microRNA-627-5p inhibits colorectal cancer cell proliferation,migration and invasion by targeting Wnt2

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.

Proteomics-based identification of proteins in tumor-derived exosomes as candidate biomarkers for colorectal cancer

BACKGROUND Colorectal cancer(CRC)is the second leading cause of cancer-related death,with high morbidity worldwide.There is an urgent need to find reliable diagnostic biomarkers of CRC and explore the underlying molecular mechanisms.Exosomes are involved in intercellular communication and participate in multiple pathological processes,serving as an important part of the tumor microenvironment.AIM To investigate the proteomic characteristics of CRC tumor-derived exosomes and to identify candidate exosomal protein markers for CRC.METHODS In this study,10 patients over 50 years old who were diagnosed with moderately differentiated adenocarcinoma were recruited.We paired CRC tissues and adjacent normal intestinal tissues(>5 cm)to form the experimental and control groups.Purified exosomes were extracted separately from each tissue sample.Data-independent acquisition mass spectrometry was implemented in 8 matched samples of exosomes to explore the proteomic expression profiles,and differentially expressed proteins(DEPs)were screened by bioinformatics analysis.Promising exosomal proteins were verified using parallel reaction monitoring(PRM)analysis in 10 matched exosome samples.RESULTS A total of 1393 proteins were identified in the CRC tissue group,1304 proteins were identified in the adjacent tissue group,and 283 proteins were significantly differentially expressed between them.Enrichment analysis revealed that DEPs were involved in multiple biological processes related to cytoskeleton construction,cell movement and migration,immune response,tumor growth and telomere metabolism,as well as ECM-receptor interaction,focal adhesion and mTOR signaling pathways.Six differentially expressed exosomal proteins(NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28)were validated by PRM analysis and evaluated by receiver operating characteristic curve(ROC)analysis.The area under the ROC curve was 0.93,0.96,0.97,0.78,0.75,and 0.88(P<0.05)for NHP2,OLFM4,TOP1,SAMP,TAGL,and TRIM28,respectively,indicating their good ability to distinguish CRC tissues from adjacent intestinal tissues.CONCLUSION In our study,comprehensive proteomic profiles were obtained for CRC tissue exosomes.Six exosomal proteins,NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28,may be promising diagnostic markers and effective therapeutic targets for CRC,but further experimental investigation is needed.

Correlation between Helicobacter pylori-associated gastric diseases and colorectal neoplasia

AIM: To explore the correlation between Helicobacter pylori(H. pylori)-associated gastric diseases and colorectal neoplasia.METHODS: Patients included in this study underwent a colonoscopy and esophago-gastro-duodenoscopy(EGD) along with histopathological measurement between March 2012 and March 2015 at Qi-Lu Hospital of Shandong University, who also had results of H. pylori detection. A total of 233 cases were selected. Demographic data, H. pylori infection status(including results of rapid urease tests and gastric mucosa pathological examinations) and histopathological examination results of gastric and colorectal mucosa were gathered and analyzed. The statistical analysis focused on the prevalence of colorectal neoplasms among patients with various histopathological categories of the stomach. ORs and their 95%CI were calculated to describe the strengths of the associations.RESULTS: The incidence rates of colorectal adenoma without high-grade intraepithelial neoplasia(HGIEN)(OR = 2.400, 95%CI: 0.969-5.941), adenoma with HGIEN(5.333, 1.025-27.758) and adenocarcinoma(1.455, 0.382-5.543) were all higher for patients with H. pylori-associated gastritis than for those in the control group. The incidence rate of colorectal adenoma with HGIEN(3.218, 0.767-13.509) was higher in patients with intestinal metaplasia than in the control group, while the incidence rates of adenoma without HGIEN(0.874, 0.414-1.845) and adenocarcinoma(0.376, 0.096-1.470) were lower in the intestinal metaplasia group than in the control group. The incidence rate of colorectal adenoma without HGIEN(3.111, 1.248-7.753) was significantly higher in the gastric intraepithelial neoplasia group than in the control group, while the rates of adenoma with HGIEN(1.481, 0.138-15.941) and adenocarcinoma(2.020, 0.561-7.272) were higher in the gastric intraepithelial neoplasia group. Incidence rates of colorectal adenoma without HGIEN(1.067, 0.264-4.314), adenoma with HGIEN(2.667, 0.231-30.800) and adenocarcinoma(2.182, 0.450-10.585) were all higher in the gastric adenocarcinoma group than in the control group.CONCLUSION: H. pylori infection as well as H. pylori-associated gastric diseases are risk factors for colorectal neoplasia.

