lincRNA Cox2 Regulates BCG-infected Macrophages Glycolysis by miR-129-5p/AMPK
[Objective]The aim of this study was to investigate the regulatory role of lincRNA Cox2 in the glycolysis of RAW264.7 macrophages infected by Bacillus Calmette-Guerin(BCG),and to elucidate the interaction between Mtb and macrophages,so as to provide a new target for the diagnosis and treatment of tuberculosis.[Method]RNA interference technique was used to knock down the expression of lincRNA Cox2,and miR-129-5p mimics were used to overexpress miR-129-5p.QPCR was performed to measure the lincRNA Cox2,miR-129-5p and proinflammatory cytokine(IL-1β,TNF-α,and IL-6)expression after BCG infection.The expression of Lactic Acid was detected by Lactic Acid assay kit.The bacterial load was measured bacterial load in BCG-infected macrophages.Dual luciferase reporter gene system validation experiments were carried out on lincRNA Cox2 and miR-129-5p,or miR-129-5p and AMPK relationships.The expression of AMPK(AMP activated protein kinase),HK1(Hexokinase 1),PKM2(pyruvate kinase M2),and LDHA(Lactate dehydrogenase A)were detected by Western blotting.[Result]The expression of lincRNA Cox2 was significantly upregulated(P=0.000013)after BCG infection in RAW264.7 macrophages for 12 h.Compared with the BCG group,the siRNA+BCG group had significantly upregulated the expression of AMPK(P=0.000771),HK1(P=0.00323),PKM2(P=0.000135),LDHA(P=0.002532),and the secretion of LD(P=0.020802),but the expression of IL-1β(P=0.000451),TNF-α(P=0.000147),IL-6(P=0.0001)was significantly reduced.The lincRNA Cox2 knockdown caused a significant reduce of bacterial load in BCG-infected macrophages(P=0.000127).Dual luciferase reporter gene system were performed to the co-localized of lincRNA Cox2 and miR-129-5p,and targeting AMPK.The expression of miR-129-5p was significantly reduced(P=0.000156)after BCG infection in RAW264.7 macrophages for 12 h.Compared with the BCG group,the miR-129-5p mimics+BCG group had significantly reduced the expression of AMPK(P=0.000262),HK1(P=0.019524),PKM2(P=0.001658),LDHA(P=0.000887),and the secretion of LD(P=0.044952).[Conclusion]lincRNA Cox2 promoted BCG-infected RAW264.7 macrophages glycolysis process by sponging miR-129-5p and targeting AMPK.
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