The Research Of HIF-1α Silence By SiRNA Reversing The Resistance Of Ovarian Cancer

Ovarian cancer which is one of three most serious female reproductive system malignant tumors threaten female healthy seriously. It is accepted that cancer chemoprevention is an essential approach to controlling cancer, yet, the clinical usefulness of this strategy is very limited due to primary drug-resistance and acquired drug-resistance. In order to solve the resistance and improve chemotherapy effects, it is essential to implore the mechanism of resistance which is very complex and always related with a serial of gene changing.Hypoxia-inducible factor-1 (HIF-1) is a transcription factor, which is activated in response to intratumoural hypoxia and as a result of genetic alterations. It plays a key role in the adaptation of tumour cells to hypoxia by activating the transcription of genes, which regulate several biological processes including angiogenesis, cell proliferation and survival, glucose metabolism, proliferation and migration. It is a heterodimers composed of HIF-1αand HIF-1βand its activity mainly depends on HIF-1α. Tumour cell can promote downstream target gene to induce chemotherapy resistance through HIF-1αoverexpression. HIF-1αexpression is increased in ovarian cancer and clinical studies have shown a correlation between HIF-1 overexpression and drug-resistance degree. Therefore, it may to get goals of treatment through suppressing HIF-1αexpression in ovarian cancer cell.RNA interference(RNAi) is the regulating mode of gene expression existing in biosystem generally. It is the process that exogenous or endogenous double strands RNA(dsRNA)cause homologous mRNA degradation specially and refrain corresponding gene expression consequently. RNAi technique has been applied in function genomics, medicine target selection, cell signaling analysis, and so on, due to its significant specificity, efficacy, stability, transmissibility and hereditability. In this experiment, HIF-1αsiRNA eukaryotic expression vector was constructed to silence HIF-1αexpression in SKOV3/DDP(DDP resistance strain). Both vivo and vitro investigation about the relationship between HIF-1αand drug resistant were explored to find a effective molecule target.In vitro, we constructed HIF-1αsiRNA eukaryotic expression vector (pshRNA-HIF) using pSuper.gfp/neop. To obtain SKOV3/DDP cell lines stably expressing HIF-1αsiRNAs, the pSUPER-HIF-1 plasmid were transfected. pshRNA-HIF transfect-SKOV3/DDP cells were treated by gradient concentration cis-diamminedichloroplatinum(DDP): 0,0.25μg/ml,1.25μg/ml,2.5μg/ml,5μg/ml,12.5μg/ml. To observe the effects of HIF-1αon silence on SKOV3/DDP resistant ,the inhibition ratio and apoptosis were detected by MTT and flow cytometry. SKOV3 (sensitive strain), SKOV3/DDP(resistance strain), pshRNA-Control transfect-SKOV3/DDP cells were control. Meanwhile, to explore the mechanism of HIF-1αsilence induced- resistance. RT-PCR analyze HIF-1α, MDR1, Bcl-2 mRNA expression; Western blot and immunohistochemistry detect HIF-1α, P-gp, Bcl-2 protein expression. The results showed that HIF-1αexpression of pshRNA-HIF transfect-SKOV3/DDP decreased 75%. Apoptosis of pshRNA-HIF transfect-SKOV3/DDP was higher than that of SKOV3/DDPand pshRNA-Control transfect-SKOV3/DDP, which is near to that of SKOV3 (sensitive strain). MTT results showed pshRNA-HIF transfect-SKOV3/DDP was more sensitive to DDP than SKOV3/DDPand pshRNA-Control transfect-SKOV3/DDP, which indicated HIF-1αsilence can reverse drug resistance of SKOV3/DDP. The detection of HIF-1α,P-gp,Bcl-2 showed HIF-1αsilence decrease MDR1,Bcl-2gene expression, which indicated resistance reversion-induced by HIF-1αmay be related with down regulation of MDR1and Bcl-2 gene expression.In vivo, athymic mice model bearing cancer were established using SKOV3(SKOV3 mice ) and SKOV3/DDP(SKOV3/DDP mice ) respectively. SKOV3/DDP mice were treated with pshRNA-HIF plasmids combining DDP;SKOV3 mice with DDP, SKOV3/DDP mice with DDP alone, and SKOV3/DDP mice with pshRNA-Control plasmids combining DDP were control. gross tumor volume and tumor weight were recorded. RT-PCR analyze HIF-1α, MDR1, Bcl-2 mRNA expression; immunohistochemistry detect HIF-1α, P-gp, Bcl-2 protein expression. Our objective is to reveal the mechanism of resistance reversion-induced by HIF-1αinitially in vivo. The results showed that pshRNA-HIF plasmids decreased HIF-1αexpression obviously and lightened tumor volume and tumor weight when combined DDP. The results also showed that HIF-1αsilence decrease MDR1,Bcl-2expression, coincident with vitro results. These results indicated that pshRNA-HIF plasmids can increase the sensitivity of SKOV3/DDP to DDP and the resistance reversion-induced by HIF-1αmay be related with down regulation of MDR1and Bcl-2 gene expression.In short, our research indicated pshRNA-HIF-mediated by plasmid vector is a potential effective methods to down regulate gene expression. HIF-1 silence can reversed drug resistance through decreasing MDR1and BCL-2 ,which makes it an attractive therapy target for ovarian.