Elsevier

Tuberculosis

Volume 131, December 2021, 102123
Tuberculosis

Circ_0001490/miR-579-3p/FSTL1 axis modulates the survival of mycobacteria and the viability, apoptosis and inflammatory response in Mycobacterium tuberculosis-infected macrophages

https://doi.org/10.1016/j.tube.2021.102123Get rights and content

Abstract

Background

Macrophages play an important role in the host immune response against mycobacterial infection, and this process is regulated by various factors, including circular RNAs (circRNAs). We intended to explore the role of circ_0001490 in tuberculosis (TB) using Mycobacterium tuberculosis (M.tb)-infected THP-1 macrophages.

Methods

Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were conducted to measure RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to analyze the viability of THP-1 macrophages. Flow cytometry was performed to analyze the apoptosis rate of THP-1 macrophages. Enzyme-linked immunosorbent assay (ELISA) was conducted to assess the release of inflammatory cytokines. Colony-forming unit (CFU) assay was conducted to analyze the survival of M.tb in THP-1 macrophages. Intermolecular target interaction was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay.

Results

Circ_0001490 expression was down-regulated in the serum samples of TB patients and M.tb-infected THP-1 macrophages. Circ_0001490 overexpression suppressed M.tb survival and promoted the viability and inflammatory response of THP-1 macrophages. Circ_0001490 interacted with microRNA-579-3p (miR-579-3p), and circ_0001490 overexpression-induced protective effects in M.tb-infected THP-1 macrophages were largely overturned by the overexpression of miR-579-3p. miR-579-3p interacted with the 3′ untranslated region (3′UTR) of follistatin-like protein 1 (FSTL1). FSTL1 silencing largely overturned miR-579-3p knockdown-induced effects in M.tb-infected THP-1 macrophages. Circ_0001490 acted as miR-579-3p sponge to up-regulate FSTL1 in THP-1 macrophages.

Conclusion

In conclusion, our results demonstrated that circ_0001490 suppressed M.tb survival and promoted the viability and inflammatory response of M.tb-infected THP-1 macrophages partly by regulating miR-579-3p/FSTL1 axis.

Introduction

Tuberculosis (TB) is a common systemic infectious disease, and is mainly caused by the infection of Mycobacterium tuberculosis (M.tb) [1]. M.tb acts as the intracellular pathogen to infect host macrophages [2]. In turn, macrophages play a crucial role in suppressing the survival and replication of M.tb by initiating innate immune responses [3,4]. However, M.tb can still evade from the defense of macrophages [5]. Illustrating macrophages-M.tb interactions is vital to find novel effective strategy for TB therapy.

Circular RNAs (circRNAs) are a special class of non-coding RNA molecules without 5′ or 3′ end, and circRNAs have been identified to be diagnostic bio-markers for TB. For instance, circ_0001953 and circ_0009024 are reported to be markedly up-regulated in the plasma of active TB patients, and the levels of circ_0001953 and circ_0009024 in plasma are positively correlated with TB severity [6]. Huang et al. found that circ_0001204 and circ_0001747 might be novel plasma diagnostic markers for active TB [7]. Luo et al. found that circ_0001380 level is decreased in the blood samples of active pulmonary TB patients, and it might be a diagnostic marker for active pulmonary TB patients [8]. However, the biological significance underlying the dysregulation of circRNAs in TB is largely unknown. Circ_0001490 is reported to be notably down-regulated in TB patients [6]. Circ_0001490 is generated from the back-splicing of its host gene kinesin family member 2A (KIF2A). Here, we analyzed function of circ_0001490 in TB and its associated molecular mechanism.

