MiR-532-3p Suppresses The Proliferation,Invasion And Metastasis Of Colorectal Cancer

Background and ObjectionColorectal cancer is one of the most common clinical malignant tumors in recent years.It has high morbidity and mortality in many countries and regions.China has high incidence of colorectal cancer.The incidence is the third highest and the mortality rate ranked fourth in malignant tumors.Therefore,elucidating the molecular mechanism of colorectal cancer,looking for relevant molecular markers,has become a major focus of colorectal cancer research.MicroRNAs(miRNAs)are a class of small non-coding single-stranded RNAs containing 18-25 bases.MiRNAs specifically bind to the 3’UTR of the target gene by base-pairing site binding,inhibit the translation of transcription products,thereby regulating the target gene expression,affecting cell development,proliferation,differentiation and apoptosis processes.MiRNAs are dysregulated in many tumors and act as oncogenes or tumor suppressor genes.MiRNAs play an important role in the development of colorectal cancer.At present,researchers have found that many miRNAs are abnormally expressed in colorectal cancer tissues and can influence the activation of colorectal cancer signal pathways by inhibiting the expression of target genes,thus regulating the occurrence,development and metastasis of colorectal cancer.Our group used GEO database GSE41655 gene differential expression analysis,and found that miR-532-3p expression was down-regulated in colorectal cancer.This suggests miR-532-3p may play as a tumor suppressor gene in colorectal cancer.In this research,we will clarify the function of miR-532-3p in the development and metastasis of colorectal cancer.Methods and materials1.Expression of miR-532-3p in colorectal cancer tissues and cells1)Through the analysis of GSE41655 in the GEO database,the differentiated miR-532-3p was screened out.2)The expression of miR-532-3p was detected by RT-PCR in 51 cases of colorectal cancer and paired normal mucosa,7 colorectal cancer cell lines and 1 normal colon cell line.3)The clinical data of 51 patients with colorectal cancer were collected to analyze the correlation between the expression level of miR-532-3p and clinical indexes.2.Effect of miR-532-3p on biological behaviors of colorectal cancer cells in vitro1)The efficiency of miR-532-3p overexpression and interference was detected by RT-PCR by transfection of miR-532-3p mimics/inhibitor.2)The effects of miR-532-3p mimics/inhibitor on the proliferation and migration ability of colorectal cancer cells in vitro were detected by CCK8 cell proliferation assay,colony formation assay,transwell cell migration assay and would-healing assay.3.Effect of miR-532-3p on biological behaviors of colorectal cancer cells in vivo1)Lentivirus was used to construct miR-532-3p stably overexpressing and interfering cell lines.The subcutaneous tumor model in nude mice was performed to assess the effect of miR-532-3p on tumor genesis and growth of colorectal cancer cells.2)Take the subcutaneous tumor tissue samples,extract protein,Ki-67 index was detected by immunohistochemistry.4.Bioinformatics analysis and identification of miR-532-3p target gene1)The bioinformatics software was used to predict the target genes of miR-532-3p.The predicted results were taken as intersection and the candidate target genes were screened out by literature and RT-PCR.2)The target genes were predicted and the expression of miR-532-3p target gene was detected by Western blot.Results1.MiR-532-3p expression is down-regulated in colorectal cancer tissues and cells lines1)Analysis of GSE41655 in the GEO database showed that miR-532-3p was reduced in colorectal cancer.RT-PCR results showed that miR-532-3p expression in colorectal cancer tissues was lower than that in paired normal tissues.The expression of miR-532-3p in colorectal cancer cell lines was lower than that in normal colorectal epithelial cell line.2)The expression level of miR-532-3p in 51 cases of colorectal cancer tissues had no significant correlation with clinical indexes.2.MiR-532-3p inhibits the proliferation and migration ability of colorectal cancer cells in vitro1)The results of RT-PCR showed that the expression of miR-532-3p in miR-532-3p mimics group was significantly higher than that in NC group;the expression of miR-532-3p in miR-532-3p inhibitor group was significantly lower than that in NC group.2)CCK8 cell proliferation assay,colony formation assay,transwell cell migration assay and would-healing assay showed that miR-532-3p overexpression inhibited cell proliferation and migration ability in vitro.After miR-532-3p was knocked down,proliferation and migration ability in vitro significantly increased.3.MiR-532-3p inhibits the proliferation ability of colorectal cancer cells in vivo1)Subcutaneous tumor formation in nude mice showed that the volume of subcutaneous tumor in miR-532-3p overexpression group was significantly smaller than that of the control group,while the volume of miR-532-3p interfered group was significantly larger than that of the control group.2)The tumor in miR-532-3p overexpression group was significantly lower in Ki-67 index than that of the control group,while this index was significantly higher in miR-532-3p interfered group compared with the control group.4.MiR-532-3p targets β-catenin and inhibits Wnt/β-catenin signaling pathway1)Four publicly available candidate bioinformatics algorithms were used to analyze target genes of miR-532-3p and 24 target genes were selected as candidate target genes.2)RT-PCR was performed on 24 target genes and it was found that β-catenin was the most down-regulated.3)Western blot results suggest that miR-532-3p inhibits β-catenin and Wnt/β-catenin signaling pathway related protein molecules.Conclusion1.MiR-532-3p is down-regulated in colorectal cancer tissues and cell lines,indicated miR-532-3p may play as a tumor supress gene in colorectal cancer.2.MiR-532-3p inhibits the proliferation and migration ability of colorectal cancer cells in vitro.3.MiR-532-3p inhibits the proliferation ability of colorectal cancer cells in vivo.4.MiR-532-3p targets β-catenin and inhibits Wnt/β-catenin signaling pathway.