miR‐18a promotes Mycobacterial survival in macrophages via inhibiting autophagy by down‐regulation of ATM

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of leading causes of global deaths. This study aimed to explore the role of miR‐18a in RAW264.7 cells response to Mtb infection. Exosomes derived from Mtb‐infected cells were isolated and further validated by size, transmission electron microscopy and Western blot. RT‐PCR was utilized to measure miR‐18a expression. Cell viability and ultrastructure were examined by CFU counting, CCK‐8 and electron microscope, respectively. Potential target genes of miR‐18a were predicted with bioinformatics and further confirmed using RT‐PCR, Western blot and laser confocal microscope analysis, respectively. LC3, AMPK and mTOR were measured using Western blot. We found that miR‐18a was induced both in Mtb‐infected RAW264.7 cells and its derived exosomes compared with the controls. In addition, up‐regulation of miR‐18a promoted intracellular Mtb survival, attenuated cell viability and reduced LC3‐II level, while its down‐regulation had the opposite effect. miR‐18a overexpression suppressed level of ATM, one possible target of miR‐18a, while its underexpression enhanced ATM. We also found that inhibition of ATM induced LC3‐II decrease in Mtb‐infected cells and could reverse the increase of LC3‐II caused by inhibition of miR‐18a. Moreover, down‐regulation of miR‐18a increased p‐AMPK level while reduction of ATM could reverse the change. Taken together, our results suggest that miR‐18a is up‐regulated in macrophages response to Mtb infection, and it promotes intracellular Mtb survival through repressing autophagic process by down‐regulation of ATM pathway. This provides new thought for TB pathogenesis, diagnosis and treatment.


| 2005
YUAN et Al. microRNA (miRNA) is an endogenous, non-coding small RNA, which plays a significant role in regulating gene expression at the post-transcriptional level, including host immune responses. 5,6 It has been reported that many miRNAs, such as miR-125, miR-144 and miR-155, are involved in modulation of innate immunity and adaptive immunity including B cell differentiation, antibody production as well as T cell development. [7][8][9][10] Moreover, growing evidence has suggest that many miRNAs play important regulatory roles in the immune response against TB. [11][12][13][14][15] miR-18a is a member of the miR-17 family, which encodes for six individual miRNAs including miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a. 16 Studies have shown that many of them are related to Mtb infection: miR-17-5p regulates autophagy by targeting Mcl-1and STAT3 in Mtb-infected RAW264.7 cells, miR-20a inhibits autophagy by targeting ATG16L1 and ATG7 to facilitate BCG survival in macrophages, and miR-92a is increased in serum from pulmonary TB patients. [17][18][19] Many studies have reported that miR-18a, the most prominent miRNA in the miR-17-92 family, is up-regulated in activated T cells, can activate rapamycin-induced autophagy and is involved in regulation of autophagy in colon cancer cells. 20,21 However, little is known about the impact of miR-18a on Mtb survival and its possible underlying mechanism. In the current study, we first investigated the role of miR-18a in macrophages response to Mtb infection.

| Infection of RAW264.7 cells with Mtb
Mtb strain H37Rv, grown on Lowenstein-Jensen (LJ) medium, was collected and then dispersed into single bacterial suspension in RPMI 1640 by vortex and needle aspiration, which was further confirmed by acid-fast staining. RAW264.7 cells were cultured in RPMI 1640 medium added with 10% foetal bovine serum in an incubator with 5% CO 2 at 37°C. Cells were infected with single Mtb at the indicated multiplicity of infection (MOI = 1, 5 and 10) and further cultured for required time period (6, 12, 24 and 48 hours).

| RT-PCR
Total RNA was extracted from RAW264.7 cells or its derived exosomes using TRIzol reagent (Invitrogen), and cDNA was then synthesized by cDNA Synthesis Kit (Takara La Taq). Polymerase chain reaction (PCR) was run under the cycling conditions for miR-18a: 95°C for 5 minutes, followed by 35 cycles of 95°C for 10 seconds, 60°C for 20 seconds and 72°C for 20 seconds, and for ataxia telangiectasia mutated (ATM): 95°C for 4 minutes, followed by 30 cycles of 95°C for 10 seconds, 58°C for 20 seconds, 72°C for 60 seconds and 72°C for 5 minutes. U6 or β-actin was used as internal controls. The primers used above were shown in Tables S1 and S2. Meantime, cells were seeded on cover slides in 24-well plates for acid-fast staining.

