Association between active pulmonary tuberculosis and miRNA-146a: A preliminary study from Serbia

Introduction: Tuberculosis (TB) continues to be a significant public health problem. The role of small non-coding RNAs, such as microRNAs (miRNAs), was investigated extensively in Mycobacterium tuberculosis (MTB) infection as well as in a variety of other pathophysiological processes in recent years. It was found that miRNAs act as regulators of both early reaction to MTB infection and in process of adaptation of the host immune cells during latent course of the disease. Molecule miRNA-146a is expressed exclusively in immune cells and it has the most prominent role in modulation of innate immunity. Methodology: We investigated the level of expression of miRNA-146a using an RT-qPCR technique in peripheral blood mononuclear cells of 44 patients with active pulmonary TB and 17 healthy individuals. We also analyzed the significance of miRNA-146a rs2910164 SNV for expression profile of miRNA-146a, in order to investigate potential usage of miRNA-146a as a biomarker for TB. Results: There was statistically significant decrease of expression of miRNA-146a in TB group compared to control group. When gender cohorts were analyzed, the expression levels in TB male and TB female subgroup were significantly lower than the expression levels in the same gender control subgroups. Conclusions: Our results indicate that miRNA-146a plays a significant role in the pathogenesis of TB, suggesting that miRNA-146a could be used as a biomarker for active pulmonary TB.


Introduction
The role of small non-coding RNAs, such as microRNAs (miRNAs), was investigated extensively in a variety of physiological and pathophysiological processes in recent years. Studies showed that the fate of cells during fundamental biological processes such as proliferation, differentiation, and apoptosis is tightly regulated by these specific molecules [1]. Growing data reveal their importance in immune response [2], embryogenesis [3], and tumorigenesis [4]. The regulatory role of various miRNAs in the immune system appears to be significant in both infectious and autoimmune diseases [5,6]. In this respect, they act as susceptibility factors as well as a regulatory component of innate and adaptive immunity [7] and they play a crucial role in bacterial or viral infectious diseases [8,9].
One-third of the human population is infected by Mycobacterium tuberculosis, but due to differences in host immunity, only about 10% of infected individuals actually develop active tuberculosis, while the remaining 90% exhibit latent infection [10]. Immunity in all of its complexity has been extensively investigated in respect to TB. It was found that miRNAs act as regulators of early reaction to MTB infection, but also in the process of host immune cells adaptation during the latent course of infection [7]. Several miRNAs are proven to regulate T cell differentiation and function, and play a critical role in regulating the innate function of macrophages, dendritic cells, and NK cells [7]. Macrophages are the first line of defense against MTB infection, using phagocytosis for the destruction of mycobacteria and initiating an inflammatory response. On the other hand, MTB has adopted several mechanisms to survive within the host cells and attenuate the efficacy of the phagolysosome compartment. Experimental studies showed that MTB is able to control the expression of miRNAs and to use miRNAs for modulation of inflammation in host tissues, thus ensuring its own survival within the host cells [11].
The role of miRNA-146a in TB miRNA-146a is one of the most investigated immunity-related miRNAs. It is exclusively expressed in immune cells, with the most prominent role in the modulation of the innate immunity [12]. It belongs to the group of early-response factors after exposition to various microbial components and proinflammatory mediators [9]. In TB, the expression of miRNA-146a is triggered by MTB-induced inflammation, followed by an escalation of proinflammatory mediators [11]. Early expression of miRNA-146a can be explained by activation of the proinflammatory TLR-TRAF6-IRAK1-NF-κB signaling pathway by MTB [13]. NF-κB binding site has been found in the promoter region of miRNA-146a gene, while the expression of IRAK1 gene is directly negatively regulated by miRNA-146a [9]. The significant role of IRAK1 gene in TB was previously shown [14].
So far, the role of miRNAs in TB was mostly investigated using in vitro model system. Recently, the assessment of circulating miRNAs has emerged as an option for their research. They are represented in sputum, blood, serum, plasma, and other body fluids, which enables their utilization as noninvasive diagnostic and prognostic biomarkers for a plethora of diseases -cancer, autoimmune and infective diseases, including TB [11,15].
To investigate the possibility of using miRNA-146a as a biomarker for TB, here we analyzed its expression level in patients who are in the acute phase of the disease. We also investigated the significance of the miRNA-146a rs2910164 SNV for its expression.