Untargeted metabolomics characteristics of nonobese nonalcoholic fatty liver disease induced by high-temperature-processed feed in Sprague-Dawley rats

BACKGROUND Nonalcoholic fatty liver disease(NAFLD)has become one of the most common chronic liver diseases in the world.In our early clinical data and questionnaire analysis of NAFLD,it was found that the body mass index of some patients did not meet the diagnostic criteria for overweight or obesity.The consumption of high-temperature-processed foods such as fried food,hot pot and barbecue is closely related to the occurrence of nonobese NAFLD.Reducing the intake of this kind of food can reduce disease severity and improve prognosis.AIM To explore the untargeted metabolomics characteristics of nonobese nonalcoholic fatty liver disease in Sprague-Dawley rats induced by high-temperatureprocessed feed.METHODS Fifty-four male Sprague-Dawley rats were divided into three groups:The control group received a standard diet;the nonfried soybeans(NDFS)group received 60%NDFS and 40%basic feed and the dry-fried soybeans(DFS)group received 60%DFS and 40%basic feed.Six rats were sacrificed at week 4,8,and 12 in each group.The food intake,body weight,Lee’s index,liver index,serological index and hepatic histopathology were assessed.Untargeted metabolomics characteristics were used to analyze the changes in liver metabolites of rats at week 12.Correlations between metabolites and pathology scores between the DFS and control groups and between the DFS and NDFS groups were analyzed.We selected some of the metabolites,both within the pathway and outside of the pathway,to explain preliminarily the difference in liver pathology in the three groups of rats.RESULTS There were no statistically significant differences in the food intake,body weight,Lee's index or serological index between the DFS group and the control group(P>0.05).At week 8 and week 12,the steatosis scores in the DFS group were significantly higher than those in the other two groups(P<0.05).At week 12,the liver index of the DFS group was the lowest(NDFS group vs DFS group,P<0.05).The fibrosis score in the DFS group was significantly higher than those in the other two groups(P<0.05).The correlation analysis of the liver pathology score and differential metabolites in the DFS and NDFS groups showed that there were 10 strongly correlated substances:Five positively correlated substances and five negatively correlated substances.The positively correlated substances included taurochenodeoxycholate-3-sulfate,acetylcarnitine,20a,22bdihydroxycholesterol,13E-tetranor-16-carboxy-LTE4 and taurocholic acid.The negatively correlated substances included choline,cholesterane-3,7,12,25-tetrol-3-glucuronide,nicotinamide adenine dinucleotide phosphate,lysoPC[16:1(9Z)]and glycerol 3-phosphate.The correlation analysis of the liver pathology score and differential metabolites in the DFS and control groups showed that there were 13 strongly correlated substances:Four positively correlated substances and 9 negatively correlated substances.The positively correlated substances included 4-hydroxy-6-eicosanone,3-phosphoglyceric acid,13-hydroxy-9-methoxy-10-oxo-11-octadecenoic acid and taurochenodeoxycholate-3-sulfate.The negatively correlated substances included lysoPC[16:1(9Z)],S-(9-hydroxy-PGA1)-glutathione,lysoPC[20:5(5Z,8Z,11Z,14Z,17Z)],SM(d18:1/14:0),nicotinamide adenine dinucleotide phosphate,5,10-methylene-THF,folinic acid,N-lactoylglycine and 6-hydroxy-5-methoxyindole glucuronide.CONCLUSION We successfully induced liver damage in rats by using a specially prepared hightemperature-processed feed and explored the untargeted metabolomics characteristics.