| Cell viability assay and ultrastructural analysis
After transfection and following infection with Mtb at MOI = 10 for 6, 12, 24 and 48 hours, the cell counting kit-8 reagent of 10 μL (CCK-8; Dojindo) was added into each well and continued to culture for 1.5 hours. The OD value was detected by spectrophotometer (Thermo Fisher Scientific Oy) at 450 nm wavelength. Simultaneously, RAW264.7 cells were collected and fixed in glutaraldehyde for ultrastructure analysis under an electron microscope.
β-tubulin was used as internal control.

| Prediction and confirmation of potential target gene ATM of miR-18a
Possible target genes of miR-18a were predicted by the TargetScan software. Then, the complementary sites and pairing scores between miR-18a and putative target ATM were analysed by TargetScan. In addition, the correlation analysis was performed between miR-18a expression and ATM mRNA expression in Mtb-infected RAW264.7 cells. The effect of modulation of miR-18a on ATM expression was examined by RT-PCR and Western blot. Afterwards, levels of pAMPK and pmTOR were evaluated for illustrating the potential role of miR-18a in the ATM-AMPK-mTOR autophagy pathway.

| Confocal microscopy assay
RAW264.7 cells were fixed with 4% paraformaldehyde for 20 minutes and then treated with 0.5% TritonX-100 for 10 minutes. After being blocked with 5% bovine serum albumin (BSA) for 30 minutes, cells were incubated with anti-ATM followed by Cy3-labelled goat anti-rabbit IgG (Boster Biological Technology) and were further stained with DAPI for 5 minutes. Cells were observed under confocal laser scanning microscope (OLYMPUS).

| Statistical analysis
All the data were displayed as mean ± standard deviation (SD) of three independent experiments. The Student's t test was used to compare two groups, and the one-way ANOVA was used to compare multiple samples. Significant statistical differences were deemed to P < .05.

| Mtb infection induced miR-18a overexpression both in RAW264.7 cells and its derived exosomes
In order to evaluate the effect of Mtb infection on miR-18a expression in macrophages, we assessed its level in Mtb-infected RAW264.7 cells. The results showed that intracellular miR-18a expression displayed a MOI-and time-dependent elevation upon Mtb infection ( Figure 1A,B). RAW264.7 cell-derived exosomes were successfully obtained based on the confirmation by TEM, particle size and marker protein CD63 analysis ( Figure 1C-E).
We also found that miR-18a was gradually up-regulated in the exosomes derived from Mtb-infected RAW264.7 cells in a timedependent manner ( Figure 1F). The data demonstrated that levels of both intracellular and exosomal miR-18a were mightily induced, which suggested that miR-18a may play pivotal role in the pathogenesis of TB.

| miR-18a facilitated Mtb survival in RAW264.7 cells
Then, we further explored the impact of regulation of miR-18a on intracellular Mtb survival. Modulation of miR-18a in RAW264.7 cells was achieved with its mimics or inhibitor. RT-PCR results showed that miR-18a mimics significantly increased miR-18a level and miR-18a inhibitor decreased its level in RAW264.7 cells (Figure 2A-B). Subsequently, we further determined whether miR-18a exert an effect on intracellular Mtb survival using CFU and acid-fast staining. As exhibition in Figure 3A, miR-18a up-regulation resulted in a significant increase while its down-regulation led to an obvious decrease of CFU number, which were identical with the results of acid-fast staining ( Figure 3B). The above observations implicated that miR-18a could promote Mtb survival within macrophages.

| Effect of miR-18a on Mtb-infected macrophages
We further studied the effect of miR-18a on Mtb-infected mac- inhibitor, while few autophagosomes were observed in miR-18a mimics treated ones. Therefore, we inferred that miR-18a may be involved in the regulation of autophagy ( Figure 4B).

