The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

Mycobacterium tuberculosis (M. tb) is the causative agent of tuberculosis (TB) that leads to millions of deaths each year. Extensive evidence has explored the involvement of microRNAs (miRNAs) in M. tb infection. Limitedly, the concrete function of microRNA-100-5p (miR-100-5p) in M. tb remains unexplored and largely elusive. In this study, using Bacillus Calmette–Guérin (BCG) as the model strain, we validated that miR-100-5p was significantly decreased in BCG-infected THP-1 cells. miR-100-5p inhibition effectively facilitated the apoptosis of infected THP-1 cells and reduced BCG survival by regulating the phosphatidylinositol 3-kinase/AKT pathway. Further, SMARCA5 was the target of miR-100-5p and reduced after miR-100-5p overexpression. Since BCG infection down-regulated miR-100-5p in THP-1 cells, the SMARCA5 expression was up-regulated, which in turn increased apoptosis through caspase-3 and Bcl-2 and, thereby, reducing BCG intracellular survival. Collectively, the study uncovered a new molecular mechanism of macrophage to suppress mycobacterial infection through miR-100-5p and SMARCA5 pathway.


Introduction
Mycobacterium tuberculosis (M. tb) is the leading etiological agent of tuberculosis (TB) in humans, with 9.9 million new cases and 1.5 million mortalities in 2020 worldwide [1]. Although one-third of the population has been infected [2], only about 10% possibly become active infection. In the other 90% of the latently infected population, this pathogen remains dormant by escaping clearance of host cells but could recover to be active form and cause TB at an unpredictable time. Therefore, it is ideal to clear the intracellular M. tb. Macrophages are the main host cells of M. tb. Although it is known that the macrophages can develop various strategies to kill M. tb, such as modulating cell death programs, regulating inflammatory responses, and inducing autophagy, during M. tb infection [3,4], M. tb can persist in the cells by overcoming the microbicidal mechanisms of macrophages. Therefore, more studies are needed to dissolve the mystery of M. tb intracellular survival.
Apoptosis is one of the important strategies of host resistance to M. tb and response to bacilli survival and transmission [5,6]. Studies have shown that host-mediated apoptosis leads to apoptosis and reduce the number of bacteria and the effective cross-presentation of the bacterial antigen [7,8]. When infected with mycobacteria, attenuated virulent strains, such as Bacillus Calmette-Guérin (BCG) and M. tb H37Ra, can better induce apoptosis than strong strains [9].

Cell Infection with BCG
Bacterial cultures at the logarithmic growth stage were centrifuged at 3800× g for 10 min. The precipitates were collected and resuspended in Hank's balanced salt solution (Gibco) and dispersed into a single-cell suspension. The concentrations were estimated by OD 600 values [20,21]. THP-1 cells were infected with BCG at a multiplicity of infection (MOI) of 10 for 8 h [21,22], followed by washing with 1 mL of 10% FBS medium plus 100 µg/mL gentamycin. This time point was set as 0 h. The samples were collected at the designated time points.

Total RNA Extraction and Sequencing
Total RNA was extracted and sequenced as previously described [23]. Briefly, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the instructions of the manufacturer. RNA purity and integrity were checked by agarose gel electrophoresis and quantified by determining the absorbance of A260 on SmartSpec (Bio-Rad, Hercules, CA, USA). A Balancer NGS Library Preparation Kit for small RNA/miRNA (GnomeGen, San Diego, CA, USA) was used for small RNA cDNA library preparation, in accordance with the instructions of the manufacturer. The purified small RNA libraries were quantified for cluster generation and 36 nt single-end sequencing analysis using Illumina GAIIx (Illumina, San Diego, CA, USA).

