miR-20a-5p调控结核分枝杆菌诱导巨噬细胞凋亡相关基因表达的研究

Effect of miR-20a-5p on the expression of apoptosis-related genes in Mycobacterium tuberculosis-infected human macrophages

目的明确小非编码RNA(microRNA,miR-20a-5p)对结核分枝杆菌(MTB)诱导人巨噬细胞凋亡相关基因表达的调控作用。方法构建可表达或抑制miR-20a-5p、转染miR-20a-5p抑制剂(miR-20a-5p-inhibitor,简称“miR-20a-5p-inh”)、作为慢病毒载体的阴性对照(LV1-NC)的慢病毒载体,由oligo-dT引物通过固相亚磷酰胺法合成茎环寡核苷酸,并克隆到含绿色荧光蛋白(GFP)的慢病毒载体pGLV3-GFP内,将构建的miR-20a-5p、miR-20a-5p-inh、LV1-NC转染THP-1人巨噬细胞3h,培养72h后用流式细胞仪分选出GFP+THP-1细胞,并分别采用减毒MTB菌株(H37Ra)感染8h和过氧化氢(H2O2)处理30min 两种方法诱导。通过实时定量PCR技术测定细胞中线粒体相关抗凋亡基因Bcl-2,以及促凋亡基因Bax、Bim和Bad的转录水平。同时,用蛋白免疫印迹法检测细胞裂解物中相关蛋白的表达。结果慢病毒转染细胞后,在无刺激的THP-1细胞中miR-20a-5p的相对荧光强度值为12.21±1.29、miR-20a-5p-inh为9.68±1.38、LV1-NC为10.64±0.96,三者的表达差异均无统计学意义(q=1.815,P=0.385;q=2.072,P=0.602);但在H37Ra和H2O2的刺激后,miR-20a-5p过表达时其相对荧光强度值分别为7.20±0.53、8.55±0.82,明显高于LV1-NC (4.46±0.07、5.49±0.44)(q=50.250,P=0.007;q=1.041,P<0.01),miR-20a-5p受抑制时(1.88±0.08、1.44±0.21)明显低于LV1-NC(q=3.457,P=0.031;q=4.384,P=0.001)。在感染miR-20a-5p慢病毒的THP-1细胞中,H37Ra诱导Bcl-2的表达增加(相对荧光强度值由10.67±0.89增至14.98±0.88)(q=1.064,P=0.008),而感染miR-20a-5p-inh慢病毒时Bcl-2表达减少(由10.67±0.89降至6.49±0.47)(q=3.518,P=0.003);在miR-20a-5p过表达的THP-1细胞中Bim的转录水平减少(由1.22±0.05降至0.98±0.04)(q=1.240,P=0.011),当miR-20a-5p受抑制时Bim的转录水平增加(由1.22±0.05增至1.51±0.08)(q=2.460,P=0.021)。蛋白印迹法检测凋亡相关基因Bcl-2和Bim的蛋白表达水平,结果与基因转录水平相符。结论 miR-20a-5p的表达水平影响MTB诱导的巨噬细胞凋亡相关基因Bcl-2和Bim的表达,并且与促凋亡基因Bim的水平呈负相关,提示miR-20a-5p可能通过调控Bim的表达而影响细胞凋亡。

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.