| miR-18a suppressed autophagy in Mtbinfected macrophages by inhibiting ATM
In order to validate the impact of miR-18a on autophagy, autophagyrelated protein LC3 was assessed by Western blot. Results showed that miR-18a mimics significantly decreased while its inhibitor increased LC3-II expression in Mtb-infected RAW264.7 cells compared with the controls ( Figure 5A). The data indicated that miR-18a restrained autophagy in macrophages response to Mtb. Next, we furthered investigated the underlying autophagy mechanisms triggered by miR-18a.
Target genes of miR-18a were predicted by TargetScan (Table   S3). ATM, a protein kinase, is known for its critical roles in activation of autophagy. 22 We found that it was one potential target of miR-18a ( Figure 5B). However, the function of ATM in TB remains unknown until now. Hence, we decided to detect ATM expres- C, Observation of exosomal characteristic by TEM. D, Estimation of the exosomal diameter by particle size analysis. E, Detection of the exosomal protein marker CD63 by Western blot. F, The expression of miR-18a at different time-points in exosomes derived from Mtbinfected RAW264.7 macrophages. Data represent the means ± SD from three independent experiments. *P < .05, **P < .01, ***P < .001. Relative miR-18a expression, miR-18a/ internal reference U6 F I G U R E 2 Identification of models of overexpression and inhibit expression of miR-18a. A, The transfection efficiency of RAW264.7 macrophages transfected with FAM-siRNA was detected by fluorescence microscope. B, The expression of miR-18a in RAW264.7 macrophages transfected with miR-18a mimics, NC-mimics, miR-18a inhibitor or NC-inhibitor by RT-PCR. Data represent the means ± SD from three independent experiments. **P < .01, ***P < .001. Relative miR-18a expression, miR-18a/ internal reference U6 was no significant change for expression of p-mTOR ( Figure 6D).
These indicates that miR-18a can attenuate autophagy in Mtbinfected macrophages via regulation of ATM-AMPK pathway. have been proved to be an effective tools for miRNA delivery. 27 In the study, we also investigated miR-18a expression in exosomes derived from macrophages response to Mtb infection. Our data revealed that consistent with the variation tendency in Mtb-infected macrophages, miR-18a in exosomes was also increased.

