Effect Of Hypoxia Inducible Factor-1 A Short Hairpin RNA Expressing Plasmid On Ovarian Cancer SKOV3 Cell

Background and purposeWith the over-proliferation of malignant tumors with disorganized, insufficient blood supply,hypoxic and or anoxic regions will inevitably develop. Hypoxia inducible factor-1(HIF-1) plays a key role in the adaptation of tumor cells to hypoxia by activating the transcription of the targeted genes. HIF-1 is a heterodimer composed of HIF-1αand HIF-1β. The expression of HIF-1αis regulated by various factors. Some studies have revealed a significant association between the expression of HIF-1αand VEGF within the ovarian cancer tissues and the poor prognosis of the patients with ovarian cancer. To investigate the effect of HIF-1αshort hairpin RNA(shRNA) expressing plasmid on ovarian cancer in vitro and in vivo.Methods1. Oligos for hairpin RNA expression which targeted HIF-1αgene were designed and selected based on the well-known principle using online software and synthesized and annealed. Annealed oligos were inserted into the down stream of human U6 promoter of the pGPU6/GFP/Neo plasmid to construct H1,H2,H3 and H4 recombinant plasmids.The recombinant vectors were verified by sequencing.2. Ovarian cancer SKOV3 cells were cultured in mimic hypoxia condition using N2-O2-Air Mixing chamber.3. The plasmids were transfected into SKOV3 cells using lipofectmine. 4. The expression of HIF-1αmRNA and protein was assayed by RT-PCR andWestern-bloting respectively. Cell apoptosis was evaluated by TUNEL.Cell viability and chemotherapy drug sensitivity were determined by methylthiazoly- ltetrazolium(MTT) assay.5. The nude mice with SKOV3 cell subcutaneous xenograft tumor were treated with the recombinant plasmids and DDP. Tumor sizes were measured every week.The HIF-1αand VEGF expression of xenograft tumor tissue were evaluated with immunohistochemistry.Results:1. Recombinant plasmids were confirmed successful by sequencing analysis.2. There are low level of expression of HIF-1αmRNA and protein in normoxic SKOV3 cells. The hypoxic environment significantly induced the expression of protein HIF-1α(P<0.05),while the HIF-1αmRNA expression were not significantly changed(P>0.05) .3. Compared to that of lipofectmine(Lipo) group, negative control(NC) group,H1group and H2 group , HIF-1αmRNA and protein expressions of H3 and H4 group were significantly downregulated at 48 h after transfection with the recombinant plasmids (P<0.05), and that were not significantly defference among Lipo group, NC group,H1group and H2 group (P>0.05). H3 plasmid(named H plasmid)was selected for the latter experiment.4. Compared with that of Lipo group and NC group ,growth and clone formation of SKOV3 cell was markedly inhibited after transfection with H plasmid and viable cells were significantly declined (P<0.05).And the apoptosis rate of SKOV3 cell was19.2%,33.09 % at 48h,72h after transfection with H plasmid respectively,which was significantly increased (p<0.05), apoptosis rate of SKOV3 cell at 72h posttransfection was significantly higher than that of SKOV3 cell at 48h posttransfection (p<0.05).5. Compared with that of Lipo group and NC group, IC50 of H plasmid group SKOV3 cells to paclitaxel,cisplatin and doxorubicin is significantly lower (P<0.05),and there were not significantly difference among the IC50 of Lipo group , NC group and the corresponding paclitaxel,cisplatin and doxorubicin group (P>0.05).6. In vivo tests,the tumor volume growth rates of H plasmid +DDP group ,DDP group and H plasmid group were significantly lower than that of NS group,Lipo group and NC group within 3 weeks after treatment(P<0.05) ;and compared with that of DDP group and H plasmid group,the tumor growth rate in H plasmid +DDP group was significantly lower (P<0.05). The expression of HIF-1αand VEGF protein of H plasmid +DDP group ,DDP group and H plasmid group xenograft tumor tissue detected by immunohistochemistry were all significantly reduced (P<0.05) when compared with those of NS group,Lipo group and NC group.Conclusions:1. These data indicate that the HIF-1αprotein expression of SKOV3 cells is significantly up-regulated by the hypoxic environment.2. RNAi expressing plasmid directed against HIF-1αcan effectively inhibit the growth and enhance the apoptosis of SKOV3 cells through down-regulating the expression of HIF-1αmRNA and protein. After selectively silencing HIF-α, hypoxia-induced expression of its target genes such as P-glycoprotein were markedly attenuated. Moreover, HIF-1αknockdown was found to increase drug sensitivity of SKOV3 cells to paclitaxel,cisplatin and doxorubicin.3. Intratumoral injection of HIF-1αRNAi expression plasmid can effectively activate RNAi-mediated down-regulation of HIF-1αand VEGF in vivo.HIF-1a knockdown can effectively curb the growth of SKOV3 cells xenograft and increase the sensitivity of SKOV3 cells xenograft tumor to DDP, and which suggest a promising combination of both downregulating HIF-1αexpression by RNA interference and traditional chemotherapy to improve cancer treatment.