Quantitative Real-Time Polymerase Chain Reaction
The miR-100-5p and SMARCA5 expression was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The concentration was detected by NanoDrop 2000 (Thermo Fisher, Waltham, MA, USA). Total RNA (1 µg) was used to synthesize cDNA using the All-in-One TM miRNA qRT-PCR Detection Kit (for miRNA; Vazyme, Nanjing, China) or HiScript ® II qRT SuperMix (for mRNA; Vazyme). After reverse transcription, qRT-PCR was used to detect miRNA and mRNA expression by AceQ qPCR SYBR Green Master Mix on an ABI ViiA 7 Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA). miRNA was normalized to U6 and mRNA was normalized to β-actin. The 2 −∆∆Ct method was used to calculate the relative expression. All primers are listed in Table S1.

Cell Death Assay
Cell death was tested by assaying the medium lactate dehydrogenase (LDH) content by the LDH Release Assay Kit (Beyotime, Shanghai, China). At the designated time points, the medium was incubated with a detection reagent in the dark for 30 min and LDH values were recorded at OD 450 .

Apoptosis Assay
Apoptosis was evaluated using the Annexin V-Fluorescein Isothiocyanate/Propidium Iodide (PI) Apoptosis Detection Kit (Vazyme) following the manufacturer's instructions. Briefly, the supernatant was discarded, and cells were washed once with precooled phosphatebuffered saline (PBS; HyClone). Cells were digested with trypsin without EDTA. The precipitates were collected by centrifugation at 800× g for 5 min at 4 • C. Cells were washed using PBS, centrifuged again, and resuspended using 1× binding buffer. Finally, cells were stained using PI and Annexin V in the dark. Flow cytometry (CytoFLEX; Beckman Coulter, Brea, CA, USA) was used to analyze the stained cells.

Assay on Intracellular Bacterial Number
THP-1 cells were infected with BCG at an MOI of 10, as described previously, to assess bacterial survival in vitro [21]. THP-1 cells were lysed with 1 mL sterile distilled water at the indicated time points. Tenfold serial dilutions of the lysates were plated on 7H11 agar plates and the colony-forming unit (CFU) was counted 21 days later.

Statistical Analysis
Data were the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Student's t-test for one comparison and analysis of variance (ANOVA) for more than one comparison in GraphPad Prism version 7.0. The p < 0.05 was determined to be statistically significant difference, and p < 0.05, 0.01, and 0.001 were expressed as *, **, and ***, respectively, in figures.

miR-100-5p Promoted BCG Intracellular Survival via Suppression of Apoptosis
BCG number was assessed in vitro to investigate whether miR-100-5p-dependent repression apoptosis enhances BCG survival. THP-1 cells were infected with BCG for 48 h at 24 h after transfection of miR-100-5p mimic or control mimic. Compared with the control mimic group, the miR-100-5p mimic group had significantly higher CFU (p < 0.001) counts ( Figure 5A) and the CFU counts in the miR-100-5p inhibitor group were significantly less than in the control group (p < 0.01; Figure 5B). A ratio analysis based on 0 h also proved the above results ( Figure 5C), indicating that miR-100-5p can promote BCG intracellular survival in macrophages.

miR-100-5p Promoted BCG Intracellular Survival via Suppression of Apoptosis
BCG number was assessed in vitro to investigate whether miR-100-5p-dependent repression apoptosis enhances BCG survival. THP-1 cells were infected with BCG for 48 h at 24 h after transfection of miR-100-5p mimic or control mimic. Compared with the control mimic group, the miR-100-5p mimic group had significantly higher CFU (p < 0.001) counts ( Figure 5A) and the CFU counts in the miR-100-5p inhibitor group were significantly less than in the control group (p < 0.01; Figure 5B). A ratio analysis based on 0 h also proved the above results ( Figure 5C), indicating that miR-100-5p can promote BCG intracellular survival in macrophages.

