The Effect Of Pll-DIONP-mediated PHIF-1α-shRNA Transfection On Cisplatin-resistant Lung Adenocarcinoma Cell Line In Vitro And In Vivo

Objective 1.To evaluate the feasibility of using dextran coated iron oxide modified nanoparticles with Poly-L-Lysine (pll-DCIONP) as gene carrier and transfecting with it in cisplatin-resistant lung adenocarcinoma cells A549/DDP.And to study effect of pll-DCIONP-mediated and Lipofectamine 2000 Reagent (lipo2000)-mediated pHIF-1α-shRNA delivery on sustained antiproliferative activity and its efficacy of reversing resistance in cisplatin-resistant human lung adenocarcinoma.2.To explore the feasibility of pll-DCIONP-mediated pHIF-1α-shRNA delivery results in targeting gene treatment with cisplatin-resistant human lung adeno-carcino maxeno -graft in nude mice.Methods 1.The pll-DCIONP was evaluated as a kind of pHIF-1α-shRNA carrier by transfecting human lung adenocarcinoma cell line A549/DDP in vitro while compareing to lipo2000.The intracellular mRNA expression of HIF-1α,MRP and LRP was analyzed by RT-PCR. The expression of protein was analyzed by immunocytochemistry.The mortality rate of cells treated by cisplatin was analyzed by MTT.2. A549/DDP were cultured routinely and implanted to subcutaneous in nude mice to establish cisplatin-resistant xenograft animal model. Magnetic field was used to direct movement of pll-DCIONP-mediated delivery,pHIF-1α-shRNA-loaded pll-DCIONP and lipo2000 was injected intravenously and the tumor growth was then observed.Volumes of tumor mass were detected respectively Then tumor growth index was thereby calculated.Then specimens were fixed by paraformaldehyde and then paraffin section,Hematoxylin and eosin(HE) staining and histological examination performed.The expression of HIF-1α,MRP and LRP protein was analyzed by immunohistochemistry.Results 1.Magnetic field was used to direct movement of pll-DCIONP capable of transferring plasmid into cells even more efficiently than the lipids examined in vitro(P＜0.01).2.The expressions of mRNA and protein of HIF-1α,MRP and LRP of cells had descended after being transfected with pHIF-1α-shRNA.The group of pll-DCIONP-mediated was lower than the lipo2000 group(P＜0.01).The changes of HIF-1αmRNA and protein were distinctly relate with MRP and LRP(r=0.900,0.915 P＜0.01),(r=0.901,0.896 P＜0.01).3.The cisplatin-resistan of A549/DDP had descended after being transfected with pHIF-1α-shRNA(P＜0.05).4.Volumes of tumor mass and the growth index of tumor were descended after being transfected with pHIF-1α-shRNA(P＜0.05).5 The expressions of mRNA and protein of HIF-1α,MRP and LRP of tumor tissues were descended after being transfected with pHIF-1α-shRNA.The group of pll-DCIONP-mediated was lower than the lipo2000 group(P＜0.01).The changes of HIF-1αprotein were distinctly relate with MRP and LRP (r=0.918,0.906 P＜0.01).6.There was no obvious side-effect for treatment following the pHIF-1α-shRNA -loaded pll-DCIONP and lipo2000 to be injected intravenously and liver and kidney tissues was proved no distinct injured by HE staining assay.Conclusion 1.pll-DCIONP could transfer reporter plasmid into cells even more efficiently than the lipids examined in vitro.2. pll-DCIONP and lipo2000 mediated pHIF-1α-shRNA delivery resulted in a reversal effect of cisplatin resistance in cisplatin-resistant human lung adenocarcinoma cell lines A549/DDP.pll-DCIONP and lipo2000 mediated pHIF-1α-shRNA delivery resulted in sustained antiproliferative activity effects on cisplatin-resistant human lung adenocarcinoma cell lines A549/DDP.3.pll-DCIONP-mediated pHIF-1α-shRNA gene delivery resulted in targeting anti-tumor with cisplatin resistant human lung adenocarcinoma xenograft in vivo and reversed the resistance in cisplatin resistant human lung adenocarcinoma xenograft in vivo.The effect grew stronger than xenograft treated with the pHIF-1α-shRNA -lipofectamine complex.The down-regulating level of HIF-1αmay cause the descending the cellular expression of MRP and LRP,and this may be one of mechanism of resistance reversal.4.There was no significant in vivo toxicity of delivery systems of pll-DCIONP.