The Mechanism Of MiR-16 Suppresses The Growth Of Colorectal Cancer By Targeting KRAS

Background and objective:The incidence rate of colorectal cancer(CRC)is high all over the world.In China,the incidence and mortality rates of colorectal cancer are increasing year by year,and there are a large number of patients die from colorectal cancer every year.So,the search for new treatments and drugs is in intense.KRAS,one member of Ras family,plays an important role in the occurrence and progression of intestinal cancer,but the underlying mechanism is poorly known.MicroRNAs(miRNAs)involving in many cancers by negatively regulating their target genes.This study aimed to explore the underlying mechanism of KRAS in CRC,select miRNAs which can directly target KRAS and further discuss the role of miRNA-KRAS pathway in the biological behavior of CRC,which provide a new basis for the treatment of colorectal cancer,and it's possible to develop new drugs by targeting this pathway to inhibitor tumors.Methods:Clinical tissue specimen experiments: Firstly,we collected sixteen paired CRC tissues and the adjacent non-tumor tissues from patients who underwent surgical resection at the Tianjin Medical University Cancer Institute and Hospital.Then we used Western blot and qRT-PCR to examin the KRAS protein and it's mRNA expression level respectively.We predicted that miR-16 can directly target the 3'-UTR of KRAS mRNA by employing bioinformatics tools.Lastly,we detected the exprsssion levels of miR-16 in the above-mentioned clinical tissues.In vitro experiments: We predicted that miR-16 can directly bond to the 3'-UTR of KRAS mRNA and we need futher verify that miR-16 can directly regulate the expression of KRAS by employing the luciferase report experiments.The expression levels of miR-16 and KRAS were determined after transfecting SW480 cells and HT-29 cells with pre-miR-16 and the Caco2 cells with anti-mi R-16.And the CCK8 assays,transwell and cell flow assays were performed to verify the role of miR-16 in CRC cells.The overexpression or knockdown of KRAS was achieved by transfected CRC cells with overexpressed KRAS plasmid or KRAS siRNA,then the functional role of KRAS in biological behavior of CRC cells was analysised.Furthermore,we simultaneously overexpressed miR-16 and KRAS to determine the effect of miR-16-KRAS pathway in the CRC cells.In vivo experiments: Untreated SW480 cells or SW480 cells transfected with miR-16 overexpressing lentivirus or SW480 cells transfected with KRAS overexpressing plasmid or SW480 simultaneously transfected with miR-16 overexpressing lentivirus and KRAS overexpressing plasmid were injected subcutaneously into nude mice respectively.After 4 weeks,mice were sacrificed,and the tumors were separated.Then we took the picture and measured the diameter of tumors.We used a part of tumor tissues to examin the expression level of KRAS and miR-16 and the other tissues were used for HE staining experiments and immunohistochemistry.Results:Clinical tissue specimen experiments: compared with the adjacent non-tumor tissues,the expression level of KRAS protein showed obviously higher in CRC tissues;however,its mRNA levels did not differ significantly.We predicted that miR-16 can directly target the 3'-UTR of KRAS mRNA by employing bioinformatics tools,and the binding energy between miR-16 and KRAS mRNA was-24.2kcal/mol.And the miR-16 expression levels were obviously decreased in the above-mentioned CRC tissues.In vitro experiments: The luciferase assay showed that miR-16 can directly bind to the 3'UTR of KRAS mRNA and regulate the expression of KRAS.Overexpression of miR-16 by transfecting SW480 cells and HT-29 cells with pre-miR-16 leads to the clear suppression of KRAS protein,but not KRAS mRNA.While transfected Caco2 cells with anti-miR-16 enhances the expression of KRAS protein.The subsequent functional experiments demonstrated that mi R-16 suppressed the proliferation and invasion while induced the apoptosis of CRC cells.Whereas the KRAS has the opposite effect.When we simultaneously overexpressed miR-16 and KRAS,overexpression of KRAS can partly reduced mi R-16-induced CRC cells growth.In vivo experiments: overexpression of miR-16 suppressed tumor growth,showed decreased tumor size and weight;while the overexpression of KRAS strongly promoted tumor growth.And the overexpression of KRAS can partly rescue the effect of miR-16 on the tumor growth.The KRAS protein was significantly inhibited in the miR-16 overexpression group,while the KRAS mRNA was not changed.The results of HE staining experiments and the immunohistochemistry experiments were as expected.Conclusion:In colorectal cancer,miR-16 function as a tumor suppressor by directely regulate the expression of KRAS.