Identification of the circRNA-miRNA-mRNA regulatory network and its prognostic effect in colorectal cancer

BACKGROUND The high morbidity and mortality of colorectal cancer(CRC)have posed great threats to human health.Circular RNA(CircRNA)and microRNA(miRNA),acting as competing endogenous RNAs(ceRNAs),have been found to play vital roles in carcinogenesis.However,the biological function of ceRNAs in CRC pathogenesis and prognosis remains largely unexplored.AIM To identify the CRC-specific circRNA-miRNA-mRNA regulatory network and uncover the subnetwork associated with its prognosis.METHODS CircRNAs,miRNAs and mRNAs differentially expressed(DE)in CRC tissues were selected by expression file analysis in the Gene Expression Omnibus(GEO)database,and the downstream target molecules of circRNAs and miRNAs were predicted.Then,the intersection of differentially expressed RNA molecules with the predicted targets was determined to obtain a ceRNA network.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were conducted to elucidate the possible mechanism of pathogenesis.A survival analysis using the gene profiles and clinical information in The Cancer Genome Atlas(TCGA)database was performed to identify the mRNAs associated with the clinical outcome of CRC patients and construct a prognostic subnetwork.RESULTS We downloaded three datasets(GSE126095,GSE41655 and GSE41657)of largescale CRC samples from the GEO database.There were 55 DEcircRNAs,114 DEmiRNAs and 267 DEmRNAs in CRC tissues compared with normal tissues.After intersecting these molecules with predicted targets,19 circRNAs,13 miRNAs and 28 mRNAs were chosen to develop a circRNA-miRNA-mRNA network.GO and KEGG functional enrichment analyses indicated that the retinol metabolic process,leukocyte chemotaxis,extracellular matrix remodeling,endoplasmic reticulum stress,alcohol dehydrogenase activity,gastric acid secretion,nitrogen metabolism and NOD-like receptor signaling pathway might participate in the tumorigenesis of CRC.After verifying the identified mRNA effect in the TCGA database,we finally recognized 3 mRNAs(CA2,ITLN1 and LRRC19)that were significantly associated with the overall survival of CRC patients and constructed a ceRNA subnetwork including 5 circRNAs(hsa_circ_0080210,hsa_circ_0007158,hsa_circ_0000375,hsa_circ_0018909 and hsa_circ_0011536)and 3 miRNAs(hsa-miR-601,hsa-miR-671-5p and hsa-miR-765),which could contain innovative and noninvasive indicators for the early screening and prognostic prediction of CRC.CONCLUSION We proposed a circRNA-miRNA-mRNA regulatory network closely associated with the progression and clinical outcome of CRC that might include promising biomarkers for carcinogenesis and therapeutic targets.

Research progress on chemopreventive effects of phytochemicals on colorectal cancer and their mechanisms

Colorectal cancer(CRC) is a type of cancer with high morbidity and mortality rates worldwide and has become a global health problem. The conventional radiotherapy and chemotherapy regimen for CRC not only has a low cure rate but also causes side effects. Many studies have shown that adequate intake of fruits and vegetables in the diet may have a protective effect on CRC occurrence, possibly due to the special biological protective effect of the phytochemicals in these foods. Numerous in vitro and in vivo studies have demonstrated that phytochemicals play strong antioxidant, anti-inflammatory and anti-cancer roles by regulating specific signaling pathways and molecular markers to inhibit the occurrence and development of CRC. This review summarizes the progress on CRC prevention using the phytochemicals sulforaphane, curcumin and resveratrol, and elaborates on the specific underlying mechanisms. Thus, we believe that phytochemicals might provide a novel therapeutic approach for CRC prevention, but future clinical studies are needed to confirm the specific preventive effect of phytochemicals on cancer.

Identification of circ_0000375 and circ_0011536 as novel diagnostic biomarkers of colorectal cancer

