotent gene markers Oct4,Sox2,and Nanog in recultured hUC-MSC revealed no significant differences.The immunophenotypic markers CD90,CD73,CD105,CD44,vimentin,CD29,Stro-1,and Lin28 were positively expressed by these recultured expanded MSCs,and were found negative for CD34,CD11b,CD19,CD45,and HLA-DR.The recultured hUC-MSC population continued to expand through passage 15.Proliferative gene expression of Pax6,BMP2,and TGFb1 showed no significant variation between recultured hUC-MSC groups.Nevertheless,a significant increase(P<0.001)in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs.Cellular senescence markers(hTERT expression andβ-galactosidase activity)did not show any negative effect on recultured hUC-MSCs.Additionally,quality control assessments consistently confirmed the absence of mycoplasma,cytomegalovirus,and endotoxin contamination.CONCLUSION This study proposes the development of a novel protocol for efficiently expanding stem cell population.This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

Diagnostic and prognostic role of LINC01767 in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is a primary contributor to cancer-related mortality on a global scale.However,the underlying molecular mechanisms are still poorly understood.Long noncoding RNAs are emerging markers for HCC diagnosis,prognosis,and therapeutic target.No study of LINC01767 in HCC was published.AIM To conduct a multi-omics analysis to explore the roles of LINC01767 in HCC for the first time.METHODS DESeq2 Package was used to analyze different gene expressions.Receiver operating characteristic curves assessed the diagnostic performance.Kaplan-Meier univariate and Cox multivariate analyses were used to perform survival analysis.The least absolute shrinkage and selection operator(LASSO)-Cox was used to identify the prediction model.Subsequent to the validation of LINC01767 expression in HCC fresh frozen tissues through quantitative real time polymerase chain reaction,next generation sequencing was performed following LINC01767 over expression(GSE243371),and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes/Gene Set Enrichment Analysis/ingenuity pathway analysis was carried out.In vitro experiment in Huh7 cell was carried out.RESULTS LINC01767 was down-regulated in HCC with a log fold change=1.575 and was positively correlated with the cancer stemness.LINC01767 was a good diagnostic marker with area under the curve(AUC)[0.801,95% confidence interval(CI):0.751-0.852,P=0.0106]and an independent predictor for overall survival(OS)with hazard ratio=1.899(95%CI:1.01-3.58,P=0.048).LINC01767 nomogram model showed a satisfied performance.The top-ranked regulatory network analysis of LINC01767 showed the regulation of genes participating various pathways.LASSO regression identified the 9-genes model showing a more satisfied performance than 5-genes model to predict the OS with AUC>0.75.LINC01767 was down-expressed obviously in tumor than para-tumor tissues in our cohort as well as in cancer cell line;the over expression of LINC01767 inhibit cell proliferation and clone formation of Huh7 in vitro.CONCLUSION LINC01767 was an important tumor suppressor gene in HCC with good diagnostic and prognostic performance.

Subcellular distribution of prohibitin 1 in rat liver during liver regeneration and its cellular implication

BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.