lncRNA260 siRNA Accelerates M2 Macrophage Polarization and Alleviates Oxidative Stress via Inhibiting IL28RA Gene Alternative Splicing

Abstract

The macrophage transformation of inflammatory M1 to anti-inflammatory M2 could be promoted by activating PI3K/AKT signaling pathway. In our previous study, it was found that downregulation of lncRNA260 could ameliorate hypoxic cardiomyocyte injury by regulating IL28RA through the activation of PI3K/AKT signaling pathways. It was suggested that lncRNA260 siRNA could promote the macrophages toward M2 polarization by regulating IL28RA. In this study, lncRNA260 siRNA was used to observe its effect on the polarization of murine bone marrow-derived macrophages (BMDM) and investigate its related mechanisms. lncRNA 260 specific siRNA were designed and synthesized which were transfected into murine BMDM with liposomes. The experiment was divided into three groups: Hypoxia group, Hypoxia+lncRNA 260-specific siRNA transfection group, and Normoxia group. The CD206-APC/CD11b-FITC or CD206-FITC/CD107b (Mac-3) double positive proportions were used to compare the M2 polarization proportions in the hypoxia process by using the immunofluorescence staining method. The p-AKT, Arg 1, PI3KCG, IL28RAV1, and IL28RAV2 protein expression changes were observed by using the western blot method. Compared with the Normoxia group, the M2 proportions were significantly decreased in the Hypoxia group ( $P<0.05$ ). Compared with the hypoxia group, the M2 proportions were significantly increased in the Hypoxia+lncRNA260 siRNA transfection group ( $P<0.05$ ). In the Hypoxia group, the ratios of Arg 1/β-Actin, p-AKT/β-Actin, PI3KCG/β-Actin, and IL28RAV1/β-Actin were significantly lower than those in the Normoxia group ( $P<0.05$ ). After transfection with lncRNA260 siRNA, the ratios of Arg1/β-Actin, p-AKT/β-Actin, PI3KCG/β-Actin, and IL28RAV1/β-Actin were significantly higher than those in the Hypoxia group ( $P<0.05$ ). Compared with the Normoxia group, the IL28RAV2/β-Actin in the Hypoxia group was significantly increased ( $P<0.05$ ). After transfection with lncRNA260 siRNA, the ratio of IL28RAV2/β-Actin was significantly decreased than that in the Hypoxia group ( $P<0.05$ ). lncRNA260 siRNA could promote the M2 polarization of the hypoxia macrophages by reducing the IL28RAV2 alternative splicing variant, which might be related to the activation of the JAK-STAT and PI3K/AKT signaling pathways. It will provide a new strategy for the anti-inflammation, antioxidative stress therapy, and cardiac remodeling after AMI.