Down-Regulation of miR-378d Increased Rab10 Expression to Help Clearance of Mycobacterium tuberculosis in Macrophages.

Yifan Zhu,Aizhen Guo,Huanchun Chen,Delai Kong,Yao Xiao,Si Zhu Suo Lang,Yingyu Chen,Jiaqiang Niu,Wenjun Zhai,Changmin Hu,Tingting Zhu,Yongchong Peng,Xi Chen,Han Liu

doi:10.3389/fcimb.2020.00108

Abstract

Mycobacterium tuberculosis (M. tb) can survive in the hostile microenvironment of cells by escaping host surveillance, but the molecular mechanisms are far from being fully understood. MicroRNAs might be involved in regulation of this intracellular process. By RNAseq of M. tb-infected PMA-differentiated THP-1 macrophages, we previously discovered down-regulation of miR-378d during M. tb infection. This study aimed to investigate the roles of miR-378d in M. tb infection of THP-1 cells by using a miR-378d mimic and inhibitor. First, M. tb infection was confirmed to decrease miR-378d expression in THP-1 and Raw 264.7 macrophages. Then, it was demonstrated that miR-378d mimic promoted, while its inhibitor decreased, M. tb survival in THP-1 cells. Further, the miR-378d mimic suppressed, while its inhibitor enhanced the protein production of IL-1β, TNF-α, IL-6, and Rab10 expression. By using siRNA of Rab10 (siRab10) to knock-down the Rab10 gene in THP-1 with or without miR-378d inhibitor transfection, Rab10 was determined to be a miR-378d target during M. tb infection. In addition, a dual luciferase reporter assay with the Rab10 wild-type sequence and mutant for miR-378d binding sites confirmed Rab10 as the target of miR-378d associated with M. tb infection. The involvement of four signal pathways NF-κB, P38, JNK, and ERK in miR-378d regulation was determined by detecting the effect of their respective inhibitors on miR-378d expression, and miR-378d inhibitor on activation of these four signal pathways. As a result, activation of the NF-κB signaling pathway was associated with the down-regulation of miR-378d. In conclusion, during M. tb infection of macrophages, miR-378d was down-regulated and functioned on decreasing M. tb intracellular survival by targeting Rab10 and the process was regulated by activation of the NF-κB and induction of pro-inflammatory cytokines IL-1β, TNF-α, IL-6. These findings shed light on further understanding the defense mechanisms in macrophages against M. tb infection.

Highlights

Mycobacterium tuberculosis (M. tb) is the etiologic agent of tuberculosis (TB)
The results showed that down-regulation of miR-378d induced by M. tb infection enhanced expression of its target Rab10, activation of NF-κB signaling and increased the generation of some inflammatory cytokines which might contribute to decrease of M. tb survival in THP-1 cells
M. tb Infection Down-Regulates miR-378d in Macrophages. Previous data from this lab from a RNA-seq study discovered that miR-378d was significantly down-regulated in M. tb-infected, phorbol 12-myristate 13acetate (PMA)-differentiated THP-1 cells at 6 and 24 h post infection (PI) (Figure 1A)

Summary

Introduction

Mycobacterium tuberculosis (M. tb) is the etiologic agent of tuberculosis (TB). In 2018, TB caused an estimated 1.2 million deaths and 10 million new cases (World Health, 2019). Macrophages are major host cells for M. tb intracellular growth and its persistence during all phases of TB and contribute to stimulation of both innate and acquired immune responses, playing an essential role in protection against the infection (Rajaram et al, 2014). MicroRNAs (miRNAs) belong to a class of small non-coding conservative RNAs within eukaryotic species They usually function as critical regulators of gene expression through posttranscriptional modification of its target mRNAs for translation inhibition or for degradation (Bartel, 2004). MiR-378 belongs to a large family of evolutionarily conserved miRNAs with eight members (miR-378 a/b/c/d/e/f/h/i) Because they share similar seed sequences, a sequence of 6–8 nucleotides that is most critical for mRNA target recognition, they are believed to have similar activities and targets (Ganesan et al, 2013). A more recent paper about esophageal squamous cell carcinoma reported that miR-378a-3p targeted Rab during the process of this cancer development (Ding et al, 2018)

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