DosR抗原Rv2626c通过lncRNA MacORIS诱导巨噬细胞活化的结核潜伏感染机制研究

doi:10.3969/j.issn.1673-8640.2024.10.001

检验医学 ›› 2024, Vol. 39 ›› Issue (10): 923-932.DOI: 10.3969/j.issn.1673-8640.2024.10.001

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DosR抗原Rv2626c通过lncRNA MacORIS诱导巨噬细胞活化的结核潜伏感染机制研究

汪兆慧¹, 郑振鲁², 李瑞娜², 马媛媛², 徐辉³, 魏振宏⁴, 贾彦娟⁴, 齐晓明⁴, 高小玲³()

1.洛阳市妇幼保健院医学遗传与产前诊断科，河南洛阳 471099
2.甘肃中医药大学，甘肃兰州 730000
3.海南省人民医院检验科，海南海口 570311
4.甘肃省人民医院临床研究与转化医学研究所，甘肃兰州 730000

收稿日期:2023-12-16 修回日期:2024-06-10 出版日期:2024-10-30 发布日期:2024-11-08
通讯作者: 高小玲，E-mail：gaoxl008@hotmail.com。
作者简介:汪兆慧，女，1997年生，硕士，初级检验师，主要从事结核分枝杆菌感染机制研究。
基金资助:
国家自然科学基金项目(81830372)

Study of latent tuberculosis infection mechanism of DosR antigen Rv2626c inducing macrophage activation through lncRNA MacORIS

WANG Zhaohui¹, ZHENG Zhenlu², LI Ruina², MA Yuanyuan², XU Hui³, WEI Zhenhong⁴, JIA Yanjuan⁴, QI Xiaoming⁴, GAO Xiaoling³()

1. Department of Medical Genetics and Prenatal Diagnostics，Luoyang Maternal and Child Health Care Hospital，Luoyang 471099，Henan，China
2. Gansu Chinese Medical University，Lanzhou 730000，Gansu，China
3. Medical Laboratory Center，Hainan Provincial People's Hospital，Haikou 570311，Hainan，China
4. The Institute of Clinical Research and Translational Medicine，Gansu Provincial People's Hospital，Lanzhou 730000，Gansu，China

Received:2023-12-16 Revised:2024-06-10 Online:2024-10-30 Published:2024-11-08

摘要/Abstract

摘要：

目的探讨结核潜伏感染（LTBI）表达的DosR抗原Rv2626c在巨噬细胞活化中的作用和长链非编码RNA（lncRNA）MacORIS对其的调控作用。方法将人急性单核细胞白血病细胞系THP-1诱导为巨噬细胞，根据转染物的不同分为4组：阴性对照（NC）组[转染NC-小干扰RNA（siRNA） 24 h]、MacORIS-siRNA组（转染MacORIS-siRNA 24 h）；NC-siRNA+重组蛋白Rv2626c（rRv2626c）组（转染NC-siRNA 24 h，再用3 μg·mL^-1 rRv2626c刺激24 h）、MacORIS-siRNA+rRv2626c组（转染MacORIS-siRNA 24 h，再用3 μg·mL^-1 rRv2626c刺激24 h）。检测各组细胞lncRNA MacORIS、Toll样受体（TLR）2、TLR4、表面共刺激分子（CD80、CD86、CD163、CD206）、吲哚胺2，3-双加氧酶1（IDO1）、肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-8、IL-1β的表达情况。采用生物信息学方法预测并构建lncRNA MacORIS的竞争内源RNA（ceRNA）网络，对lncRNA MacORIS潜在的靶微小RNA进行验证。结果 rRv2626c可有效促进巨噬细胞lncRNA MacORIS和IDO1上调（P<0.05）。rRv2626c可依赖于lncRNA MacORIS诱导THP-1细胞中TLR2的表达（P<0.05）。rRv2626c可依赖于lncRNA MacORIS上调巨噬细胞中CD80、CD86、TNF-α、IL-1β的表达（P<0.001）。与NC组比较，MacORIS-siRNA组miR-141-3p、miR-150-5p、miR-33-5p、miR-200-3p和miR-203-3p相对表达量均显著升高（P<0.05）。结论 rRv2626c可促进巨噬细胞极化到M1表型，并促进TLR2和炎症因子表达，这些变化均依赖于lncRNA MacORIS。lncRNA MacORIS可负调控miR-141-3p、miR-150-5p、miR-33-5p、miR-200-3p和miR-203-3p的表达。

关键词: 长链非编码RNA MacORIS, Rv2626c, 巨噬细胞, 结核潜伏感染

Abstract:

Objective To investigate the regulation role of dormancy survival regulator（DosR） antigen Rv2626c expressed in latent tuberculosis infection（LTBI） in the activation of macrophage and the regulation influence of long non-coding RNA（lncRNA） MacORIS. Methods Human acute monocytic leukemia cell line THP-1 cells were differentiated into macrophage and categorized into 4 groups based on different transfections：negative control（NC） group [transfected with NC-small interfering RNA（siRNA） for 24 h] and MacORIS-siRNA group [transfected with MacORIS-siRNA for 24 h]，NC-siRNA+recombinant protein Rv2626c（rRv2626c） group [transfected with NC-siRNA for 24 h，then stimulated with 3 μg·mL^-1 rRv2626c for 24 h]，MacORIS-siRNA+rRv2626c group [transfected with MacORIS-siRNA for 24 h，then stimulated with 3 μg·mL^-1 rRv2626c for 24 h]. The expression of lncRNA MacORIS，Toll-like receptor（TLR）2，TLR4，surface co-stimulatory molecules（CD80，CD86，CD163，CD206），indoleamine 2，3-dioxygenase 1（IDO1），tumor necrosis factor-alpha（TNF-α），interleukin（IL）-6，IL-8 and IL-1β were determined. The competitive endogenous RNA（ceRNA） networks of lncRNA MacORIS were predicted and constructed by bioinformatics methods，and the potential target microRNA of lncRNA MacORIS was verified. Results The rRv2626c effectively promoted the upregulation of lncRNA MacORIS and IDO1 in macrophage（P<0.05）. The rRv2626c induced the expression of TLR2 in THP-1 cells，which was dependent on lncRNA MacORIS（P<0.05）. Furthermore，rRv2626c depended on lncRNA MacORIS to upregulate the expressions of CD80，CD86，TNF-α and IL-1β in macrophage（P<0.001）. Compared to the NC group，the relative expression levels of miR-141-3p，miR-150-5p，miR-33-5p，miR-200-3p and miR-203-3p in the MacORIS-siRNA group were increased（P<0.05）. Conclusions The rRv2626c promotes the polarization of macrophage toward M1 phenotype，and it enhances the expression of TLR2 and inflammatory factors，which depends on lncRNA MacORIS. The lncRNA MacORIS could negatively regulate the expression of miR-141-3p，miR-150-5p，miR-33-5p，miR-200-3p and miR-203-3p.

Key words: Long non-coding RNA MacORIS, Rv2626c, Macrophage, Latent tuberculosis infection

中图分类号:

R446.7

汪兆慧, 郑振鲁, 李瑞娜, 马媛媛, 徐辉, 魏振宏, 贾彦娟, 齐晓明, 高小玲. DosR抗原Rv2626c通过lncRNA MacORIS诱导巨噬细胞活化的结核潜伏感染机制研究[J]. 检验医学, 2024, 39(10): 923-932.

WANG Zhaohui, ZHENG Zhenlu, LI Ruina, MA Yuanyuan, XU Hui, WEI Zhenhong, JIA Yanjuan, QI Xiaoming, GAO Xiaoling. Study of latent tuberculosis infection mechanism of DosR antigen Rv2626c inducing macrophage activation through lncRNA MacORIS[J]. Laboratory Medicine, 2024, 39(10): 923-932.

图/表 10

表1 基因引物序列

基因名称	正向引物（5'~3'）	反向引物（5'~3'）
MacORIS	GCCGCTAGTATTCAGCGAGA	AAGTCTTTTTGGGGAAAGCGTTG
GAPDH	ACAACTTTGGTATCGTGGAAGG	GCCATCACGCCACAGTTTC
TLR2	AGGTCCAGAGTTCCCTCCG	ACACTGTTAAAAGAGAAACTGGGAG
TLR4	AGTTGATCTACCAAGCCTTGAGT	GCTGGTTGTCCCAAAATCACTTT
IDO1	GTGTTTCACCAAATCCACGATCA	AGACCGTCTGATAGCTGGGG
IL-6	AACCTGAACCTTCCAAAGATGGC	TCTGTGCCCAGTGGACAGGTTTC
IL-8	GCGCCAACACAGAAATTATTGTAA	TGGTCATGAGTACAACAAACTCAC
TNF-α	CTGTAGCCCATGTTGTAGCAAAC	TCTGGTAGGAGACGGCGATG
IL-1β	GAAATGATGGCTTATTACAGTGGCA	CCTTGCTGTAGTGGTGGTCG
CD80	GTCGCCTCTCTGAAGATTACCC	TTGTGGATTTAGTTTCACAGCTTGC
CD86	ATTTGGAACCAAGCAAGAGCA	TACTCAGTCCCATAGTGCTGTCAC
CD163	AGACAGCGGCTTGCAGAAAAT	GCCTCCTTTTCCATTCCAGAAATA
CD206	ACCTCACAAGTATCCACACCATC	CTTTCATCACCACACAATCCTC
iNOS	GTGATGGCAAGCACGACTTC	CGAAACCACTCGTATTTGGGATG
RNU6A	GCTCGCTTCGGCAGCACATATAC	CGAATTTGCGTGTCATCCTTGCG
miR-141-3p	CGCGTAACACTGTCTGGTAAAG	通用下游引物（货号B661601-0002）
miR-150-5p	TCTCCCAACCCTTGTACCAG	通用下游引物（货号B661601-0002）
miR-33-5p	CGTGCATTGTAGTTGCATTGC	通用下游引物（货号B661601-0002）
miR-200-3p	GCTAACACTGTCTGGTAACGAT	通用下游引物（货号B661601-0002）
miR-203-3p	GCGTGAAATGTTTAGGACCAC	通用下游引物（货号B661601-0002）