BACKGROUND Colorectal cancer(CRC)imposes a tremendous burden on human health,with high morbidity and mortality.Circular ribonucleic acids(circRNAs),a new type of noncoding RNA,are considered to participate in cancer pathogenesis as microRNA(miRNA)sponges.However,the dysregulation and biological functions of circRNAs in CRC remain to be explored.AIM To identify potential circRNA biomarkers of CRC and explore their functions in CRC carcinogenesis.METHODS CircRNAs and miRNAs differentially expressed in CRC tissues were identified by analyzing expression profiles from the Gene Expression Omnibus(GEO)database.Circ_0000375 and circ_0011536 were selected as CRC biomarker candidates.Quantitative real-time polymerase chain reaction was utilized to evaluate the expression of these 2 circRNAs in CRC tissues,serums and cell lines.Receiver operating characteristic curves were generated to assess the diagnostic performances of these 2 circRNAs.Then,functional experiments,including cell counting kit-8,wound healing and Transwell invasion assays,were performed after the overexpression of circ_0000375 and circ_0011536 in CRC cell lines.Furthermore,candidate target miRNAs of circ_0000375 and circ_0011536 were predicted via bioinformatics analysis.The expression levels of these miRNAs were explored in CRC cell lines and tissues from GEO datasets.A luciferase reporter assay was developed to examine the interactions between circRNAs and miRNAs.Based on the target miRNAs and downstream genes,functional enrichment analyses were applied to reveal the critical signaling pathways involved in CRC carcinogenesis.RESULTS Downregulated circ_0000375 and circ_0011536 expression was observed in CRC tissues in GSE126095,clinical CRC tissue and serum samples and CRC cell lines.The areas under the curve for circ_0000375 and circ_0011536 were 0.911 and 0.885 in CRC tissue and 0.976 and 0.982 in CRC serum,respectively.Moreover,the serum levels of these 2 circRNAs were higher in patients at 30 d postsurgery than in patients before surgery,suggesting that the serum expression of circ_0000375 and circ_0011536 is related to CRC tumorigenesis.Circ_0000375 and circ_0011536 overexpression inhibited the proliferation,migration and invasion of CRC cells.Furthermore,miR-1182 and miR-1246,which were overexpressed in CRC tissues in GSE41655,GSE49246 and GSE115513,were verified as target miRNAs of circ_0000375 and circ_0011536,respectively,by luciferase reporter assays.The downstream genes of miR-1182 and miR-1246 were enriched in some CRC-associated pathways,such as the Wnt signaling pathway.CONCLUSION Circ_0000375 and circ_0011536 may function as tumor suppressors in CRC progression,serving as novel biomarkers for CRC diagnosis and as promising candidates for therapeutic exploration.

Comprehensive proteomic signature and identification of CDKN2A as a promising prognostic biomarker and therapeutic target of colorectal cancer

BACKGROUND The carcinogenesis of colorectal cancer(CRC)involves many different molecules and multiple pathways,and the specific mechanism has not been elucidated until now.Existing studies on the proteomic signature profiles of CRC are relatively limited.Therefore,we herein aimed to provide a more comprehensive proteomic signature profile and discover new prognostic markers and therapeutic targets by performing proteomic analysis of CRC and paired normal tissues.AIM To investigate the proteomic signature and identify novel protein prognostic biomarkers of CRC.METHODS Cancer tissues and paired normal tissues were collected from 48 patients who underwent surgical removal at the China-Japan Friendship Hospital from January 2020 to June 2021.Data independent acquisition(DIA)quantitative proteomic analysis was performed using high-performance liquid chromatography–mass spectrometry/mass spectrometry(nano-UHPLC–MS/MS)to identify differen tially expressed proteins,among which those with a P adj value(t test,BH correction)<0.05 and an absolute fold change(|log2FC|)>2 were identified as potential markers.Differentially expressed proteins were selected by bioinformatics analysis and validated by immunohistochemical tissue microarrays,and their association with prognosis was further analyzed with the Gene Expression Profiling Interactive Analysis database to identify prognostic protein biomarkers of CRC.RESULTS Significantly differential protein expression was observed between cancer tissues and normal tissues.Compared with normal tissues,1115 proteins were upregulated and 705 proteins were downregulated in CRC based on P adj<0.05 and|log2FC|>2,and bioinformatics analysis revealed that the differentially expressed proteins were involved in multiple biological processes associated with tumorigenesis,including ribosome biogenesis in eukaryotes,focal adhesion,extracellular matrix-receptor interactions and other tumor metabolism processes.Moreover,cyclin-dependent kinase inhibitor 2A(CDKN2A)expression was markedly upregulated in CRC,as validated by immunohistochemistry(0.228 vs 0.364,P=0.0044),and was significantly enriched in tumor proliferation and signal transduction pathways such as the cell cycle and p53 signaling pathways.High CDKN2A expression was significantly correlated with poor prognosis(P=0.021).These results demonstrated that CDKN2A functions as a driver of CRC.CONCLUSION Our study provides a comprehensive proteomic signature of CRC and highlights CDKN2A as a potential powerful prognostic marker and precision therapeutic target.

