Function Study On The Molecular Mechanism Of MiRNA-6857-3p Inhibiting Autophagy Through AKT/mTOR In Mycobacterium Tuberculosis Infected Macrophages

Purpose 1.Observe the expression of miRNA-6857-3p in BCG-infected macrophages.2.Verify the targeting relationship between miRNA-6857-3p and Sp110 gene m RNA in BCG-infected macrophages.3.Verify the relationship between siRNA and Sp110 gene in BCG-infected macrophages.4.Explore the relationship between miRNA-6857-3p and the occurrence of autophagy in BCG-infected macrophages.5.Observe the regulation of miRNA-6857-3p by Akt/mTOR pathway in rapamycin-activated BCG-infected macrophages.6.Observe the effect of overexpression of miRNA-6857-3p on Akt/mTOR pathway in macrophages.7.Detect the effects of miRNA-6857-3p mimics and inhibitors on the proliferation of BCG-infected macrophages.Methods(1)Using BCG to infect macrophages according to different infection time and different multiplicity of infection,real-time fluorescent quantitative PCR was used to detect the expression of miRNA-6857-3p under different conditions.(2)Use Lipofectamin TM3000 Reagent to wrap miRNA-6857-3p mimic and miRNA-6857-3p inhibitor to transfect BCG-infected cells RAW264.7,and use western blot to detect the expression of Sp110 protein.(3)Use Lipofectamin TM3000 Reagent to wrap siRNA to transfect BCG-infected RAW264.7 cells,and use western blot to detect the relative expression of Sp110 protein.(4)Wrap miRNA-6857-3p mimic and miRNA-6857-3p inhibitor with Lipofectamin TM3000 Reagent,and transfect BCG-infected RAW264.7 cells,and use western blot to detect the expression of LC3 protein.(5)Wrap miRNA-6857-3p mimic and miRNA-6857-3p inhibitor with Lipofectamin TM3000 Reagent,and transfect BCG-infected RAW264.7 cells,and use western blot to detect Akt/mTOR pathway related proteins.(6)Rapamycin and 3-MA were used to induce BCG-infected RAW264.7 cells,and real-time fluorescent quantitative PCR was used to detect the relative expression of miRNA-6857-3p.(7)Use CCK-8 to carry out cell proliferation experiments,and measure the OD values??of different test substances in the 450 nm region with a microplate reader.Understand the proliferation of miRNA-6857-3p inhibitor group and mimic group at different time points of BCG-infected macrophages,and explore the relationship between miRNA-6857-3p and its inhibitor on the proliferation of BCG-infected macrophages.Results(1)BCG-infected macrophages can cause an increase in the expression of miRNA-6857-3p.(2)There is a targeting relationship between miRNA-6857-3p and Sp110 gene in BCG-infected macrophages.miRNA-6857-3p mimics transfected BCG-infected RAW264.7 cell line can inhibit the protein expression of Sp110,and in the miRNA inhibitor+BCG group,we found that miRNA-6857-3p inhibitor can increase the expression of SP110 protein and make The above results have been reversed.(3)In RAW264.7 cells 48 hours after siRNA transfection and BCG infection,the Sp110 protein of the BCG+siRNA group was significantly lower than that of the BCG group(P<0.01).(4)In BCG-infected macrophages,overexpression of miRNA-6857-3p with miRNA-6857-3p mimic can regulate the expression of autophagy-related proteins and reduce the ratio of LC3 Ⅱ /I,which is performed in miRNA-6857-3p inhibitor.After blocking,the LC3II/I ratio increased.(5)Activation of the Akt/mTOR pathway in BCG-infected macrophages can inhibit the expression level of miRNA-6857-3p.(6)miRNA-6857-3p is involved in the signal regulation of Akt/mTOR pathway.(7)The results of CCK-8 showed that the proliferation of macrophages in the BCG+inhibitor group was weaker than that in the BCG+mimic group.Conclusion1.There is a targeting relationship between miRNA-6857-3P and Sp110 gene.2.miRNA-6857-3p can regulate macrophage autophagy by regulating Sp110 gene.3.miRNA-6857-3p modulates the Akt/mTOR signaling pathway through the regulation of Sp110 gene to achieve the regulation of autophagy,thereby affecting the growth and proliferation of BCG.