miR-100-5p Directly Targets SMARCA5
A series of transcripts were found as potential targets of miR-100-5p using the base alignment approach. Accordingly, seven and 25 genes were predicted by five and four software, respectively ( Figure 6A). Among them, SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 were chosen for the next step; all of them were found to have specific binding sites for miR-100-5p in its 3 -UTR ( Figures 6B and S2A). Luciferase reporter constructs were generated by cloning either WT or mut 3 -UTR of targets into a pSicheck vector to confirm whether they are direct targets of miR-100-5p. These plasmids were co-transfected with miR-100-5p mimic into HEK 293T cells and the lysates were analyzed 24 h later. Transfection with miR-100-5p mimic markedly inhibited the luciferase activity for the WT 3 -UTR of all five genes (p < 0.01) but showed no significant repressive effects on the mutated 3 -UTR of genes compared to control double-stranded RNA (Figures 6C and S2B). These results suggested that miR-100-5p may suppress SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 expression by binding to the 3 -UTR in a direct and sequence-specific manner.

miR-100-5p Directly Targets SMARCA5
A series of transcripts were found as potential targets of miR-100-5p using the base alignment approach. Accordingly, seven and 25 genes were predicted by five and four software, respectively ( Figure 6A). Among them, SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 were chosen for the next step; all of them were found to have specific binding sites for miR-100-5p in its 3′-UTR ( Figures 6B and S2A). Luciferase reporter constructs were generated by cloning either WT or mut 3′-UTR of targets into a pSicheck vector to confirm whether they are direct targets of miR-100-5p. These plasmids were cotransfected with miR-100-5p mimic into HEK 293T cells and the lysates were analyzed 24h later. Transfection with miR-100-5p mimic markedly inhibited the luciferase activity for the WT 3′-UTR of all five genes (p < 0.01) but showed no significant repressive effects on the mutated 3′-UTR of genes compared to control double-stranded RNA (Figures 6C and  S2B). These results suggested that miR-100-5p may suppress SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 expression by binding to the 3′-UTR in a direct and sequence-specific manner. SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 mRNA levels were much higher in BCG-infected cells than in their representative controls (Figures 6D and S2C). THP-1 cells were transfected with miR-100-5p mimic or inhibitor and infected with BCG to examine the effects of miR-100-5p on SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 expression during BCG infection. However, only SMARCA5 showed stable results. Treatment with miR-100-5p mimic significantly inhibited SMARCA5 at both mRNA and protein levels, whereas miR-100-5p inhibitor markedly enhanced SMARCA5 at mRNA and protein levels in BCG-infected macrophages ( Figure 6E-H). These results suggested that Binding site of miR-100-5p with SMARCA5 WT or mut 3 -UTR as predicted by online prediction software. (C) Dual-luciferase reporter assay performed in HEK 293T cells. miR-100-5p mimic or NC mimic was cotransfected with dual-luciferase reporter plasmids, such as psiCHECK2 control, psiCHECK2-SMARCA5-wt, and psiCHECK-SMARCA5-mut. The ratio represents the Renilla to firefly (Rluc/Fluc) ratio in miR-100-5p mimic versus NC mimic. (D) Differential SMARCA5 expression between BCG-infected (MOI = 10) and uninfected cells measured by qRT-PCR at 0, 12, and 24 hpi. (E,F) THP-1 cells were transfected with NC mimic, miR-100-5p mimic, NC inhibitor, or miR-100-5p inhibitor for 24 h and infected with BCG at an MOI 10 for 8 h. SMARCA5 mRNA expression was detected by qRT-PCR at 12, 24, and 48 hpi. (G-H) SMARCA5 protein expression detected by Western blotting assay. The expression ratio was analyzed using ImageJ with normalization to β-actin. Data are from three independent experiments with biological duplicates in each ((C-F); mean ± s.e.m. of n = 3 duplicates) or representative of three independent experiments ((G,H), mean ± s.e.m). * p < 0.05; ** p < 0.01; ***p < 0.001. SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 mRNA levels were much higher in BCG-infected cells than in their representative controls (Figures 6D and S2C). THP-1 cells were transfected with miR-100-5p mimic or inhibitor and infected with BCG to examine the effects of miR-100-5p on SMARCA5, MTMR3, HS3ST3B1, EPDR1, and TRIB2 expression during BCG infection. However, only SMARCA5 showed stable results. Treatment with miR-100-5p mimic significantly inhibited SMARCA5 at both mRNA and protein levels, whereas miR-100-5p inhibitor markedly enhanced SMARCA5 at mRNA and protein levels in BCG-infected macrophages ( Figure 6E-H). These results suggested that miR-100-5p expression suppresses SMARCA5 expression during BCG infection.

