MiR-139-3p Functions As A Tumor Suppressor In Colorectal Cancer By Targeting RAb1A

ObjectiveThis paper was aimed to explore the expression level and mechanism of action of miR-139-3p in colorectal cancer and search for target gene RAb1 A to further analyze the regulation mechanism of RAb1 A for colorectal cancer cells and the possible signaling pathways,which would provide scientific experimental basis for finding new targets for colorectal cancer detection and treatment.MethodsThis article was divided into three parts,four chapters to complete the experimental content,including clinical,cellular and in vivo tumor experiments.1 Clinical trial: RT-PCR was adapted to detect the differential expression of miR-139-3p in colorectal cancer,the RAb1 A protein level was examined by Western Blot,and immunohistochemical assay was used to text the differential expression of KI67,?-SMA and Bcl-2.2 Cellular experiments: After transfecting miR-139-3p and silencing RAb1 A into HT29 and SW620,first,MTT and colony forming assays were used to examine the cell proliferation.Second,wound healing test and transwell invasion assay were adapted to evaluate the activities of cell migration and invasion.Third,cell apoptosis and cycle were detected by flow cytometry.Fourth,Western blot was carried out to test related proteins.Last,we used the dual luciferase reporter gene assay system to confirm the target gene.3 Nude mouse tumorigenicity assay: Stable cell SW620 was obtained after transfection of miR-139-3p,and subcutaneously injected into nude mice for colorectal cancer xenograft model.The volume and weight of tumor volume were recorded and the growth curve was drawn to determine the effect of miR-139-3p on tumor formation.Moreover,immunohistochemistry was used to detect the differential expression of proteins in the transplanted tumors.Results1 From the results of RT-PCR,we knew that miR-139-3p was significantly lower in colorectal cancer tissues and colorectal cancer cell lines HT29 and SW620 compared with the adjacent tissues and normal colorectal epithelial cell line CCD841.Con(p< 0.01).2 After overexpression of miR-139-3p or silencing RAb1 A,the cell proliferation ability decreased significantly from the results of MTT and colony formation,and wound healing and Transwell experimental results verified the activities of migration and invasion also showed a downward trend.Besides,most cells were arrested at the G1 phase and elevated apoptotic levels.3 Dual-luciferase target tests confirmed that RAb1 A was the target gene of miR-139-3p,and the expression level of RAb1 A in colon cancer decreased with the increase of miR-139-3p.4 Overexpression of miR-139-3p could significantly reduce the tumorigenic ability of SW620,and inhibit the expression of KI67,Bcl-2,?-SMA and target protein RAb1 A,which further confirmed the inhibitory activity of miR-139-3p on the growth of colorectal cancer.Western blot verified that RAb1 A was highly expressed in colorectal cancer tissues and cancer cells,and was involved in the regulation of the progress of proliferation,migration and invasion of colon cancer by mTOC1(p-S6K1)signaling pathway.ConclusionMiR-139-3p was lower expressed in colorectal cancer,and overexpression of miR-139-3p or silencing RAb1 A could significantly inhibit the proliferation,migration and invasion of colon cancer cells.Furthermore,RAb1 A was the target gene of miR-139-3p,decreased with the increase of miR-139-3p levels,and affected the development and progression of colon cancer via mTOC1(p-S6K1)signaling pathways.