RESUMO
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.
RESUMO
Objective To investigate the expression and possible function of tumor susceptibility gene 101 (TSG101) in multidrug-resistant cell lines of gastric cancer.Methods The expression of TSG101 was examined in gastric cancer cell line SGC7901 and its vincristine(VCR)-resistant subline SGC7901/VCR with semi -quantitative RT-PCR and Western blot. TSG101 eukaryotic expression vector was transfected into SGC7901 cells by lipofectamine TM 2000. The expression levels of TSG101 in SGC7901 and the transfectants were detected with Western blot. Fluorescence-activated cell sorting was applied to examine the cell cycle alteration and the intracellular mean fluorescence intensity of adriamycin (ADR). Growth curve and drug sensitivity of cells to VCR and ADR were analyzed by MTT assay.Results TSG101 was highly expressed in VCR resistant gastric cancer cells. The expression of TSG101 in TSG101 transfectants was up-regulated compared with that in SGC7901 transfected with empty vector or in SGC7901 cells. TSG101 transfectants were accumulated in S phase, with a concomitant decrease of cell population in G 1 phase. MTT assay showed that TSG101 transfectants proliferated rapidly and were more resistant to VCR and ADR than control cells. Conclusions The over-expression of TSG101 could promote the multidrug resistance phenotype of sGC7901 cells. TSG101 may play a certain role in multidrug resistance of gastric cancer.