MiR-92a Promotes The Invasion And Migration Of Colorectal Cancer By Targeting RECK

Colorectal cancer(CRC)is one of the most common malignant tumors in China.Its incidence is increasing year by year.MicroRNAs(miRNAs)are a class of numerous non-coding RNAs that regulate target gene expression by inhibiting post-transcriptional translational levels.Our previous studies have found that the expression of microRNA-92a(miR-92a)is up-regulated in colorectal cancer and its derived cell lines,and it can promote the invasion and migration of colorectal cancer by indirectly regulating matrix metalloproteinase 2(MMP2)and matrix metalloproteinase 9(MMP9).However,what kind of intermediate signalmolecules can miR-92a play a role in promoting cancer?Previous studies have shown that the Reversion inducing Cysteine-rich protein with Kazal motifs(RECK)gene is a specific inhibitor of MMP2 and MMP9.Moreover,bioinformatic analysis indicated that there is a complementary binding site between miR-92a and the 3’untranslated coding(3’-UTR)of RECK gene mRNA.Therefore,we propose the scientific hypothesis that miR-92a may promote the expression of MMP2 and MMP9 by regulating RECK gene,and then promote the invasion and migration of CRC.To verify this hypothesis,this project aims to observe the effects of altering the expression level of miR-92a in CRC cell lines on the biological behavior of CRC cells and RECK signaling pathway in vitro,and to determine the role of RECK-MMPs signaling pathway in the regulation of CRC invasion and migration by miR-92a.This study will provide a basis for exploring the feasibility of miR-92a as a diagnostic and prognostic indicator for colorectal cancer,and can provide a clue for the development of new drugs or inhibitors for this target。Objective:To analyze the correlation between expression of the miR-92a and clinicopathological features and prognosis of colorectal cancer.And to investigate the regulatory effect of miR-92a on cell invasion and migration in colorectal cancer.Methods:In situ hybridization was used to detect the expression of miR-92a in 124 colorectal cancer tissues.The median expression level was used as the threshold to divide patients into miR-92a high expression group and miR-92a low expression group.Spearman correlation test was used to analyze the correlation between miR-92a expression level and clinicopathological features of colorectal cancer.The Kaplan-Meier method was used to analyze the survival rate and survival time of patients with colorectal cancer after 3 years.Univariate and multivariate Cox regression models were used to analyze whether miR-92a expression levels can be used as independent risk factors for predicting the prognosis of colorectal cancer patients.Quantitative reverse transcription PCR was used to detect the expression of miR-92a and its target gene.Protein levels were determined by Western blotting.Luciferase assays confirmed the direct target of miR-92a.Furthermore,cell invasion and migration were detected using Transwell assays and wound healing tests.Results:The expression of miR-92a was correlated with the degree of differentiation(P=0.015),depth of invasion(P=0.031),lymph node metastasis(P=0.030),distant metastasis(P=0.011)and American Joint Committee on Cancerstaging(AJCC,eighth edition)(P=0.047).Kaplan-Meier survival analysis showed that the 3-year cumulative survival rates were 66.1%and 91.9%(χ2=12.006,P=0.001)in patients with high and low expression of miR-92a.Multivariate Cox analysis showed that the expression of microRNA-92a was an independent prognostic factor for colorectal cancer(P=0.026).The expression level of miR-92a in tumor tissues was upregulated compared with that of paired normal tissues and negatively correlated with the RECK protein level.After transfection of the miR-92a mimics,the miR-92a levels were increased in HCT116 and SW620 cell lines,while the protein expression of RECK was decreased instead of the mRNA level,along with up-regulation of matrix metalloproteinase(MMP)protein expression.Conversely,after transfection with miR-92a inhibitor,the opposite trend was achieved.The RECK gene 3’-UTR luciferase assay showed that the RECK gene is a direct target of miR-92a.Conclusion:The expression of miR-92a is significantly correlated with the clinical stage of colorectal cancer and can be used as a molecular marker to predict the long-term prognosis of patients.MiR-92a promotes the invasion and migration of colorectal cancer through the RECK-MMPs signaling pathway,and the upregulation of miR-92a was associated with poor long-term prognosis in colorectal cancer.