CircRNAs serve as molecular sponges for microRNAs (miRNAs) to regulate cell biological behaviors [9]. Accumulating evidence have demonstrated that miRNAs are important regulators in TB progression. For example, miR-155 is reported to regulate immune response to the infection of mycobacteria by regulating cellular reprogramming process [10]. miR-140 is reported to facilitate the survival of M.tb pathogen and decrease the release of inflammation-associated cytokines in macrophages partly by targeting TRAF6 [11]. miR-579-3p was one of the candidate targets of circ_0001490 via the prediction of circinteractome database. miR-579 has been reported to promote the death of M.tb-infected macrophages [12]. In this article, the functional correlation between miR-579-3p and circ_0001490 was explored in TB progression.

miRNAs post-transcriptionally regulate the expression of target messenger RNAs (mRNAs) via binding to their 3′ untranslated region (3′UTR) [13]. Based on the prediction of StarBase bioinformatics database, follistatin-like protein 1 (FSTL1) was a candidate target of miR-579-3p. FSTL1 is reported to induce the release of macrophages- and fibroblasts-derived inflammatory cytokines [14], demonstrating that FSTL1 plays a pro-inflammatory role. Zhang et al. found that miR-32-5p facilitates the survival of M.tb pathogen and restrains the release of inflammatory factors in macrophages, and FSTL1 overexpression reverses miR-32-5p-mediated effects [15]. We tested the interaction between miR-579-3p and FSTL1 and further explored the working mechanism of FSTL1 in THP-1 macrophages infected with M.tb pathogen.

In this study, we first analyzed the expression of circ_0001490 in the serum samples of TB patients and normal volunteers, and we found that circ_0001490 was markedly down-regulated in TB patients. To uncover the role of circ_0001490 in TB pathology, THP-1 macrophages were infected with M.tb to establish TB model in vitro. Bioinformatics analysis and a series of functional experiments were then conducted to explore the working mechanism of circ_0001490 in TB progression.

Section snippets

Clinical samples

A total of 30 TB patients and 23 normal volunteers were recruited at Jiangxi Chest Hospital. The diagnosis of TB patients (n = 30) was based on sputum smear examination using Ziehl-neelsen acid fast staining and clinical symptoms. TB patients were free of immunodeficiency disease and other pulmonary diseases. Normal volunteers (n = 23) without bacteriological or clinical evidence of TB were enrolled in this study. A volume of 5 mL peripheral blood samples from TB patients who had not received

Circ_0001490 expression is decreased in TB patients and M.tb-infected THP-1 macrophages

We first measured the level of circ_0001490 in the serum samples of normal volunteers (n = 23) and TB patients (n = 30), and the RT-qPCR data uncovered that circ_0001490 level was down-regulated in TB patients compared with normal volunteers (Fig. 1A). THP-1 macrophages are widely utilized as the in vitro model to analyze the interaction between the host and pathogen during M.tb infection [17]. We measured the level of circ_0001490 in THP-1 macrophages infected with M.tb at different MOIs via

Discussion

Accumulating studies have demonstrated that circRNAs play important roles in TB pathology [8,20,21]. In our study, we found that circ_0001490 was down-regulated in serum samples of TB patients compared with that in healthy volunteers. Also, M.tb infection reduced circ_0001490 expression in THP-1 macrophages in a dose-dependent manner. Through gain-of-function experiments, we found that circ_0001490 overexpression suppressed M.tb survival and elevated the viability and inflammatory response of

Author contribution

Qun Deng had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Qun Deng, Jian Huang, Jinjin Yan; acquisition of data: Erning Mao, HuiJuan Chen, Caiwen Wang; critical revision of the manuscript for important intellectual content: Qun Deng; administrative, technical or material support: Qun Deng, Jian Huang, Jinjin Yan; study supervision: Qun Deng.

Disclosure of interest

The authors declare that they have no conflicts of interest.

Funding

None.

Availability of data and materials

Please contact the correspondence author for the data request.

Ethics approval and consent participate

Written informed consent was obtained from patients with approval by the Institutional Review Board in Jiangxi Chest Hospital.

Consent for publication

Not applicable.

Acknowledgment

None.

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