The Role And Mechanism Of Mir-452-5p In The Progression Of Colorectal Cancer By Regulating An ERK/MAPK Positive Feedback Loop

ObjectiveColorectal cancer is the third most common malignant tumor in the world.Patients in advanced stages are related to poor treatment outcomes.Therefore,in depth study of the molecular mechanism of colorectal cancer occurrence and development will help find potential treatment targets for colorectal cancer and improve patient prognosis.MiRNA is a small single-stranded non-coding RNA that interacts with mRNA mainly through binding to the complementary sequence of the 3'untranslated region,thereby exerting a role in inhibiting gene expression.MiRNAs have been reported to play an important role in the genesis and development of various tumors.MiR-452-5p has been reported to play an important role in the development of a variety of tumors,but there are few researches of its role in colorectal cancer.The purpose of this study is to evaluate the expression of miR-452-5p in colorectal cancer,to explore whether miR-452-5p can affect the biological function of colorectal cancer cells and its underlying molecular mechanism.Methods1.Verification of the expression level of miR-452-5p in colorectal tissues and cell lines1)The expression of miR-452-5p in colon adenomas and tumors was analyzed by TCGA.2)RT-PCR assay was used to detect the expression of miR-452-5p in colorectal tumor tissues and paired normal tissues,colorectal cancer cell lines and normal intestinal epithelial cell lines.2.MiR-452-5p promotes malignant biological behaviors of colorectal cancer cells1)Proliferation in vitro:miR-452-5p mimics and inhibitors were transfected into colon cancer cells.CCK8 assay and colony formation assay were carried out after verifying the efficiency of transfection.2)Cell cycle:The effect of miR-452-5p on cell cycle of colorectal cancer cells was verified by cell cycle experiments.3)Chemotherapeutic resistance:The effect of miR-452-5p on cell apoptosis and chemotherapeutic resistance to 5-fluorouracil of colorectal cancer cells was verified by IC50 test,apoptosis assay and western blot.4)Proliferation in vivo:Subcutaneous tumorigenesis assay of cells with overexpression or knock-down of miR-452-5p were performed in nude mice.3.Validation of the mechanism of the miR-452-5p—PKN2/DUSP6—c-Jun positive feedback loop1)The down-regulated genes in cells with over-expression of miR-452-5p and predicted target genes were intersected to identify the proposed target genes,which were verified by RT-PCR and western blot.The direct binding of miR-452-5p with target genes was verified by dual-luciferase reporter assay.2)The effect of miR-452-5p on ERK/MAPK signaling pathway was confirmed by western blot of downstream genes.3)Recovery experiments(CCK8 and colony formation assay)were used to verify that PKN2/DUSP6 axis is involved in the regulation of colorectal cancer by miR-452-5p.4)The transcriptional regulation of c-Jun on miR-452-5p was verified by Bioinformatics analysis and prediction,RT-PCR and ChIP(Chromatin immuoprecipitation)assay.Results1.Compared with normal tissues,the expression of miR-452-5p in colorectal adenomas and tumors was significantly higher,and the expression of miR-452-5p was higher in HT29 cells with higher malignancy,lower in RKO,SW480 cells with lower malignancy and the lowest in FHC cells.2.Overexpression of miR-452-5p promotes the proliferation of colorectal cancer cells and G1/S phase transformation.4.MiR-452-5p directly targeted PKN2 and DUSP6 and participated in ERK/MAPK signaling pathway.5.Recovery experiments showed that over-expression of PKN2 and DUSP6 could counteract the promoting effect of miR-452-5p on malignant biological behaviors of colorectal cancer.ConclusionThis study confirmed that the expression of miR-452-5p was high in colorectal cancer,and that the overexpression of miR-452-5p could promote the activation of ERK/MAPK signaling pathway by targeting the PKN2/DUSP6 axis.The miR-452-5p—PKN2/DUSP6—c-Jun positive feedback loop promotes the maglinant behavior of colorectal cancer.