Silencing PI3Kγ Gene via Magnetic Nanoparticles Regulates the Effect of Tumor Associated Macrophages on Mouse Lung Cancer Cells

The purpose of this research was to explore the effect of silencing PI3Kγ (phosphatidylinositol 3 kinase γ) gene via SPIONs (superparamagnetic iron oxide nanoparticles) to regulate TAM (tumor-associated mac rophages) on the proliferation and apoptosis of mouse LLC (Lewis lung carcinoma) cells. The pSilencer-EGFP-SPp110 plasmid which can specifically promote MФ (macrophages) to express siRNA of PIK3CG (PI3Kγ catalytic subunit p100) was constructed and loaded into SPIONs-DNA (magnetic nano-plasmid complexes) via SPIONs which was transfected into macrophages under strong magnetism. The distribution of SPIONs-DNA in the cells was detected by Prussian blue staining, and the expression of PI3Kγ p110 of transfected cells was detected by Real-time PCR and Western blot. The M1 and M2 MФ models were established, and SPIONs-DNA was transfected into M2 MФ under strong magnetism. The phenotype of the transfected cells was identified by Real-time PCR and Western blot, and the intensity of M2 MФ transformed into M1 was determined. The Transwell system was used to establish the co-culture models of SPIONs-DNA transfected M2 MФ 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 apoptosis level of LLC cells was detected by flow cytometry. The results showed that the prepared SPIONs-DNA complex was successfully transfected into MФ under strong magnetism and distributed in a large amount around the cell nucleus. The mRNA and protein levels of PI3Kγ p110 in SPIONs-DNA transfected cells were significantly lower than those in the blank control group (P<0.05). The established M1 MФ highly expressed iNOS (P<0.001), and the M2 MФ highly expressed ARG-1 (P<0.001). After SPIONs-DNA transfection, the expression levels of iNOS mRNA and protein in M2 cells increased significantly (P<0.01), and the expression of ARG-1 mRNA and protein decreased significantly (P<0.01). Among the co-culture groups, SPONs-DNA transfected M2 type cell group was able to secrete a large amount of NO, the growth and proliferation capacity of LLC cells were significantly reduced (P<0.05), and the apoptosis rate of LLC cells were significantly increased (P<0.01).These results confirm that magnetic nanoparticles loaded with pSilencer-EGFP-SP-p110 plasmids could specifically inhibit the expression of PI3Kγ p110 in MФ, and then induce the transformation of M2 type MФ into M1 type. The SPIONs-DNA transfected M2 type MФ can significantly inhibit the growth and proliferation of LLC cells, and promote the apoptosis of LLC cells, which is related to its large amount of NO secretion. The magnetic nano-plasmid complex can induce TAM to play an anti-tumor role what laying a foundation for the research and development of effective gene therapy for lung cancer.

CSTR: 32200.14.cjcb.2020.02.0015