The Effects Of MiR-145 Transfected With PMSC-PPDL-co-PDMAEMA On H1299 Cells

Background:Nanotechnology gene delivery is a branch of nanomedicine that involves the synthesis,characterization,and functionalization of nanomaterials for target gene delivery.Compared with viral vectors,nanocarriers play an increasingly important role due to their low immunogenicity,low toxicity,and high efficiency in terms of gene therapy.In recent years,cancer has become one of the biggest problems that endanger social health.In tumor gene therapy,microRNAs have been studied as the targets for the reason that they plays an important role in regulating the pathogenesis of cancer.The expression of microRNA-145(miR-145)is reduced in various tumor cells and regulated the biological function of tumor cells,including regulating cell proliferation,differentiation,apoptosis,invasion,and metastasis.Lung cancer is the most common cancer and the main cause of deaths with cancer of men and women in worldwide.Studies have found that miR-145 expression is reduced in lung cancer cells and miR-145 is associated with the development of lung cancer.Therefore,miR-145 may become a potential target to suppress the tumor characteristics of lung cancer by increasing the intracellular expression content.Objective:In this study,a novel gene delivery vector,PMSC-PPDL-co-PDMAEMA,was synthesized by the enzymatic aggregating reaction.This vector has higher transfection efficiency less toxicity.Using this vector,we transferred miR-145 into H1299 to explore the toxicity and the transfection efficiency of PMSC-PPDL-co-PDMAEMA compared to PEI25K;we also aim to investigate the effects and mechanisms of regulation miR-145 expression on growth,proliferation,migration,invasion,and apoptosis of non-small cell lung cancer cell H1299.Methods:1.Synthesis of PMSC-PPDL-co-PDMAEMAFirst,Novozym 435,a lipase,was used to catalyze ring-opening and polymerization reactions of ethyl sebacate,N-methyldiethanolamine and?-pentadecanolide to form the copolyeser,and then bromoisobutyric acid was added to catalyze end capping to obtain copolymer PMSC-PPDL-Br.This copolymer material can stimulate glycidyl methacrylate to undergo atom transfer radical polymerizationto generate PMSC-PPDL-co-PDMAEMA under the action of ethanolamine.2.The effects of miR-145 transfected by PMSC-PPDL-co-PDMAEMA on H1299 cellsThe PEI25K vector was used as a positive control,and the toxicity of PMSC-PPDL-co-PDMAEMA to H1299 cells was detected using the CCK8 kit.Gene vectors of different concentrations were used to form a complex with miR-145 mimics,and the complex was transfected into cells for 48 hours.Then,real-time quantitative PCR was used to test the transfection efficiency;CCK8 was added to the wells to detect the cell proliferation after gene transfection;The effects of miR-145 on migration and invasion ability of H1299 cells was analyzed by scratch test and cell migration invasion test technology(Transwell).Flow cytometry was used to detect the effect of miR-145 gene on apoptosis of H1299 cells;Western blot analysis was used to explore the mechanism of miR-145 gene in combating the characteristics of tumor cells.ResultsThe copolyester PMSC-PPDL-co-PDMAEMA successfully synthesized by enzymatic method.The toxicity of the copolyesters with different concentrations were lower than that of the PEI25K group.When the ratio of copolyester and miR-145mimics gene mass was 20:1,the transfection efficiency of copolyesters groups to H1299 cells was higher than that of the PEI25K group.After transfection with complex of miR-145 and Nanocarrier,H1299 cells showed lower capabilitie of proliferation,invasion,and migration than those of the control group(P<0.05);After cultured for 72 hours,the expression levels of miR-145 was negatively correlated with the protein content of PDK1 in H1299 cells transfected with miR-145.Conclusion:1.In this study,we successfully synthesized a new gene vector PMSC-PPDL-co-PDMAEMA,which has the characteristics of low toxicity and high transfection efficiency,by using an enzyme-catalyzed method;2.Co-polyester deliver miR-145 into H1299 cells induced apoptosis of tumor cells and inhibit the proliferation,migration,and invasion of H1299 cells by down-regulating PDK1 expression.