Study On The Potential Of MicroRNA As A Molecular Marker For Colorectal Tumors And Its Mechanism Of Actio

Part I Circulating microRNA are promising diagnostic biomarkers of colorectal neoplasiaBackground: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:The objective of this study was to identify candidate circulating miRNAs as screening biomarkers in colorectal neoplasm patients.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 collected and detected by quantitative real-time polymerase chain reaction(qRT-PCR)for miRNAs,and their diagnostic power was detected by receiver operating characteristic(ROC)analysis;(3)the origin of circulating miRNAs in cancer patients were investigated.Fifteen pairs of serum samples obtained from cancer patients after and before surgery were used to identify the expression alterations of miR-199a-5p and miR-627-5p between preoperative and postoperative patients.Results:Based on bioinformatics analysis,miR-627-5p and miR-199a-5p were differentially expressed in both the plasma and tissues of patients with colorectal neoplasms and HCs and were selected for further study.Further validation in an independent set of CRC patients,AA patients and HCs 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 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.Conclusion:Serum miR-199a-5p and miR-627-5p were markedly increased in patients with CRC and AA and showed strong potential as non-invasive biomarkers for the early detection of colorectal neoplasms.Part II miR-627-5p inhibits colorectal cancer cell proliferation,migration and invasion by targeting Wnt2Background:Dysregulation of miR-627-5p has been observed in several solid tumors.such as gastric cancer,oral squamous cell carcinoma,hepatocellular carcinoma,and glioblastoma multiforme.However,the mechanistic details of the function of miR-627-5p in CRC progression remains unclear yet.Aim:The objective of this study was to explore the effects of miR-627-5p on the biological behavior of CRC cells by targeting Wnt2.Methods:CCK-8,wound healing,cell apoptosis,and transwell assays were performed to investigate the effects of miR-627-5p on the growth,migration,apoptosis,and invasion of CRC cells.Candidate target genes of miR-627-5p were screened by using TargetScan database,and the results showed that the 3’untranslated region(3’UTR)of Wnt2 contained a potential miR-627-5p binding site.Luciferase reporter assay was used to determine whether Wnt2 was a target of miR-627-5p.qRT-PCR analysis was applied to investigate the gene expressions of miR-627-5p and Wnt2 in CRC tissues and cell lines.The mRNA and protein expressions of Wnt2 were examined by qRT-PCR and Western blot analysis in CRC cells with high miR-627-5p expression.Cotransfection with the overexpression vector of Wnt2 and miR-627-5p mimics in CRC cells was utilized to verify whether overexpression of Wnt2 could reverse the effects of miR-627-5p in CRC progression.Western blot and qRT-PCR analysis were performed to investigate the effects of miR-627-5p on Wnt/β-catenin signaling pathway.Results:In vitro gain of function assays demonstrated that upregulated of miR-627-5p weakened the motility,viability,and invasion of CRC cells and promoted apoptosis,suggesting its inhibitory role in CRC progression.Dual luciferase reporter assay confirmed that miR-627-5p targeted the 3’UTR of Wnt2 directly.miR-627-5p was significantly downregulated in CRC cell lines and tissue samples while the gene expression of Wnt2 was significantly increased.Western blot and qRT-PCR analysis revealed that overexpression of miR-627-5p markedly suppressed the protein and gene expressions of Wnt2 in tumor cells.These results suggested that Wnt2 was a direct target of miR-627-5p.The upregulation of Wnt2 reversed the suppressive effects of miR-627-5p in CRC.Moreover.miR-627-5p reduced the gene and protein expressions of the downstream Wnt/β-catenin signaling.such as β-catenin,c-myc.CD44,and cyclinD1.Conclusion:These results demonstrated that miR-627-5p acted as a critical tumor suppressor in CRC possibly by directly targeting Wnt2 and negatively regulating Wnt/β-catenin signaling pathway,revealing that miR-627-5p might be a potential therapeutic target in CRC.