Study On The Expression And The Roles Of MiR-338-5p In Colorectal Cancer

ObjectiveThe aims of this study was to evalute the expression of miR-338-5p in colorectal cancer tissues and colon cell lines, and explore its role in colon cancer cell proliferation and apoptosis, and identify its target genes. We hope that this study would provide a new taget for the diagnosis and treatment of colorectal cancer by investigativing the relationship between miR-338-5p and the pathogenesis of colorectal cancer.MethodThe expression of miR-338-5p in40colorectal cancer tissues and corresponding adjacent to cancer tissue samples was detected by real-time PCR (RT-PCR). Then, analyze the relationship between miR-338-5p and clinicopathological characteristics of these colorectal cancer tissues. And the expression of miR-338-5p in colon cancer cell lines HCT116, HT29, SW480and SW620were also detected for the further functional study. Based on the expression of miR-338-5p in colorectal cancer tissues and colon cancer cell lines, we chose two colon cancer cell lines in which shown lower expression of miR-338-5p for rescue restoration of miR-338-5p in further functional study. We then verified the rescue restoration of miR-338-5p for which tranfected with miR-338-5p mimics or Negative control by RT-PCR in colon cancer cells. The cell proliferation was measured by CCK-8, and the cell apotosis and cell cycle analysis were performed by flow cytometry, in the biological functions study of miR-338-5p respectively. Using the available publicly programs to predict the potential targets of miR-388-5p in colorectal cancer. We then performed a luciferase reporter assay to verify the candidate genes. Western blot was also performed to dectect the expression levels of protein.Resultsl.miR-338-5p expression was significantly downregulated in the colorectal cancer tissues as compared with corresponding adjacent to cancer tissue samples(P<0.01). However, the miR-338-5p expression level had no correlation with clinicopathological features. And the colon cancer cell lines HCT116and SW620were chose for further study owing to their lower expression levels.2.Compared with the negative control, the expression of miR-338-5p in colon cancer cells HCT116and SW620increased significantly after transfected with miR-338-5p mimics (p<0.001). The cell function assay shown that the proliferation ability of colon cancer cells HCT116and SW620was significantly decreased(P<0.01), and the cell apoptosis ration was significantly increased(HCT116:11.43±0.67%versus7.98±0.36%, P<0.01; SW620:10.5±0.2%versus7.93±0.5%, P<0.01), and the cell cycle was arrested G1(HCT116:80.41±1.34%versus64.87±1.83%, P<0.01; SW620:68.76±0.41%versus54.89±0.78%, P<0.01) when ectopic expression of miR-338-5p.3.Using the available publicly programs, DIANA LAB, Miranda, TargetScan and so on, indicated that miR-338-5p potentially targeted KRAS. And the luciferase reporter assay demonstrated that KRAS was a direct target of miR-338-5p. And miR-338-5p could suppress the expression of KRAS and regulate its downstream singnaling pathway Raf/MEK/ERK and PI3K/AKT by decreasing the expression of p-AKT and p-ERK1/2of which dectected by Western blot.ConclusionAll the results revealed that miR-338-5p may act as a tumor suppressor by targeting KRAS to inhibit cell proliferation, promote cell apoptosis in the pathogenesis of colorectal cancer. And it may function as a new target for diagnosis and treatment in colorectal cancer.