Patients and control subjects
This study was performed on 44 TB patients (21 females, 23 males), with an average age of 58.4 ± 18.2 years. Patients who had diabetes, cancer, and other pulmonary diseases or co-infection with other pathogens were excluded. Study also included 17 healthy individuals (8 females and 9 males, 59.1 ± 8.5 years) free of active or latent TB infection and who did not display any clinical symptoms of any other infectious or noninfectious diseases. They were recruited as controls. Demographic characteristics of TB patients and control group are shown in Table 1.
Informed consent was obtained from all participants and the protocol for the research project was approved by the Ethics Committee of the Clinical Centre of Serbia. We described 44 TB patients having the clinical form of pulmonary tuberculosis. The TB diagnoses were established between October and December 2018 in Clinic for pulmonology, Clinical Centre of Serbia and in the Special hospital for lung diseases Ozren. All patients were HIV-negative, and were receiving antituberculous therapy at the time of blood sampling. All TB patients were drug responders to the first-line treatment (isoniazid, rifampin, ethambutol, pyrazinamide). The exclusion criteria for the healthy participants also included a history of TB and a pulmonary infection. The TB diagnosis was made if acid-fast bacilli had been positive in search and/ or culture in each case.

Blood sampling, DNA preparation, and genotyping
Venous blood was collected into two 4.5-mL sodium citrate anticoagulant tubes (Vacutainer, Becton-Dickinson, Plymouth, UK). Genomic DNA was isolated from whole peripheral blood with QIAampDNA Mini Kit (Qiagen GmbH, Hilden, Germany), and stored at -20 °C until analysis. Analysis of genetic variant in miRNA-146a (rs2910164 C > G) was performed by direct sequencing of PCR product using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on 3130 Genetic Analyzer (Applied Biosystems, USA). The PCR of the miRNA-146a C > G SNP was performed using the following primers to generate a 233-bp DNA product: forward: 5'-GACAGGCCTGGACTGCAAG-3' and reverse 5'-CTGGAGACAGAAGGCAGAGTC-3'.

RNA extraction
Fresh peripheral blood collected on 4.5-mL sodium citrate was used for isolation of peripheral blood mononuclear cells (PBMCs) using Ficoll densitygradient centrifugation (GE Healthcare, Sweden). TRI Reagent Solution (Ambion, USA) was used for total RNA extraction. The quantity of RNA was determined using a Nanodrop spectrophotometer (ThermoFisher Scientific, USA).

Quantification of miRNA-146a expression
Twenty nanograms of total RNA were reverse transcribed using TaqMan miRNA-146a Gene Expression Assays (ThermoFisher Scientific, USA) and miRNA-specific stem-loop primers. Normalization was performed using RNU6B (TaqMan microRNA Control Assay RNU6B, Thermo Fisher Scientific, USA) and TaqMan

Statistical analysis
Genotype frequencies in patient and control groups were tested for the Hardy-Weinberg equilibrium (HWE) using the χ 2 test. The differences in the allele and genotype distributions between patients and controls were performed by Chi-square test by Fisher exact test. The recessive model was used for comparisons between genotypes, where the frequency of homozygous for minor allele was compared to grouped homozygous major allele plus heterozygous genotype. For the relative expression analyses data are presented as medians with range, means ± SD, or as absolute numbers with percentages. Distributions of continuous data were tested with the Shapiro-Wilk and Kolmogorov-Smirnov tests. Differences in these variables were analyzed using a nonparametric Mann-Whitney U test for distribution between two groups. Correlations between variables within the group of TB patients were analyzed using Spearman's rank-order correlation coefficient (r). The statistical analyses were performed using the SPSS computer software 20.0 (SPSS, Inc, Chicago, IL, USA). For all analyses, the p values were 2-tailed, and p < 0.05 was considered statistically significant.

Results
This study included 44 patients with TB and 17 controls. Baseline characteristics of patients are presented in Table 1. Patients and controls were matched by age and gender, and there was no statistical difference in genotypes between two groups.

Association between miRNA-146a rs2910164 C > G variant and susceptibility to TB
Comparison of variant in miRNA-146a gene in TB patients and healthy controls was performed through recessive model. Both patient's and control group were in HWE for miRNA-146a rs2910164 C > G (χ 2 = 0.937, p = 0.645 and χ 2 = 0.006, p = 0.212, respectively). There was no statistically significant difference between TB patients and control group regarding genotype and allele distribution ( Table 2).
Next, the correlation between miRNA146a and age was examined in the whole study cohort (patients and controls, n = 61). No significant relationship between these two variables was observed (r = 0.077, p = 0.575).

