hsa_circ_0007460通过调节巨噬细胞的自噬和凋亡影响细胞内结核分枝杆菌的存活

doi:10.16288/j.yczz.23-160

遗传 ›› 2023, Vol. 45 ›› Issue (11): 1039-1051.doi: 10.16288/j.yczz.23-160

hsa_circ_0007460通过调节巨噬细胞的自噬和凋亡影响细胞内结核分枝杆菌的存活

章金怡¹(), 何禹墨¹, 周晶雨², 翁术锋¹, 马慧霞¹, 林太玥¹, 徐颖¹()

1.复旦大学生命科学学院，上海 200433
2.复旦大学附属华山医院感染科，上海 200040

收稿日期:2023-06-30 修回日期:2023-08-31 出版日期:2023-11-20 发布日期:2023-09-18
通讯作者: 徐颖 E-mail:20210700011@fudan.edu.cn;yingxu2520@fudan.edu.cn
作者简介:章金怡，硕士研究生，专业方向：微生物学。E-mail: 20210700011@fudan.edu.cn。
基金资助:
国家自然科学基金(81971900)

Hsa_circ_0007460 affects the survival of intracellular Mycobacterium tuberculosis by regulating autophagy and apoptosis of macrophages

Jinyi Zhang¹(), Yumo He¹, Jingyu Zhou², Shufeng Weng¹, Huixia Ma¹, Taiyue Lin¹, Ying Xu¹()

1. School of Life Science, Fudan University, Shanghai 200433, China
2. Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai 200040, China

Received:2023-06-30 Revised:2023-08-31 Published:2023-11-20 Online:2023-09-18
Contact: Ying Xu E-mail:20210700011@fudan.edu.cn;yingxu2520@fudan.edu.cn
Supported by:
National Natural Science Foundation of China(81971900)

摘要/Abstract

摘要：

环状RNA (circular RNA, circRNA)是一类缺乏5′-帽子和3′-poly(A)尾巴的非编码RNA，可以参与多种人类疾病的生物学过程。然而，对其在活动性肺结核(active pulmonary tuberculosis，ATB)中的诊断和功能价值却知之甚少。本研究旨在研究hsa_circ_0007460是否能作为ATB患者的潜在诊断生物标志物，并对其功能进行初探。通过实时荧光定量PCR (real-time quantitative fluorescent PCR，RT-qPCR)发现hsa_circ_0007460在32例ATB患者的外周血以及牛结核分枝杆菌的减毒株——BCG (bacillus Calmette-Guerin)感染的THP-1人源巨噬细胞中显著上调。受试者工作特征曲线(receiver operating curve, ROC)显示曲线下面积(the area under ROC curve, AUC)为0.7474，灵敏度76.67%，特异度78.13%。通过RNase R消化和放线菌素D抑制实验证实hsa_circ_0007460相较于其线性mRNA更加稳定，提示其有作为ATB的诊断生物标志物的潜力。通过蛋白质印迹(Western blot)、CCK-8 (cell counting kit-8)、平板计数、免疫荧光等实验表明hsa_circ_0007460能调控巨噬细胞凋亡和自噬。最后，通过生物信息学分析预测下游的miRNA和mRNA，构建了hsa_circ_0007460/hsa-miR-3127-5p/PATZ1轴。上述研究结果表明，hsa_circ_0007460在ATB患者外周血中显著上调，可以作为潜在的诊断生物标志物，且hsa_circ_0007460能促进巨噬细胞凋亡、抑制巨噬细胞自噬从而促进胞内BCG的存活。

关键词: hsa_circ_0007460, 自噬, 凋亡, TB

Abstract:

Circular RNA (circRNA) is a category of non-coding RNAs characterized by the absence of a 5'-cap and 3′-poly(A) tail, and participates in the physiological processes of various human diseases. Nonetheless, the diagnostic and functional significance of circRNAs in active pulmonary tuberculosis (ATB) remains uncertain. Consequently, the purpose of this study is to investigate whether hsa_circ_0007460 can be employed as a potential diagnostic biomarker in ATB patients and explore its function. The result of real-time quantitative fluorescent PCR (RT-qPCR) validated a notable increase in the expression of hsa_circ_0007460 in the peripheral blood of 32 ATB patients, as well as in THP-1 human macrophages infected with Bacillus Calmette Guerin (BCG) which is an attenuated strain of Mycobacterium bovis. Additionally, the receiver operating curve (ROC) illustrated that the area under the ROC curve (AUC), sensitivity and specificity were 0.7474, 76.67%, and 78.13% respectively. RNase R, Actinomycin D and other experiments confirmed that hsa_circ_0007460 was stabler than its linear mRNA, indicating that hsa_circ_0007460 has potential as a diagnostic biomarker of ATB. Furthermore, Western blot (WB), Cell Counting Kit-8 (CCK-8), plate counting, and immunofluorescence experiments revealed that hsa_circ_0007460 could regulate apoptosis and autophagy of macrophages. The downstream miRNAs and mRNAs were subsequently predicted using bioinformatics, and the hsa circ 0007460/hsa-miR-3127-5p/PATZ1 axis was built. These above results suggest that hsa_circ_0007460 is substantially up-regulated in the peripheral blood of patients with ATB and can be utilized as a potential diagnostic biomarker. In addition, hsa_circ_0007460 can promote apoptosis of macrophages and inhibit autophagy of macrophages, thereby promoting the survival of BCG.

Key words: hsa_circ_0007460, autophagy, apoptosis, TB

章金怡, 何禹墨, 周晶雨, 翁术锋, 马慧霞, 林太玥, 徐颖. hsa_circ_0007460通过调节巨噬细胞的自噬和凋亡影响细胞内结核分枝杆菌的存活[J]. 遗传, 2023, 45(11): 1039-1051.

Jinyi Zhang, Yumo He, Jingyu Zhou, Shufeng Weng, Huixia Ma, Taiyue Lin, Ying Xu. Hsa_circ_0007460 affects the survival of intracellular Mycobacterium tuberculosis by regulating autophagy and apoptosis of macrophages[J]. Hereditas(Beijing), 2023, 45(11): 1039-1051.

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特征	ATB (n=32)	HC(n=30)
年龄(岁, 平均值)	41.88 ± 19.57 (20~70)	40.76 ± 12.42 (25~66)
性别
女	16	14
男	16	16
痰涂片
阳性	18	0
阴性	14	30
ELISPOT
阳性	32	0
阴性	0	30

目的基因	引物序列(5′→3′)		扩增长度(bp)
目的基因	上游序列	下游序列	扩增长度(bp)
hsa_circ_0007460	GTGGGTCTGGAGTAAACAACAG	CCCTGTAAGCCTTGCCAAAA	161
GAPDH	TCTCTGCTCCTCCTGTTCGA	GCGCCCAATACGACCAAATC	122

名称	序列(5′→3′)
hsa_circ_0007460- siRNA-1	ACCUUCGAAAUUCUGAUACAATT
hsa_circ_0007460- siRNA-2	GACCACCUUCGAAAUUCUGAUTT
hsa_circ_0007460- siRNA-3	CACCUUCGAAAUUCUGAUACATT

hsa_circ_0007460通过调节巨噬细胞的自噬和凋亡影响细胞内结核分枝杆菌的存活

Hsa_circ_0007460 affects the survival of intracellular Mycobacterium tuberculosis by regulating autophagy and apoptosis of macrophages

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