miR-146a通过调节IPr1基因对结核分枝杆菌感染的AEC Ⅱ型细胞活性作用机制  

The mechanism of miR-146a regulating the activity of IPr1 gene on type II cells infected by Mycobacterium tuberculosis

作　　者：许明昭 林清香 寇昌伟 汪求真 XU Mingzhao;LIN Qingxiang;KOU Changwei;WANG Qiuzhen(Department of Tuberculosis,Shandong Clinical Center of Public Health,Jinan 250100,China)

机构地区：[1]山东省公共卫生临床中心结核科,山东济南250100

出　　处：《西部医学》2024年第3期333-337,共5页Medical Journal of West China

基　　金：国家自然科学基金委员会资助项目(81473268)。

摘　　要：目的 探讨miR-146a通过调节IPr1基因观察对结核分枝杆菌(Mtb)感染的肺泡II型上皮细胞(AEC II)活性的作用机制。方法 采用Mtb感染AECⅡ细胞。将AECⅡ细胞分为结核组(感染Mtb)、 NC组(模型+转染miR-146a NC)、转染组(模型+转染miR-146a mimics)。RT-PCR检测AECⅡ细胞miR-146a、Ipr1基因表达;CCK8检测AECⅡ细胞增殖;流式细胞仪检测AECⅡ细胞凋亡;双荧光素酶报告测定Ipr1与miR-146a靶向关系。结果 DIO荧光染色的Mtb在感染人AECⅡ细胞12 h内会进入细胞质基质中,并围绕细胞核分布,表明建立的结核分枝杆菌感染模型成功;结核组与NC组AECⅡ细胞中miR-146a、Ipr1基因表达比较无差异(P>0.05),与NC组相比,转染组AECⅡ细胞中miR-146a、Ipr1基因表达水平升高(P<0.05);结核组与NC组AECⅡ细胞在不同时间点的OD值比较均无差异(P>0.05),与NC组相比,转染组AECⅡ细胞在不同时间点的OD值均升高(P<0.05);结核组AECⅡ细胞凋亡率与NC组相比无差异(P>0.05),与NC组相比,转染组AECⅡ细胞凋亡率显著降低(P<0.001)。双荧光素酶报告结果显示,转染miR-146a后野生型Mtb感染的AECⅡ细胞中Ipr1活性升高(P<0.05),突变型Mtb感染的AECⅡ细胞中Ipr1活性无明显变化(P>0.05),表明Ipr1是miR-146a的靶基因。结论 过表达的miR-146a可增加Mtb感染的AECⅡ细胞活性,降低凋亡,研究机制认为可能与通过激活Ipr1水平相关。Ipr1是miR-146a靶基因,可通过激活表达而发挥减少Mtb对AECⅡ细胞活性的损伤。Objective To study the activity of miR-146a on type II cells infected with Mycobacterium tuberculosis(Mtb) by regulating IPr1 gene.Methods Alveolar type Ⅱ epithelial cells(AECⅡ) were infected with Mtb.AECⅡ cells were divided into tuberculosis group(infected nodule Mtb),NC group(model + transfection with miR-146a NC),and transfection group(model + transfection with miR-146a mimics).The expression of miR-146a and IPr1 genes in AECⅡ cells was detected by RT-PCR.AECⅡ cell proliferation was detected by CCK8.AECⅡ cell apoptosis was detected by flow cytometry.Dual luciferase assay determined the targeting relationship between IPr1 and miR-146a.Results DIO fluorescently stained mycobacterium tuberculosis entered the cytoplasmic matrix and distributed around the nucleus within 12 h after infection with human alveolar type II epithelial cells,indicating that the established mycobacterium tuberculosis infection model was successful.There was no difference in the expression of miR-146a and IPr1 genes in AECⅡ cells between tuberculosis group and NC group(P>0.05),but the expression levels of miR-146a and IPr1 genes in AECⅡ cells in transfection group were increased compared with NC group(P<0.05).OD value of AECⅡ cells in tuberculosis group and NC group was not different at different time points(P>0.05),OD value of AECⅡ cells in transfection group was increased at different time points compared with NC group(P<0.05).There was no difference in the apoptosis rate of AECⅡ cells in tuberculosis group compared with NC group(P>0.05),but the apoptosis rate of AECⅡ cells in transfection group was significantly decreased compared with NC group(P<0.001).The results of dual luciferase report showed that IPr1 activity was increased in wild-type Mtb infected alveolar type II epithelial cells after transfection of miR-146a(P<0.05),but there was no significant change in IPr1 activity in mutant Mtb infected alveolar type II epithelial cells(P>0.05),indicating that IPr1 was the target gene of miR-146a.Conclusion Overexpr

关 键 词：MIR-146A 肺结核 胞内病原体抗性基因1 结核分枝杆菌 肺泡Ⅱ型上皮细胞 增殖 凋亡 

分 类 号：R521[医药卫生—内科学] R446.6[医药卫生—临床医学]