MiR‐140 modulates the inflammatory responses of Mycobacterium tuberculosis‐infected macrophages by targeting TRAF6

Abstract This study aimed to examine miR‐140 expression in clinical samples from tuberculosis (TB) patients and to explore the molecular mechanisms of miR‐140 in host‐bacterial interactions during Mycobacterium tuberculosis (M tb) infections. The miR‐140 expression and relevant mRNA expression were detected by quantitative real‐time PCR (qRT‐PCR); the protein expression levels were analysed by ELISA and western blot; M tb survival was measured by colony formation unit assay; potential interactions between miR‐140 and the 3′ untranslated region (UTR) of tumour necrosis factor receptor‐associated factor 6 (TRAF6) was confirmed by luciferase reporter assay. MiR‐140 was up‐regulated in the human peripheral blood mononuclear cells (PBMCs) from TB patients and in THP‐1 and U937 cells with M tb infection. Overexpression of miR‐140 promoted M tb survival; on the other hand, miR‐140 knockdown attenuated M tb survival. The pro‐inflammatory cytokines including interleukin 6, tumour necrosis‐α, interleukin‐1β and interferon‐γ were enhanced by M tb infection in THP‐1 and U937 cells. MiR‐140 overexpression reduced these pro‐inflammatory cytokines levels in THP‐1 and U937 cells with M tb infection; while knockdown of miR‐140 exerted the opposite actions. TRAF6 was identified to be a downstream target of miR‐140 and was negatively modulated by miR‐140. TRAF6 overexpression increased the pro‐inflammatory cytokines levels and partially restored the suppressive effects of miR‐140 overexpression on pro‐inflammatory cytokines levels in THP‐1 and U937 cells with M tb infection. In conclusion, our results implied that miR‐140 promoted M tb survival and reduced the pro‐inflammatory cytokines levels in macrophages with M tb infection partially via modulating TRAF6 expression.


Tuberculosis (TB) is a serious infectious disease and is caused by
Mycobacterium tuberculosis (M tb) infection, and TB has imposed great threats to human health worldwide. 1 According to the epidemic statistics, around 9 million individuals were diagnosed as TB patients, and TB caused around 1 million deaths in 2016. 2

In
China, owing to the spread of TB with drug resistance and the dual infection of TB and human immunodeficiency virus, the cases of TB infection have remarkably increased in recent years. 3 Mechanistic studies have shown that M tb is an intracellular pathogen, which can parasitize the host's macrophages, 4 11 Further study by Zheng et al, revealed that in the pulmonary TB, miR-155 and miR-132 could serve as important diagnostic biomarkers. 12 Liu et al, showed that TB induced miR-582-5p up-regulation in TB patients and miR-582-5p inhibited monocytes apoptosis by targeting forkhead box protein O1. 13 In addition, miR-223 was found to exert positive effect on the recruitment of lung neutrophil, which may be related to TB susceptibility, 14 and miR-124 could negatively regulate toll-like receptor signalling in M tb-infected alveolar macrophages. 15 Recently, studies from Lin et al, demonstrated that miR-140 was overexpressed in human macrophages infected with M tb using microarray analysis, 16 suggesting the potential actions of miR-140 in TB. Unfortunately, the role of miR-140 in TB has not been examined in detail so far.
In our current investigations, miR-140 up-regulation was found in the human peripheral blood mononuclear cells (PBMCs) from patients with TB, and further in vitro functional assays were performed to determine the miR-140 actions on the M tb survival and the proinflammatory cytokine levels in the human macrophages with M tb infection. In addition, the underlying molecular mechanisms were also assessed by the in vitro functional assays. The current investigations may imply the novel role of miR-140 in the host-bacterial interactions during M tb infections.

| Cell lines and culture
Two human macrophage cell lines (THP-1 and U937) were commercially purchased from the ATCC company (Manassas, VA). The THP-1 and U937 cells were both cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) with the supplement of 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific), sodium pyruvate (1 mmol/L; Sigma, St. Louis, MO), L-glutamine (2 mmol/L; Sigma), penicillin (100 U/mL; Sigma) and streptomycin (100 mg/mL; Sigma). THP-1 and U937 cells were maintained in the humidified conditions with 5% CO 2 at 37°C. The cells were treated with 100 nmol/L phorbol 12-myristate 13-acetate (PMA; Sigma) for overnight and then washed for three times. Following PMA stimulation, cells were rested for 3 days before further in vitro studies.  17,18 Briefly, 1 mL cultures of H37RV mycobacterial strain was pelleted for 2 minutes followed by re-suspension in RPMI 1640 medium, and the suspension was then vortexed for 2 minutes and sonicated in a bath sonicator (Thomas Scientific, Swedesboro, USA) for 5 minutes. After sonication, dispersed mycobacterial suspensions were allowed to rest for 5 minutes, and the upper 500 μL was used for infecting macrophages. For the infections, the H37RV and macrophages were cultured onto the 24-well plates. H37RV with different multiplicity of infections (MOIs) were allowed to infect the macrophages for different durations (3, 6, 12, 24 and 48 hours).

