The role of miR-99a-5p in the immune regulation of host macrophages infected by Mycobacterium tuberculosis

doi:10.19982/j.issn.1000-6621.20220532

Abstract

Abstract:

Objective: To explore the role of specific microRNA in THP-1 cells mediated host defense against Mycobacterium tuberculosis (MTB) infection. Methods: Total RNA were extracted from THP-1 cells with or without MTB (H37Rv) infection, and then the miRNA expression profiles in THP-1 cells with or without MTB infection were identified using Illumina NovaSeq6000 next-generation sequencing platform. Four health controls were recruited and the human monocyte-derived macrophage (hMDM) were separated. Validation of differentially expressed miRNA between MTB infected and uninfected THP-1 cells, as well as that between MTB infected and uninfected hMDM, were performed by quantitative real-time PCR (qRT-PCR). The miRNA overexpression vector was constructed and transfected into THP-1 macrophages by lentivirus. The expression level of tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ) and colony-forming unit (CFU) in H37Rv infected THP-1 cells which were transfected with miRNA overexpression vector or control empty vector was tested using qRT-PCR. Results: miR-99a-5p was selected as the study target gene (P=0.021) by not previously studied and most significantly different, from 16 miRNA molecules with significant expression differences in second-generation sequencing results. By qRT-PCR validation in THP-1 cells and primary macrophages, miR-99a-5p expression within THP-1 infected groups (0.482±0.148) and primary macrophages (0.433±0.072) were significantly lower than the uninfected group (1.536±0.290 and 1.113±0.218)(t=6.476,P<0.001; t=3.167,P=0.019). CFU results of the THP-1 cells infected by MTB showed that the intracellular bacteria amount of miR-99a-5p overexpression cells at 24 h after infection was 64.0×10⁴ (52.0×10⁴,87.0×10⁴), which was significantly higher than that in the control cells (17.0×10⁴ (16.0×10⁴,24.0×10⁴))(Z=-2.323, P=0.029). Furthermore, the expression levels of TNF-α and IFN-γ in MTB infected miR-99a-5p over expression cells were 1.018±0.310 and 1.687±0.135, which were significantly lower than TNF-α (0.740±0.001) and IFN-γ (0.631±0.374) those in the MTB infected control cells (t=4.631, P=0.010; t=3.349, P=0.010). Conclusion: miR-99a-5p can inhibit the release of TNF-α and IFN-γ, promote the growth of MTB in macrophages. Therefore, the decreased miR-99a-5p expression after MTB infection may play a protective role in host innate immunity against MTB infection.

Key words: Mycobacterium tuberculosis, Microarray technology, MicroRNAs, Macrophages, Immunomodulation

CLC Number:

Shi Yuting, Dong Jing, Jia Hongyan, Zhu Chuanzhi, Yang Bin, Li Zihui, Sun Qi, Du Boping, Xing Aiying, Zhang Zongde, Pan Liping. The role of miR-99a-5p in the immune regulation of host macrophages infected by Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2023, 45(5): 464-471. doi: 10.19982/j.issn.1000-6621.20220532

Figures/Tables 4

References 37

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样本名称	测序类型	碱基序列长度(bp)	碱基质量 (%)	结果
THP-1-1640-1	miRNA seq	21 289 140	98.73	Pass
THP-1-1640-2	miRNA seq	26 075 024	98.66	Pass
THP-1-1640-3	miRNA seq	21 649 327	98.71	Pass
THP-1-Rv-1	miRNA seq	26 065 198	98.59	Pass
THP-1-Rv-2	miRNA seq	24 885 359	98.65	Pass
THP-1-1-Rv-3	miRNA seq	22 724 008	98.73	Pass

miRNA名称	Log2FC	P值	miRNA名称	Log2FC	P值
has-miR-216a-5p	4.861	<0.001	has-miR-217	1.843	0.031
has-miR-31-5p	2.998	0.000	has-miR-301b-5p	3.082	0.045
has-miR-409-3p	Inf	<0.001	has-miR-3190-3p	Inf	0.031
has-miR-125b-5p	1.017	0.007	has-miR-33a-5p	1.210	0.047
has-miR-127-3p	2.795	0.006	has-miR-411-5p	2.275	0.045
has-miR-1307-5p	2.335	0.012	has-miR-4518	Inf	0.032
has-miR-15a-3p	1.889	0.048	has-miR-4755-5p	Inf	0.026
has-miR-190a-3p	2.590	0.025	has-miR-99a-5p	2.191	0.021

组别	感染4h菌落形成单位计数(×10⁴)	感染24h菌落形成单位计数(×10⁴)
对照空载体组	50.0(26.0,84.0)	17.0(16.0,24.0)
miR-99a-5p过表达组	51.0(37.0,56.0)	64.0(52.0,87.0)
Z值	-0.145	-2.323
P值	0.886	0.029

组别	TNF-α相对表达量		IFN-γ相对表达量
组别	未感染组	感染组(6h)	未感染组	感染组(6h)
对照空载体组	0.499±0.005	1.018±0.310	0.530(0.476,2.082)	1.687±0.135
miR-99a-5p过表达组	0.516±0.016	0.740±0.001	0.522(0.513,1.058)	0.631±0.374
t值	-0.169	4.631	1.135	3.349
P值	0.874	0.010	0.100	0.010