TSG101-siRNA expression vector reverses anti-drug resistanse of human colon cancer cell line HT29 against oxaliplatin / 第三军医大学学报

ABSTRACT

Objective To construct an siRNA expression plasmid against tumor susceptibility gene 101(tsg101) and investigate its effect of RNA interference on oxaliplatin(L-OHP)-resistant human colon cancer cell line HT29/L-OHP.Methods The targeting fragments specifically against tsg101 were designed according to the principle of small interfering RNA designation using an online software.The sequences were cloned into siRNA expression plasmids,and the plasmids were transfected into HT29/L-OHP cells.The expression of tsg101 mRNA after the transfection of TSG101-siRNA plasmid was detected by RT-PCR.The expression of TSG101 after TSG101-siRNA transfection was detected by Western blotting.MTT test was applied to measure the inhibition combined with L-OHP on the proliferation of HT29/L-OHP cells.Distribution of cell cycle was analyzed using flow cytometry after RNA interference to HT29/L-OHP cells.Results The expected fragments were designed,and the TSG101-siRNA plasmid was confirmed by DNA sequencing.The TSG101-siRNA plasmid inhibited the expression of tsg101 mRNA,and the expression of TSG101 protein,and suppressed the proliferation of HT29/L-OHP cells obviously when combined with oxaliplatin.The analysis of cell cycle indicated that the TSG101-siRNA plasmid reduced the cells in G2/M phases and increased the cells in G0/G1 and S phases.Conclusion The expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP cells and increases the sensitivity to chemotherapy.