Association of genotype variants with miRNA-146a expression level
To investigate the influence of the C and G allele of rs2910164 on the production of the mature form of miRNA-146a, carriers of C allele were compared to carriers of G allele in both TB and healthy control groups. No significant difference was observed between groups.

Discussion
Specific miRNAs have been shown to be associated with pulmonary TB and the first study that identified SNPs in precursor miRNAs was conducted in 2005. New and more effective biomarkers are needed to establish early diagnosis of TB and thus enhance its treatment and prevention.
It has been observed that miRNA-146a plays an important role in MTB infection and in the pathogenesis of pulmonary TB. Several experimental studies have provided evidence that C allele in rs2910164 of miRNA-146a may reduce the production of mature miRNA-146a, which results in modification of the inflammatory process [16]. Although the role of miRNA-146a rs2910164 C > G SNV in the pathogenesis of pulmonary TB is unclear, this SNV may have an impact on the immune response in MTB infection, thereby increasing the incidence of pulmonary TB. miRNA-146a has previously been described as a negative regulator of the immune response and its systemic down-regulation may be associated with an escalated inflammatory response in TB patients [17,18]. SNV rs2910164 C > G , is encoded on chromosome 5q33 and located in pre-miRNA-146a in the precursor stem region, + 60 relatives to the first nucleotide pre-miRNA-146a, as opposed to the mature miRNA-146a sequence [17,19]. This transversion results in C: U mismatch in the structure of the miRNA-146a precursor, which may reduce the stability and processing efficiency of pre-miRNA, and decrease the expression of pre-miRNA-146a and mature miRNA-146a [20].
Three studies described an association between rs2910164 C > G in miRNA-146a and TB. One study was conducted in the Kazak population [16], another in the Tibetan/Han population [17], and a third in the Chinese Han population [18].
Zhang et al. investigated the potential link between the four precursors of miRNA SNPs (miRNA-146a rs2910164 C > G, miRNA-149 rs2292832 T > C, miRNA-196a2 rs11614913 T > C and miRNA-499 rs3746444 T > C) and susceptibility to pulmonary tuberculosis in Chinese Uygur, Kazak, and South Han. The frequencies of alleles and genotypes of miRNA-146a differed significantly between these two groups in the Kazak population. miRNA-146a SNP has been found to be associated with pulmonary tuberculosis using codominant, recessive, and additive models [16].
Li et al investigated the association of miRNA-146a rs2910164 G > C SNV with susceptibility to pulmonary tuberculosis in Chinese Tibetan and Han populations. An association with the risk of pulmonary TB in both the Tibetan and Han populations was found [17]. They speculated that this was the main reason for differences in different ethnic groups.
Wang Min et al. did not repeat previous significant discoveries in the Chinese Han population, nor did they reveal an association between miRNA-146a and pulmonary tuberculosis.
The results of these three studies suggest that susceptibility to pulmonary TB may be closely related to individual differences caused by genetic factors among different ethnic groups in China. The gene-gene interaction is often combined with genetic and environmental factors and makes the perceived difference more complicated between different ethnic groups.
Yuksen Fatma et al. also determined the diagnostic potential of miRNA-146a in patients with active TB. Their results suggest that miRNA-146a may distinguish active pulmonary TB from healthy controls based on data provided from normalization with miRNA-93 in Turkish population [24].
In our study, we found that there was a statistically significant difference in miRNA-146a expression level between TB and control groups, with values higher in healthy controls. MiRNA-146a levels compared between CC/GC and GG genotypes showed no significant influence on the production of the mature form of miRNA-146a.
The expression level of miRNA-146a in our study was higher in males than females, but without statistical significance. Comparison between males in TB and males in control group, as well as the same comparison between females, revealed significantly lower expression in TB subgroups, which indicates a common mechanism in both sexes [25,26].
The main limitation of this work is the small sample size. Since this is the first study in Serbia that investigated active pulmonary tuberculosis in correlation to miRNA-146a, the results presented here are a valuable and open new paradigm for using small noncoding RNAs as potential biomarkers and diagnostics, prognostics, and stratification tools in the future.

Conclusions
We believe that miRNA research will provide a new method of testing as well as understanding the pathogenesis and treatment of pulmonary TB. Despite the above limitations, this is the first preliminary study in Serbia to examine changes in miRNA-146a levels in patients with tuberculosis. We found that miRNA-146a may be a suitable candidate for further research as a potential biomarker for TB.