图1 rRv2626c的鉴定注：（a）rRv2626c经EcoRⅠ和HindⅢ酶切后的电泳图；1和2为pET30a-Rv2626c质粒；3和4为pET30a质粒；M为DNA 2 000 Marker；（b）rRv2626c的SDS-PAGE图；M为DNA Marker；1为空pET30a质粒；2和3为rRv2626c；（c）rRv2626c的免疫印迹图；1为空pET30a质粒；2和3为rRv2626c。

图2 MacORIS-siRNA转染效率注：（a）转染24 h；（b）转染48 h；与NC-siRNA比较，*P<0.001、**P<0.000 1。

图3 不同方式处理的THP-1细胞中lncRNA MacORIS、TLR2 mRNA和TLR4 mRNA的表达情况注：（a）不同方式处理的THP-1细胞lncRNA MacORIS相对表达量比较；与PBS（对照）比较，*P<0.01、**P<0.001；（b）各组TLR2 mRNA相对表达量比较，与NC组比较，*P<0.000 1；与NC-siRNA+rRv2626c组比较，#P<0.000 1；（c）各组TLR4 mRNA比较。

图4 各组细胞表面极化标志物表达的比较注：（a）CD80 mRNA；（b）CD86 mRNA；（c）iNOS mRNA；（d）CD163 mRNA；与NC组比较，*P<0.05、**P<0.000 1；与NC-siRNA+rRv2626c组比较，#P<0.05、##P<0.001 、###P<0.000 1。

图5 rRv2626c处理后的巨噬细胞表面极化标志物表达情况注：（a）CD80；（b）CD86；（c）CD163；（d）CD206；—同型对照、—对应抗体；与NC组比较，*P<0.000 1；与NC-siRNA+rRv2626c组比较，#P<0.001、##P<0.000 1。

图6 各组IDO1 mRNA相对表达量比较注：与NC组比较，*P<0.000 1；与NC-siRNA+ rRv2626c组比较，#P<0.000 1。

图7 各组TNF-α mRNA、IL-6 mRNA、IL-8 mRNA、IL-1β mRNA相对表达量比较注：（a）TNF-α mRNA；（b）IL-6 mRNA；（c）IL-8 mRNA；（d）IL-1β mRNA；与NC组比较，*P<0.001、**P<0.000 1；与NC-siRNA+rRv2626c组比较，#P<0.001、##P<0.000 1。

图8 各组TNF-α、IL-6、IL-8、IL-1β水平比较注：（a）TNF-α；（b）IL-6；（c）IL-8；（d）IL-1β；与NC组比较，*P<0.001、**P<0.000 1；与NC-siRNA+rRv2626c组比较，#P<0.001、##P<0.000 1。

图9 lncRNA MacORIS的生物信息学分析结果注：（a）MacORIS相关的ceRNA网络，红色为lncRNA MacORIS，蓝色为微小RNA，绿色为mRNA；（b）KEGG通路富集分析（前20个途径）；（c）GO富集分析（前20个途径）；（d）NC组和MacORIS-siRNA组miR-141-3p、miR-150-5p、miR-33-5p、miR-200-3p和miR-203-3p相对表达量比较； NC组、 MacORIS-siRNA组、与NC组比较，*P<0.05、**P<0.000 1。

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DosR抗原Rv2626c通过lncRNA MacORIS诱导巨噬细胞活化的结核潜伏感染机制研究

Study of latent tuberculosis infection mechanism of DosR antigen Rv2626c inducing macrophage activation through lncRNA MacORIS

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