RNA Interference Of ILK Gene And Its Effect On Invasion And Migration In Human Colon Cancer Cells

Objective:To explore the impact of migration and invasion by RNA interference ILK gene and investigate the role of RNAi ILK gene inducing mesenchymal-epithelial transition in human colon cancer cell lines.Method:1. The sequence of shRNA for ILK gene was designed and the plasmid of pGenesill.2-ILK shRNA was constructed. SW620, SW480and HT29human colon cancer cell lines were cultured.The mRNA and protein expression of ILK was analyzed by RT-PCR and Western Blot respectively.The colon cancer cell line with highest expression of ILK gene was selected.2.Colon cancer cell line was transfected with plasmid pGenesi11.2-ILK shRNA by cationic lipid transfection.Experiment were devided into three groups as:(1) Blank control group:non transfected cell group;(2) Experiment group:plasmid pGenesi11.2-ILKshRNA transfected cell group;(3) Negative control group:plasmid pGenesi11.2transfected cell group. Transfection efficacy was analysed by flowcytrometry. The mRNA and protein expression level of ILK gene were determined by fluorescence quantitative real time PCR and Western blot respectively.3.At the same time mRNA and protein expression level of epithelial-mescenchymal transition related gene, E-cadherin and Vimentin were also determined by fluorescence quantitative real time PCR and Western blot respectively.4.Migration and invasive analysis was done by transwell experiment.Result:1. SW620has the highest mRNA and protein expression level of ILK in the three human colon cancer cell lines. We selected SW620cell line for the further experiment.2. After ILK shRNA plasmid was transfected into colon cancer SW620cell line, the results of fluorescence quantitative PCR displayed that the experimental group ILK mRNA expression decreased significantly compared with the negative control group and blank control group, the difference was statistically significant (P<0.05). The protein expression level of ILK in experimental group compared with the negative control group and blank control group were also significantly lower, the difference was statistically significance (P<0.05).3. After transfection of ILK shRNA plasmid into SW620cell line, mRNA and protein expression level of E-cadherin in experimental group was higher than that of negative control group and control group,the difference was statistically significant(P<0.05). The mRNA and protein expression level of Vimentin in experimental group is lower than that of negative control group and blank control group,the difference was statistically significant(P<0.05).4. After transfection, the transwell migration assay in experimental group, negative group and blank control group showed the number of cell were134.94±4.76,196.76±4.97and203.35±7.31respectively. Experimental group clearly showed decreased migration of cell number in comparison to other two groups, the difference was statistically significant(P<0.05). The transwell invasion cell assay through matrigel membrane in experiment group, negative group and blank control group were36.44±3.31,86.8±2.17and88.56±4.02respectively. Experiment group invaded number of cell also clearly showed decreased through matrigel in comparison to other two groups, the difference was statistically significant (P<0.05).Conclusion:1. Human colon cancer cell line SW620has the higher ILK expression compared with SW480and HT29.2. RNA i ILK gene was able to effectively silence the ILK gene expression of SW620.3. RNAi ILK gene was able to up-regulate E-cadherin expression and down-regulate vimentin respectively, also could reverse EMT and promote MET in human colon cancer cell line SW620.4. Inhibition of ILK gene expression effectively inhibited colon cancer cell migration and invasion.