| MiRNA oligonucleotides, vectors and oligonucleotides transfection
The miRNA oligonucleotides including the mimic and inhibitor for miR-140 as well as their corresponding negative controls (NCs, the corresponding miRNAs with the scrambled sequence) were com-

| Gene expression as measured by quantitative real-time PCR
Total RNA isolation from cells was performed with TRIzol reagent

| M tb survival as determined by colony-forming unit assay
Mycobacterium tuberculosis survival after infecting into THP-1 or U937 cells was assessed by the colony-forming unit (CFU) assay.
Briefly, the transfected human macrophages were infected by M tb at 10 MOI for a duration of 48 hours followed by lysing the infected cells with sterile distilled water. Ten-fold serial dilutions were used for quantitative culturing with each dilution inoculated on Middlebrook 7H10 agar plates containing 10% oleic acid albumin dextrose catalase enrichment (Sigma). The M tb was cultured for 3 weeks at 37°C and the CFUs were determined using the standard protocols.

| Protein levels as determined by ELISA assay
The medium for culturing THP-1 and U937 cells that received different treatments was collected for the ELISA analysis of pro-inflammatory cytokine levels. The protein levels of interleukin 6 (IL-6), tumour necrosis-α (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFN-γ) were analysed using the corresponding ELISA kits (Thermo Fisher Scientific).

| Bioinformatics prediction and luciferase reporter assay
The complementary sequence between TRAF6 3′UTR and miR-140 was predicted by TargetScan tool. The wild-type fragment of TRAF6 3′UTR with the miR-140 binding sequence was amplified by PCR and inserted into the pGL3 vector (Promega, Madison, USA). The mutation of TRAF6 3′UTR was performed by mutating the relevant bases in miR-140 seed region using a site-directed mutagenesis method (Takara). For the luciferase reporter assay, HEK293T cells were cotransfected with miRNA oligonucleotides and Renilla luciferase pRL-TK vector, and wild-type/mutant TRAF6 3′UTR reporter vector. At 48 hours following transfection, luciferase activity was determined using a dual-luciferase assay system (Promega) according to the manufacturer's protocol.

| Statistical analysis
All the data analyses were performed in the GraphPad Prism Version 6.0 software (GraphPad Prism Software, La Jolla, USA).
All the experimental assays were performed in triplicates. The data were shown as mean ± SD. Student's t test or one-way ANOVA followed by Turkey's post hoc test was used to analyse the statistical significance between different treatment groups.
The differences were considered to be statistically significant when P < 0.05.

| MiR-140 promoted M tb survival in macrophages
In order to further determine the molecular mechanisms of miR-140 in TB, we manipulated the miR-140 expression levels by transfecting human macrophage cell lines with different miRNA oligonucleotides. As shown in Figure 2A

| Effects of M tb infection on the proinflammatory cytokines levels in THP-1 and U937 cells
Firstly, we performed qRT-PCR to determine the expression of IL-6, TNF-α, IL-1β and IFN-γ mRNA in the macrophages after being infected by M tb (10 MOI). Infecting THP-1 cells for 48 hours significantly increased the mRNA expression of IL-6, TNF-α, IL-1β and IFN-γ ( Figure 3A-D). Consistently, infection of M tb enhanced the mRNA expression of these pro-inflammatory cytokines in U937 cells ( Figure 3E-H). Moreover, ELISA assay was performed to determine the protein levels of these pro-inflammatory cytokines, and M tb infection significantly caused the increase in protein levels of these pro-inflammatory cytokines in both THP-1 and U937 cells (Table 1).

| MiR-140 suppressed TRAF6 expression via targeting the 3'UTR
To further explore the mechanisms of miR-140 in regulating the inflammatory responses, we performed bioinformatics analysis using Targetscan (www.targe tscan.org) prediction software.
There are many potential targets were predicted, and TRAF6 was selected from these predicted targets for further validation, as TRAF6 has been reported for its role in M tb infection. 20 The bioinformatics analysis showed that TRAF6 was one of the miR-140targeted genes, and the complementary sequences between miR-140 and TRAF6 3′UTR were shown in Figure 5A. Furthermore,       Abbreviation: IFN-γ, interferon-γ; IL-6, interleukin 6; IL-1β, interleukin-1β; TNF-α, tumour necrosis-α; NC, negative control. *P < 0.05, **P < 0.01, ***P < 0.001 relative to mimic NC group; # P < 0.05, ## P < 0.01 relative to inhibitor NC group. pro-inflammatory cytokines would be more supportive for our current findings. Fourthly, the downstream targets of miR-140 were not limited to TRAF6, and it is necessary for us to further explore other interaction between miR-140 and other targets in the future studies. In addition, future work to confirm the findings in primary human monocyte-derived macrophages will be warranted.

| CON CLUS IONS
In conclusion, we demonstrated that up-regulation of miR-140 was detected in the human PBMCs from the TB patient, and the up-regulation of miR-140 was also found in the human macrophages upon M tb infection. Further in vitro mechanistic studies indicated that miR-140 promoted M tb survival and suppressed pro-inflammatory cytokines production in M tb-infected macrophages partially via modulating TRAF6 expression.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
XL and NP conceived and designed the study. XL and SH per-

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data generated in the study are available upon reasonable request.