Silencing PI3K? Gene Via Magnetic Nanoparticles Regulates The Effect Of Tumor Associated Macrophages On Mouse Lung Cancer Cells

Objective:The purpose of this research was to explore the effect of silencing phosphatidylinositol 3 kinase ?(PI3K?)gene via superparamagnetic iron oxide nanoparticles(SPIONs)to regulate tumor-associated macrophages(TAMs)on the proliferation and apoptosis of mouse lewis lung carcinoma(LLC)cells.Methods:1.3 pSilencer-EGFP-SP-p110 plasmids which can specifically promote macrophages(M?)to express siRNA of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma(PIK3CG)were constructed and transfected into RAW264.7 cells.Fluorescence microscopy was used to detect the expression of fluorescent protein in each group of cells.The ability to specifically initiate expression of siRNA and the efficiency of PI3K? p110 gene silencing after transfection of macrophages were detected by Real-time PCR and Western blot.The recombinant plasmid with the best silencing efficiency was selected for subsequent experiments.2.The recombinant plasmid with the best silencing efficiency was loaded into magnetic nanoplasmid complexes(SPIONs-DNA)via SPIONs which were transfected into macrophages under strong magnetism.The distribution of SPIONs-DNA in the cells was detected by Prussian blue staining,and the expression of PI3K? p110 subunit of transfected cells was detected by Real-time PCR and Western blot.3.The M1 and M2 macrophages models were established,their inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1)expression levels were identified by Real-time PCR and Western blot.4.The M2 macrophages were transfected with SPIONs-DNA under strong magnetism.The phenotype of the transfected cells was identified by Real-time PCR and Western blot,and the intensity of M2 macrophages transformed into M1 was determined.5.The Transwell system was used to establish the co-culture models of SPIONs-DNA transfected M2 macrophages and RAW264.7 cells with mouse LLC cells.The number of live cells of LLC was detected by trypan blue staining and the cell growth curve was drawn.The proliferation of LLC cells was detected by CCK-8 assay.The NO concentrations in supernatant of co-culture medium was detected by Nitrate reductase method.The cell apoptosis level of LLC cells was detected by flow cytometry.Results:1.Strong fluorescence was observed in RAW264.7 cells after transfected with the three recombinant plasmids,and the fluorescence intensity of cells transfected with recombinant plasmid S1 was the highest.All three recombinant plasmids could significantly inhibit the expression levels of PI3K? p110 mRNA and protein(P<0.01),and the suppression efficiency of recombinant plasmid S1 was the best.2.The prepared SPIONs-DNA complex was successfully transfected into RAW264.7 cells under strong magnetism and distributed in a large amount around the cell nucleus.The expression levels of mRNA and protein of PI3K? p110 in SPIONs-DNA transfected cells were significantly lower than those in the blank control group(P<0.05).3.M1 and M2 macrophages models were successfully established.The M1 macrophages highly expressed iNOS(P<0.001)and the M2 macrophages highly expressed ARG-1(P<0.001).4.After SPIONs-DNA transfection,the expression levels of iNOS mRNA and protein in M2 macrophages increased significantly(P<0.01),and the expression of ARG-1 mRNA and protein decreased significantly(P<0.01).5.Among the co-culture groups,M2 macrophages group and RAW264.7 cell group transfected with SPONs-DNA and M1 macrophages group were able to secrete large amounts of NO(P<0.01).The growth and proliferation capacity of their co-cultured LLC cells were significantly lower than other control groups(P<0.05),the apoptosis rate of LLC cells was significantly higher than other control groups(P<0.01).Conclusion:1.The magnetic nanoparticle-loaded pSilencer-EGFP-SP-p110 plasmid could specifically inhibit the expression of PI3K? p110 in macrophages,and then induce the transformation of M2 type macrophages into M1 type.2.The SPIONs-DNA transfected M2 macrophages and RAW264.7 cells can significantly inhibit the growth and proliferation of LLC cells,and promote the apoptosis of LLC cells,which is related to its large amounts of NO secretion.The magnetic nano-plasmid complex can induce TAMs to play an anti-tumor role what lays a foundation for the research and development of effective gene therapy for lung cancer.