miR-100-5p Regulates Apoptosis and BCG Survival in THP-1 via SMARCA5
THP-1 cells were transfected with miR-100-5p inhibitor or siRNA SMARCA5 and infected with BCG for 24 h to determine whether miR-100-5p regulates apoptosis and BCG intracellular survival by targeting SMARCA5. Compared to the NC inhibitor + control siRNA control group, SMARCA5 expression was remarkably reduced in the NC inhibitor + siS-MARCA5 group (p < 0.01) and increased in the miR-100-5p inhibitor + control siRNA group (p < 0.001). SMARCA5 levels were significantly lower in the miR-100-5p inhibitor + siS-MARCA5 group than in the miR-100-5p inhibitor + control siRNA group (p < 0.001; Figure 7).  Based on the SMARCA5 interference model, LDH release assay, flow cytometry, and Western blotting were performed to detect apoptosis and CFU assay was performed to detect intracellular bacterial load.

Discussion
In this study, we found that miR-100-5p regulates SMARCA5 and, thus, mediates cell apoptosis and BCG survival in cells. M. tb is a persistent intracellular pathogen that survives within the hostile microenvironment of the macrophage. During the infection, host cells triggers a series immunity responds to destroy the pathogen and clear the debris including apoptosis [24], which prevents the bacterial release and the spreading of mycobacterial infection and activating the host's innate and adaptive immune response [25].
miR-100-5p is involved in multiple diseases and affects apoptosis in previous studies. In chordoma cells, miR-100-5p suppresses proliferation and induces apoptosis via insulinlike growth factor-I receptor [26]. Oncogenic miR-100-5p is associated with cellular viability, migration, and apoptosis in renal cell carcinoma and can be used as a diagnostic biomarker for renal cell carcinoma [27]. In goat endometrial interstitial cells, miR-100-5p can inhibit cell proliferation and promote apoptosis through its target HOXA1 and the molecular sponge circ-9110 [28]. Cortical neurons can elicit cell-autonomous apoptosis through miR-100-5p-induced TLR activation and contribute to neurodegeneration [15]. In the current study, we demonstrated a sharp low level of miR-100-5p in BCG-infected THP-1 cells and identified that miR-100-5p also regulate apoptosis in M. tb infection. This is in agreement with the function of miR-100-5p in other diseases.

