Effect Of MACC1 By RNA Interference(RNAi) On The Invasion, Metastasis Of Colon Cancer Cell And The Possible Upstream Signaling Pathway

Objective: To investigate the effect of MACC1 by RNA interference on the migration and invasion of colon cancer cell SW620,and to reveal its potential upstream signal path ways.Methods: MACC1-sh RNA recombinant plasmid which contains three different kinds of RNAinterference fragment was established to be transfected by using Lipofectamin 2000 medium. Three new cell lines were obtained after screening. Experiment was divided into three groups: interference fragment transfection group,negative control group, blank plasmid group. The efficiency of transfection was examined by the green fluorescent protein as a report gene.q PCR and Western Blot methods were used to detect the expression of MACC1 gene m RNA and protein of all the cell groups, aiming to choose the most effective one as the stable Interference group. Wound Sratch Assay and Transwell Invasion Assay were used to investigate the change of the migration and invasion ability of the stably transfection cell groups. All the cell groups were treated using the specificity kinase inhibitor PD98059,Curcumin,Wortmannin,Dasatinib to block the pathway: Ras/Raf/MAPK, Wnt/β-Catenin/GSK-3β, PI3K/PKB/ILK, Src/STAT3. The expression of MACC1 gene m RNA were detected by q PCR and the possible upstream signaling pathways involved in regulating effect on the migration and invasion of MACC1 gene was selected.Results:The cell transfection rate was above 80%. Transfection efficiency tests showed that compared with control group, the expression of MACC1 gene m RNA and protein reduce significantly in interference groups(P<0.05), but there is no statistic difference between control groups(P>0.05). Wound Sratch Assay suggest that the migration of interference group significantly reduce compared to control groups(P<0.05). Transwell Invasion Assay revealed that the invasion ability of cells in interference group were inhibited markedly by MACC1-sh RNA(P<0.05). q PCR experiments indicated that the expression of SW620 cell lines of MACC1 gene m RNA decreased obviously after blocking the Ras/Raf/MAPK、Wnt/β-Catinin/GSK-3β and Src/STAT3 signaling pathways, respectively. However,no obvious effects on the expression of m RNA was observed if block the PI3K/ILK/PKBConclusions: MACC1 can regulate the migration and invasion of colon cancer cell SW620.It might be a potential target for molecular-based therapy for colorectal cancer. Ras/Raf/MAPK、Wnt/β-Catenin/GSK-3β and Src/STAT3 signaling pathways may be involved in the upstream signal transduction of MACC1 gene expression.