MiRNA-20a Targeted Regulation Of ATG16l1 And ATG7 Mediated BCG Survivals By Inhibiting Autophagy

Objective Aims to study the effect of miR-20 a on expression regulation of ATG16L1 and ATG7,and on BCG survivals mediated by cell autophagy.Method 1 Real-time PCR to detect the expression levels of miR-20 a and other 5 mi RNAs in mi R-17-92 cluster which treated with Rapamycin, 3-MA and BCG, and then use bioinformatics analysis software to predict the possibility of ATG16L1 and ATG7 function as its targeted genes.2 To verify the relationship between miR-20 a,ATG16L1 3’UTR and ATG7 3’UTR, we cloned the 3’UTR of ATG16L1 and ATG7 using PCR and inserted them into pMIR-report luciferase vector. Using Dual-luciferase Report system and Western blot to detect luciferase activity and protein expression level of ATG16L1 and ATG7.3 MiR-20 a mimic and inhibitor were transfected into RAW264.7 macrophages respectively, which treated with rapamycin. Transmission electron microscopy, laser confocal microscope and Western blot method were used to detect the autophagosome formation, the expression of ATG16L1 and ATG7, and the expression of LC3II/I, to study the mechanism of miR-20 a on cell autophagy pathway.4 MiR-20 a mimic and inhibitor were transfected into RAW264.7 macrophages respectively, which treated with rapamycin, after 24 h BCG infection, Real-time PCR were used to detect the expression of BCG in each group, to reveal the effects of miR-20 a on the survival of BCG.Results 1 After 2hours treated by rapamycin,the expression of miR-17,miR-18 a, miR-20 a in RAW264. 7 cells increased(P<0.05); After treated with 3-MA for 12 hs miR-20 a was down regulated(P<0.05). After BCG infected 6h,12 h and 24 h; the expression of miR-20 a levels in RAW264.7 cells significantly increased(P< 0.01), after infected 48 hours, miR-20 a was lower than infected 6 hours,12 hours and 24 hours, but still higher than uninfected group(P < 0.05).2 The bioinformatics analysis software result showed that ATG16L1 and ATG7 are the targeted genes of mir-20 a. Compared with normal control group, relative luciferase activity of ATG16L1 in miR-20 a mimic group decreased 1.8 times(P < 0.05), and that of ATG 7 decreased 1.6 times(P < 0.05). Dual-luciferase Report system and Western Blot results showed that miR-20 a can combine the 3 ’UTR of autophagy related gene ATG16L1 and ATG7 to inhibit its expression.3 After infected with BCG for 24 hours, RAW264.7 cells treated with miR-20 a mimic can inhibit the formation of autophagosome detected by laser confocal microscope; on the contrary, after treated with miR-20 a inhibitor, the autophagosome significantly increased; meanwhile,miR-20 a can decrease the protein level of ATG16L1, ATG7 and LC3II/I ratio significantly; inhibiting the miR-20 a expression, the expression of ATG16L1 and ATG7 and LC3II/I ratio was increased(P<0.05).4 The specific gene expression of BCG in RAW264.7 cells was detected by real-time PCR, results show that the BCG gene expression in mi R-20 a mimic group was higher than that in normal group(P<0.01); on the contrary, when miR-20 a was inhibited, BCG gene expression were significantly lower than that in normal group(P<0.01).Conclusion Our results prove that expression of miR-20 a can inhibit the expression of autophagy related gene ATG16L1 and ATG7, formation of autophagosome, which benefit to BCG survival in macrophages.