Atypical Whipple’s disease with special endoscopic manifestations:A case report

BACKGROUND Whipple’s disease is a rare systemic infection caused by Tropheryma whipplei.Most patients present with nonspecific symptoms,and routine laboratory and imaging examination results also lack specificity.The diagnosis often relies on invasive manipulation,pathological examination,and molecular techniques.These difficulties in diagnosing Whipple’s disease often result in misdiagnosis and inappropriate treatments.CASE SUMMARY This paper reports on the case of a 58-year-old male patient who complained of fatigue and decreased exercise capacity.The results of routine blood tests indicated hypochromic microcytic anemia.Results of gastroscopy and capsule endoscopy showed multiple polypoid bulges distributed in the duodenal and proximal jejunum.A diagnosis of small intestinal adenomatosis was initially considered;hence,the Whipple procedure,a pylorus-preserving pancreaticoduodenectomy,was performed.Pathological manifestations showed many periodic acid-Schiff-positive macrophages aggregated in the intestinal mucosa of the duodenum,upper jejunum,and surrounding lymph nodes.Based on comprehensive analysis of symptoms,laboratory findings,and pathological manifestations,the patient was finally diagnosed with Whipple’s disease.After receiving 1 mo of antibiotic treatment,the fatigue and anemia were significantly improved.CONCLUSION This case presented with atypical gastrointestinal manifestations and small intestinal polypoid bulges,which provided new insight on the diagnosis of Whipple’s disease.

Circulating miR-627-5p and miR-199a-5p are promising diagnostic biomarkers of colorectal neoplasia

BACKGROUND Early detection of colorectal neoplasms,including colorectal cancers(CRCs)and advanced colorectal adenomas(AAs),is crucial to improve patient survival.Circulating microRNAs(miRNAs)in peripheral blood are emerging as noninvasive diagnostic markers for multiple cancers,but their potential for screening colorectal neoplasms remains ambiguous.AIM To identify candidate circulating cell-free miRNAs as diagnostic biomarkers in patients with colorectal neoplasms.METHODS The study was divided into three phases:(1)Candidate miRNAs were selected from three public miRNA datasets using differential gene expression analysis methods;(2)an independent set of serum samples from 60 CRC patients,60 AA patients and 30 healthy controls(HCs)was included and analyzed by quantitative real-time polymerase chain reaction for miRNAs,and their diagnostic power was detected by receiver operating characteristic(ROC)analysis;and(3)the origin and function of miRNAs in cancer patients were investigated in cancer cell lines and tumor tissues.RESULTS Based on bioinformatics analysis,miR-627-5p and miR-199a-5p were differentially expressed in both the serum and tissues of patients with colorectal neoplasms and HCs and were selected for further study.Further validation in an independent cohort revealed that both circulating miR-627-5p and miR-199a-5p were sequentially increased from HCs and AAs to CRCs.The diagnostic power of miR-672-5p yielded an area under the curve(AUC)value of 0.90,and miR-199a-5p had an AUC of 0.83 in discriminating colorectal neoplasms from HCs.A logistic integrated model combining miR-199a-5p and miR-627-5p exhibited a higher diagnostic performance than either miRNA.Additionally,the levels of serum miR-627-5p and miR-199a-5p in CRC patients were significantly lower after surgery than before surgery and the expression of both miRNAs was increased with culture time in the culture media of several CRC cell lines,suggesting that the upregulated serum expression of both miRNAs in CRC might be tumor derived.Furthermore,in vitro experiments revealed that miR-627-5p and miR-199a-5p acted as tumor suppressors in CRC cells.CONCLUSION Serum levels of miR-199a-5p and miR-627-5p were markedly increased in patients with colorectal neoplasms and showed strong potential as minimally invasive biomarkers for the early screening of colorectal neoplasms.