Reversal Of Multi-drug Resistance In HeLaB2/DDP Cells By Small Interference RNA

Resistance of cancer cells to chemotherapy continues to be a major clinical obstacle to the successful treatment of cancer. It is critical to reverse multidrug-resistance for improving chemotherapy effect. The mechanism of multidrug resistance is mainly relevant with the drug resistant gene mdr-1 and expression of its coding glycoprotein P-gp.The apoptosis suppressor gene bcl-2 and its coding protein Bcl -2 are also concerned with multidrug-resistance. RNA interference (RNAi) is a phenomenon in cell that double -stranded RNA mediates degradation of homologous mRNA inducing sequence-specific silence of gene expression. As a kind of post-transcriptional gene silencing (PTGS), RNAi is a potent method to reverse multidrug-resistance.The drug-resistant cell line of cisplatin (HeLaB2/DDP)was established gradually by increasing dose of cisplatin and intermittent administration.To observe the effect of small interfering RNA (siRNA)or asymmetric small interfering RNA(asiRNA) targeting multidrug resistance protein (P-gp) and bcl-2 genes in modulating drug resistance of HelaB2/DDP cells, four siRNA or asiRNA targeting respectively bcl-2 or mdr-1 genes were synthesized and transfected either alone or in combination into HeLaB2/DDP cells via Iipofectamine2000.CCK-8 assay was used to evaluate the viability of the transfected cells.Quantitative real-time PCR (Q-RT-PCR) was performed to determine the mRNA levels of bcl-2 or mdr-1. The effects of mdr-1 siRNA, bcl-2 siRNA and bcl-2 asiRNA on the protein expression of Bcl-2 or P-gp were evaluated with flow cytometry. The microRNA expression profile of HeLaB2and HeLaB2 by continuous transfection with 19/21b were detected by microRNA microarray techniques during drug-resistant process.After siRNA or asiRNA targeting mdr-1 or bcl-2 were transfected into HeLaB2/DDP, the levels of target mRNA and target protein decrease significantly. Meanwhile the IC50 decreased significantly compared with control group(P<0.05).The IC50 of the combining transfected group was the lowest among all groups(P<0.05). It provided a primary experimental basis for reversion of MDR at gene level.19/21b reversed the levels of most microRNA that changed in HeLaB2 during the drug-resistant process, microarray analyses indicated that these microRNAs may be involved in regulation of bcl-2 gene or drug-resistant. The mechanism is still pending in further study.