| D ISCUSS I ON
Macrophages are the first barrier to Mtb infection and its activation such as autophagy formation can decrease intracellular Mtb burden. 28 In this study, we explored the effect of miR-18a on Mtb survival in macrophages. Our results showed that there was a positive correlation between miR-18a expression and Mtb load in macrophages. Next, we further explored the underlying mechanism of miR-18a promotion of intracellular Mtb survival.
Autophagy is one of the effective ways for host cells to defend against Mtb, which can directly eliminate intracellular Mtb, thereby limiting the growth of Mtb. 29 In the study, electron microscopic results exhibited that more autophagosomes were shown in miR-18a inhibitor transfected cells compared with the controls, indicating that miR-18a may be associated F I G U R E 3 Effect of miR-18a on the survival of Mtb in RAW264.7 cells. A, The effect of Mtb survival in RAW264.7 macrophages transfected with miR-18a mimics, NC-mimics, miR-18a inhibitor or NC-inhibitor was measured by CFU analysis. B, The effect of Mtb survival in RAW264.7 macrophages transfected with miR-18a mimics, NC-mimics, miR-18a inhibitor or NC-inhibitor was measured by acid-fast stain. Data represent the means ± SD from three independent experiments. *P < .05, **P < .01. CFU, colony-forming unit F I G U R E 4 Effect of miR-18a on the Mtb-infected RAW264.7 macrophages. A, The viability of RAW264.7 cells was detected by CCK-8. B, The ultrastructure changes of RAW264.7 macrophages were detected by TEM. The number of autophagosomes per cross-sectioned cell was counted (20 cells per group counted by TEM). Data represent the means ± SD from three independent experiments. *P < .05, **P < .01, ***P < .001, # P < .05, ## P < .01, ### P < .001 with autophagy. To further examine the regulatory role of miR-18a in autophagy, we examined the impact of miR-18a on expression of autophagic protein LC3. Underexpression of miR-18a elevated LC3-II level while its overexpression inhibited LC3-II expression, which suggests that miR-18a negatively regulates autophagy. It also led us to further investigate the role of miR-18a in autophagy signalling pathway.
Studies have shown that ATM maintained the autophagy pathway by activating the LKB1/AMPK/TSC2 signalling pathway and suppressing the negative regulator mTORC1. 30 Bioinformatic analysis shown that ATM might be an autophagy-related gene that miR-18a may target. Moreover, we found that there was a negative correlation between ATM expression and miR-18a level in Mtb-infected macrophages. Then, we investigated the relationship between them. Indeed, our data showed that up-regulation of miR-18a suppressed ATM expression, whereas its down-regulation elevated ATM level. Next, we found that down-regulation of ATM suppressed autophagy process in macrophage response to Mtb infection and could reverse autophagy activation elicited by inhibition of miR-18a. These results imply that miR-18a can negatively regulate autophagy by inhibition of ATM.
AMPK is well known as one key signalling molecule which controls autophagy signalling pathway. Hence, we further explored the role of miR-18a in the ATM-AMPK-mTOR autophagy pathway.
Inhibition of miR-18a up-regulated p-AMPK expression while the change could be reversed by down-regulation of ATM. Combined these results, miR-18a could attenuate autophagy in Mtb-infected macrophages by modulation of ATM-AMPK autophagy pathway. F I G U R E 5 miR-18a inhibits autophagy in RAW264.7 macrophages by suppressing ATM. A, The expression of LC3-II in RAW264.7 macrophages transfected with miR-18a mimics, NC-mimics, miR-18a inhibitor or NC-inhibitor followed by Mtb infection. B, The binding sites and matching degree were predicted between miR-18a and ATM by TargetScan analysis. C, The expression of ATM mRNA at different time-points in RAW264.7 macrophages infected with Mtb by RT-PCR. D, Correlation analysis between miR-18a and ATM expression. E, The expression of ATM mRNA in RAW264.7 macrophages transfected with miR-18a mimics, NC-mimics, miR-18a inhibitor or NC-inhibitor followed by Mtb infection by RT-PCR. F, The expression of ATM protein in RAW264.7 macrophages transfected with miR-18a mimics, NCmimics, miR-18a inhibitor or NC-inhibitor followed by Mtb infection by Western blot. G, Observation of ATM immunofluorescence by laser scanning confocal microscope. Data represent the means ± SD from three independent experiments. **P < .01, ***P < .001. Relative ATM expression, ATM/β-actin F I G U R E 6 miR-18a inhibited autophagy in RAW264.7 macrophages by ATM-AMPK pathway. A, The RAW264.7 cells were infected with Mtb or without Mtb and then transfected with ATM siRNA or ATM siRNA NC. The expression of LC3-II in RAW264.7 macrophages was determined by Western blot. B, The RAW264.7 cells were infected with Mtb or without Mtb and then transfected with miR-18a inhibitor or ATM siRNA. The expression of LC3-II in RAW264.7 macrophages was determined by Western blot. C, The RAW264.7 cells were infected with Mtb or without Mtb and then transfected with miR-18a inhibitor or ATM siRNA. The expression of p-AMPK in RAW264.7 macrophages was determined by Western blot. D, The RAW264.7 cells were infected with Mtb or without Mtb and then transfected with miR-18a inhibitor or ATM siRNA. The expression of p-mTOR in RAW264.7 macrophages was determined by Western blot. Data represent the means ± SD from three independent experiments. **P < .01, *P < .05 In short, our study showed that miR-18a expression, including exosomal miR-18a, is induced upon Mtb infection, and miR-18a accelerates Mtb survival in macrophages via negatively regulating autophagic process by inhibiting ATM. To the best of our knowledge, it is the first study to explore the role and mechanism of miR-18a in Mtb infection, and this provide new ideas and theoretical basis for diagnosis of TB and the treatment of TB patients.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare related to this article.

AUTH O R CO NTR I B UTI O N S
YF and ZY designed the study. QY, HC and YY performed the experiments. YF, ZY, QY, HC and YY analysed the data. QY drafted the manuscript. YF and ZY revised the manuscript and supervised the work.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sets and materials used and/or analysed during the current study are available in the manuscript itself.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section.