Abstract:

Objective To investigate the effect of miR-20a-5p on the expression of apoptosis-related genes in Mycobacterium tuberculosis (MTB)-infected human macrophages. Methods Construction of expression or inhibition of miR-20a-5p, transfection of miR-20a-5p inhibitor (miR-20a-5p-inhibitor, referred to as “miR-20a-5p-inh”), 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-20a-5p, miR-20a-5p-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 (H37Ra) for 8 h and hydrogen peroxide (H₂O₂) 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-20a-5p expression in THP-1 cells without stimulation, the relative fluorescence intensity were 12.21±1.29 of the miR-20a-5p group and 9.68±1.38 of the miR-20a-5p-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-20a-5p could be up- or down-regulated as expected after H37Ra or H₂O₂ stimulation. The relative fluorescence intensity was 7.20±0.53 and 8.55±0.82 in the miR-20a-5p group, and they were 1.88±0.08 and 1.44±0.21 in the miR-20a-5p-inh group. The differences were all significant when compared with LV1-NC group (4.46±0.07 and 5.49±0.44)(q=50.250, P=0.007; q=1.041, P<0.01; q=3.457, P=0.031;q=4.384,P=0.001). Meanwhile, we found H37Ra-induced Bcl-2 expression was increased from 10.67±0.89 to 14.98±0.88 in THP-1 cells transfected with miR-20a-5p lentivirus(q=1.064,P=0.008), and decreased to 6.49±0.47 when the miR-20a-5p 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-20a-5p overexpressed THP-1 cells(q=1.240,P=0.011), and increased to 1.51±0.08 when miR-20a-5p 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-20a-5p influences apoptosis related genes Bcl-2 and Bim expression in MTB-infected macrophages, and the expression of miR-20a-5p is negatively correlated with the level of pro-apoptotic gene Bim, suggesting that miR-20a-5p regulates MTB-induced apoptosis through inhibiting Bim gene.

Key words: Mycobacterium tuberculosis, RNA,small untranslated, Apoptosis, Genes,bcl-2, Gene expression regulation,bacterial, Macrophages