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      《Chinese Journal of Antituberculosis》 2019-07
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      Effect of miR-20a-5p on the expression of apoptosis-related genes in Mycobacterium tuberculosis-infected human macrophages

      DING Guang-gui;HE Xing;LIANG Juan;LIU Ya-ya;OU Min;LU Jian;ZHANG Guo-liang;Department of Thoracic Surgery,Shenzhen People's Hospital;  
      Objective To investigate the effect of miR-20 a-5 p on the expression of apoptosis-related genes in Mycobacterium tuberculosis(MTB)-infected human macrophages.Methods Construction of expression or inhibition of miR-20 a-5 p,transfection of miR-20 a-5 p inhibitor(miR-20 a-5 p-inhibitor,referred to as"miR-20 a-5 pinh"),as a negative control lentiviral vector(LV1-NC).The stem-loop oligonucleotide was synthesized by oligo-dT primer by solid phase phosphoramidite method and cloned into the lentiviral vector pGLV3-GFP containing green fluorescent protein(GFP).The constructed miR-20 a-5 p,miR-20 a-5 p-inh and LV1-NC were transfected into THP-1 human macrophages for 3 h.After 72 h of culture,GFP+THP-1 cells were sorted by flow cytometry.GFP+THP-1 cells were induced by attenuating MTB strain(H37 Ra)for 8 h and hydrogen peroxide(H_2 O_2)for 30 min.The transcription levels of the mitochondria-associated anti-apoptotic gene Bcl-2 and the pro-apoptotic genes Bax,Bim and Bad were determined by real-time quantitative PCR.At the same time,the expression of related proteins in cell lysates was detected by Western blotting.Results After lentivirus transfection,no differences were observed in miR-20 a-5 p expression in THP-1 cells without stimulation,the relative fluorescence intensity were 12.21±1.29 of the miR-20 a-5 p group and 9.68±1.38 of the miR-20 a-5 p-inh group.However there was no significant difference when compared with LV1-NC group(q=1.815,P=0.385;q=2.072,P=0.602),whose relative fluorescence intensity was 10.64±0.96.But miR-20 a-5 p could be up-or down-regulated as expected after H37 Ra or H_2 O_2 stimulation.The relative fluorescence intensity was 7.20±0.53 and 8.55±0.82 in the miR-20 a-5 p group,and they were 1.88±0.08 and 1.44±0.21 in the miR-20 a-5 p-inh group.The differences were all significant when compared with LVl-NC group(4.46±0.07 and 5.49±0.44)(q=50.250,P=0.007;q=1.041,P0.01;q=3.457,P=0.031;q=4.384,P=0.001).Meanwhile,we found H37 Ra-induced Bcl-2 expression was increased from 10.67±0.89 to 14.98±0.88 in THP-1 cells transfected with miR-20 a-5 p lentivirus(q=1.064,P=0 008),and decreased to6.49±0.47 when the miR-20 a-5 p was blocked(q=3.518,P=0.003).However,the expression levels of the pro-apoptotic Bim gene was decreased from 1.22±0.05 to 0.98±0.04 in miR-20 a-5 p overexpressed THP-1 cells(q=1.240,P=0.011),and increased to 1.51±0.08 when miR-20 a-5 p was inhibited(q=2.460,P=0.021).However,there were no significant differences between Bax and Bad gene expression. The relevant protein expression was detected by using Western blotting assay,and the results were consistent with the gene expression level.Conclusion These results clearly demonstrate that miR-20 a-5 p influences apoptosis related genes Bcl-2 and Bim expression in MTB-infected macrophages,and the expression of miR-20 a-5 p is negatively correlated with the level of pro-apoptotic gene Bim,suggesting that miR-20 a-5 p regulates MTB-induced apoptosis through inhibiting Bim gene.
      【Fund】: 国家自然科学基金(81873958);; “十三五”国家科技重大专项(2017ZX10103004);; 深圳市科技计划项目(JCYJ20170412151620658 JCYJ20170307095003051)
      【CateGory Index】: R392
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      【Citations】
      Chinese Journal Full-text Database 2 Hits
      1 WANG Fei-yu;WANG Xin-min;WANG Chan;WANG Xiao-fang;ZHANG Yu-qing;WU Jiang-dong;WU Fang;ZHANG Wan-jiang;ZHANG Le;Department of Pathophysiology,Medical College of Shihezi University;Key Laboratory of Xinjiang Endemic and Ethnic Diseases Cooperated by Education Ministry with Xinjiang Province,Medical College of Shihezi University;Department of Urinary Surgery, First Affiliated Hospital,Medical College of Shihezi University;Department of Pathogen Biology and Immunology,Medical College of Shihezi University;;Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis[J];中国病理生理杂志;2015-12
      2 ZHANG Yu-qing;WANG Xin-min;WANG Chan;WANG Fei-yu;WANG Xiao-fang;ZHAO Jin;WU Fang;WU Jiang-dong;JI Rong;ZHANG Wan-jiang;ZHANG Le;Department of Pathophysiology,Medical college of Shihezi University;Laboratory of Xinjiang Endemic and Ethnic Diseases, Medical college of Shihezi University;The First Affiliated Hospital, Medical college of Shihezi University;Department of Pathogen Biology & Immunology,Medical college of Shihezi University;Department of Pathology,Medical college of Shihezi University;;Effect of Mcl-1 signaling pathway blockers on apoptosis of mouse macrophages infected with Mycobacterium tuberculosis H37Rv[J];中国病理生理杂志;2015-11
      【Co-citations】
      Chinese Journal Full-text Database 1 Hits
      1 DING Guang-gui;HE Xing;LIANG Juan;LIU Ya-ya;OU Min;LU Jian;ZHANG Guo-liang;Department of Thoracic Surgery,Shenzhen People's Hospital;;Effect of miR-20a-5p on the expression of apoptosis-related genes in Mycobacterium tuberculosis-infected human macrophages[J];中国防痨杂志;2019-07
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