Discussion
In this study, we found that miR-100-5p regulates SMARCA5 and, thus, mediates cell apoptosis and BCG survival in cells. M. tb is a persistent intracellular pathogen that survives within the hostile microenvironment of the macrophage. During the infection, host cells triggers a series immunity responds to destroy the pathogen and clear the debris including apoptosis [24], which prevents the bacterial release and the spreading of mycobacterial infection and activating the host's innate and adaptive immune response [25].
miR-100-5p is involved in multiple diseases and affects apoptosis in previous studies. In chordoma cells, miR-100-5p suppresses proliferation and induces apoptosis via insulinlike growth factor-I receptor [26]. Oncogenic miR-100-5p is associated with cellular viability, migration, and apoptosis in renal cell carcinoma and can be used as a diagnostic biomarker for renal cell carcinoma [27]. In goat endometrial interstitial cells, miR-100-5p can inhibit cell proliferation and promote apoptosis through its target HOXA1 and the molecular sponge circ-9110 [28]. Cortical neurons can elicit cell-autonomous apoptosis through miR-100-5p-induced TLR activation and contribute to neurodegeneration [15]. In the current study, we demonstrated a sharp low level of miR-100-5p in BCG-infected THP-1 cells and identified that miR-100-5p also regulate apoptosis in M. tb infection. This is in agreement with the function of miR-100-5p in other diseases.
As reported, miR-100-5p has many targets in different diseases, such as mTOR [19], IGF1R [26], BMPR2 [29], and SMARCA5 [30]. To our knowledge, the function of miR-100-5p and SMARCA5 in mycobacterium have not previously been reported. Here, we found that SMARCA5 was regulated by miR-100-5p using bioinformatics prediction and a dualluciferase reporter system during M. tb infection. SMARCA5 is an ATPase from the ISWI subfamily and acts as a molecular motor for assembling and sliding nuclear complexes of nucleosomes of basic chromatin subunits. The complex containing SMARCA5 can guide the transcription of ribosomes in NORC and B-WICH complexes and participate in the DNA repair mechanism to coordinate the formation of the high-order chromatin structure of centromeres and chromosomes [31]. Previous studies reported that SMARCA5 can affect the cell apoptosis process [32,33], lack of SMARCA5 reduced the development of thymocyte DN3 stage and proB stage of early B cells, and increased cleaved caspase-3 to promote apoptosis [31]. In this study, through cotransfection miR-100-5p and siSMARCA5, miR-100-5p negatively regulated SMARCA5 and affected apoptosis by targeting SMARCA5. This was in agreement with the previous studies. Meanwhile, SMARCA5 could also regulate DNA damage [34], which can lead to growth arrest and apoptosis [35,36]. Therefore, it was speculated that apoptosis regulated by miR-100-5p through SMARCA5 may be related to DNA damage caused by pathogen infections.
Besides apoptosis, miRNAs can also affect bacterial intracellular survival. In M. tb infection, miR-27a induction can inhibit autophagy from promoting the intracellular survival in macrophages [37]. This study also found that miR-100-5p can lead to the accumulation of intracellular bacterium. When infected with BCG, the bacterium tried to establish a persistent infection. As the host cells began their defense system to break the persistent infection, it induced apoptosis and decreased the intracellular ability of BCG by inhibiting miR-100-5p.
PI3K/AKT signaling pathway plays a major role in cell metabolism, proliferation, and apoptosis [38]. The activated PI3K/AKT pathway can increase the activity of cytochrome c oxidase to inhibit apoptosis [39]. miRNA-regulated apoptosis through the PI3K/AKT pathway has attracted extensive attention previously, such that miR-107 aggravates apoptosis by inactivating the PI3K/AKT signaling pathway by targeting FGF9 [40]. miR-100 overexpressed in osteosarcoma could decrease IGF receptor expression and inhibit PI3K/AKT signal transduction and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways from promoting apoptosis [41]. This study identified that PI3K and AKT could be phosphorylated to activate the PI3K/AKT pathway by overexpressing miR-100-5p. However, the detailed mechanism of the miR-100-5p-suppressed PI3K/AKT pathway in regulating apoptosis remains unclear. Insufficient understanding of how miR-100-5p is downregulated in BCG-infected macrophages was also noted. These are worthy of further exploration.

Conclusions
These findings indicated that, after BCG infection of THP-1 cells, miR-100-5p was downregulated and targeted to SMARCA5 and inactivated the PI3K/AKT pathway, resulting in apoptosis and ultimately reducing BCG intracellular survival in macrophages ( Figure 9). This finding offers a new insight of further understanding of host defense mechanism and supports the development of host-directed anti-TB therapeutic approaches. Figure 9. Pattern of miR-100-5p-mediated apoptosis to clear BCG in macrophages. miR-100-5p was downregulated when THP-1 cells were infected with BCG, promoting SMARCA5 expression and inhibiting the PI3K/Akt signaling pathway. Thus, caspase-3 expression increased, resulting in cell apoptosis. BCG intracellular survival was reduced finally.