The Molecular Mechanism Of MiR-145 In Mediating Metastasis Of Colorectal Cancer By Targeting PAK4

Objective: In this study, we are planning to verify PAK4 is the target gene of mi R-145 in vitro cellular level, and make clear that mi R-145 regulate the proliferation, migration, and invasion of cancer cells by mediating PAK4. Furthermore, we want to explore the regulation of mi R-145/PAK4 on downstream signal pathway LIMK1/cofilin.Methods: 1. Analysis the relationship between mi R-145 and PAK4 by using the luciferase reporter assay. 2. Constructing stable SW1116 cells that overexpressed mi R-145 and knocked down PAK4, analysis the change of proliferation, migration, and invasion of colorectal cancer cell by using MTT, Transwell, and Transwell with matrigel. 3. Using western blotting to detect the influence of overexpressing of mi R-145 and knocking down PAK4 on downstream signal pathway LIMK1/cofilin in colorectal cancer cells.Results: 1. Construting the wild type reporter gene plasmid psi CHECK2-PAK4-WT and mutant type reporter gene plasmid psi CHECK2-PAK4-MUT, construting plasmid that overexpressing mi R-145 p CDH-mi R-145 and vector plasmid p CDH-V, co-transfecting psi CHECK2-PAK4-WT or psi CHECK2-PAK4-MUT and p CDH-mi R-145 or p CDH-V into the HEK293 cells, and according to the reporter gene analysis, the luiferase activity of cells transfected with p CDH-mi R-145 and psi CHECK2-PAK4-WT were significantly reducedcompared to the cells that transfected with p CDH-V and psi CHECK2-PAK4-WT, but the repression was abolished by psi CHECK2-PAK4-MUT. Constructing p CDH-anti-mi R-145 in order to inhibit the endogenous expression of mi R-145, and then co-transfecting psi CHECK2-PAK4-WT or psi CHECK2-PAK4-MUT and p CDH-anti-mi R-145 or p CDH-V into NCM460 cells, the results showed that anti-mi R-145 increased the luiferase activity of cells transfected with psi CHECK2-PAK4-WT, but not with the wild type. 2. Constructing stable SW1116 cells that overexpressed mi R-145 and knocked down PAK4, detecting the influence of overexpressing mi R-145 and knocking down PAK4 to proliferation, migration and invasion of colorectal cancer cells by using MTT, Transwell and Transwell with matrigel. The results shows that overexpressed mi R-145 inhibited proliferation, migration and invasion of SW1116 cells compared to the control vector, and knocked down PAK4 also inhibited proliferation, migration and invasion of SW1116 cells compared to the control vector. 3. The western blotting results demonstrate that the phosphorylate level of LIMK1 and cofilin were reduced by overexpressing mi R-145 and knocking down PAK4.Conclusion: 1. PAK4 is the target gene of mi R-145. 2. mi R-145 inhibits proliferation, migration, and invasion of colorectal cancer cells by targeting PAK4. 3. mi R-145 inhibits metastasis of colorectal caner by mediating LIMK1/cofilin, the downsream signal pathway of PAK4.