The miR-125a-3p is Associated With Leprosy Phenotypes and Correlated With Bacillary Index

Background. Mycobacterium leprae infects skin and peripheral nerves causing a broad of clinical forms. Data have shown that the miR-125a-3p can inuence immune mechanisms such as autophagy as well as to target genes leading to abnormal proliferation, metastasis, and invasion of cells. Methods. Here we used quantitative real time PCR (qPCR) to evaluate the miR-125a-3p expression pattern as a marker for leprosy phenotypes in biopsies obtained from patients with and without reactions. Data were analysed according to clinical forms and bacillary index (BI). Results. Our results show a signicant increase in the miR-125a-3p expression in paucibacillary (PB) vs multibacillary (MB) (p = 0.007) and vs RR (reversal reactions) (p = 0.005), respectively; and also a higher expression in patients with erythema nodosum leprosum (ENL) vs MB without reactions (p = 0.002). In addition, there was a positive correlation between the miR-125a-3p expression and BI in patients with reactions (r=0,81; p=0,002).


Background:
Leprosy is a spectral disease, comprising a variety of clinical presentations ranging from the tuberculoid to lepromatous poles, which correspond respectively to the paucibacillary and multibacillary forms of the WHO classi cation [1]. In 1966, Ridley and Jopling created a classi cation that comprise the wide different clinical manifestations: indeterminate, tuberculoid, borderlines or lepromatous. Additionally, circa of 30-40% of patients develops sudden and acute in ammatory episodes as so-called reactions. Type 1 or RR present clinically with neuritis or erythema or edema of the skin lesions; type 2 or ENL, involves the appearance of subcutaneous nodules and symptoms such as fever, arthritis, neuritis, among others [2]. MicroRNAs are well-known small noncoding RNA molecules that control gene expression by interacting with mRNAs in practically all biological processes. By base-pairing to mRNAs, miRNAs can inhibit the translation or increase mRNA turnover [3]. Previous studies have shown that the expression levels of both miR-125a-3p/5p can alter host macrophages pro les [4] and that the miR-125a-3p can inhibit host defenses against mycobacterial infection by targeting the gene encoding autophagy UV radiation-resistance-associated protein [5]. Together, these data suggest that miR-125a may inhibit innate macrophage responses by regulating macrophage differentiation, in ammation, and autophagy [6]. Here we investigate whether miR-125a-3p are associated with leprosy phenotypes through its expression in skin biopsies taken from patients with and without reactions.

Methods:
Twenty-eight skin biopsies from leprosy patients (7 PB, 7 MB, 6 RR and 8 ENL), both sex, age mean 45.89 ± 16.30, were collected before treatment at the outpatients leprosy clinics at the Hospital Universitário Tissue RNA was obtained by the TRIzol® RNA isolation method. Gene expression of miR-125a-3p was performed using a pre-designed assay (miRCURY LNA SYBR, QIAGEN) following manufacturer's instructions and using the 7500 Real Time PCR System device (Applied Biosystems®).

Results:
The U6snRNA, miR-146a-5p and miR-511-3p normalizers were identi ed using the RefFinder program (https://www.heartcure.com.au/re nder/) to validate the qPCR. Statistical data regarding biopsies were generated by Welch's t test and for the analysis of bacillary index the Spearman's Correlation test, using GraphPad Prism8. Our analysis showed a higher expression of miR-125a-3p in the PB group as compared to MB (p = 0.007) and RR groups (p = 0.005), respectively; and also, a higher expression in the group of patients with ENL as compared to MB without reactions (p = 0.002). However, there was no difference by comparing all patients without reactions (PB + MB) versus patients with reactions (RR + ENL) as shown in Table 1, (Figure 1). Additionally, we also observed a positive correlation between the miR-125a-3p expression and bacillary index (BI) in patients with reactions ( Figure 2).

Discussion:
Members of Bcl-2 family and others involved in apoptosis are an important group of miR-125 targets [7].
In lesions of LL patients, a negative regulation of all miRNAs that act directly on the anti-apoptotic genes such as BCL2 and MCL1 was observed [8].Consistent with this, in another study with untreated patients, a low detection of apoptosis and abundant expression of BCL2 was observed in lepromatous (LL) samples where both BCL2 and MCL1 genes were induced by M. leprae in monocytes [9,10]. These data suggest that in LL patients there is a negative regulation of apoptosis favoring the persistence and multiplication of bacilli in macrophages as one of the mechanisms in favour of M. leprae [9]. In addition, the miR-125 family also targets ERBB2 [11], a gene that promotes the binding of M. leprae to myelinated Schwann cells through the Erk1/2 signaling pathway, which results in Schwann cell demyelination and creates an environment that contributes M. leprae proliferation, leading to nerve damage [12]. Taken together, it can be thought that the greater expression of miR-125a-3p in PB biopsies that we observed may be a factor that contributes to a more e cient apoptotic response characteristic of this clinical form, contributing to a milder disease phenotype. Interestingly, miR-125a-3p was also more expressed in individuals with ENL, when compared to MB individuals without reactions. Similarly, Cleverson et al., 2017, identi ed a differential expression of miR-125b-2, in the type 2 reactions [13]. However, the mechanism by which miR-a model using M.tuberculosis (Mtb), it has been shown that increased expression of miR-125a-3p inhibits the activation of autophagy and antimicrobial effects against Mtb via TLR2 and MyD88 in macrophages [14].The recruitment of MyD88 or TRIF adapter molecules by TLR after ligand engagement is crucial for the activation of a variety of in ammatory mediators in macrophages after mycobacterial infection [15]. In another study associated with human cancers, it was observed that miR-125a increases the invasive potential in urothelial carcinomas [16]. Data such as these are relevant, as they call attention to mechanisms linked to processes of dissemination and metastasis that could justify the action of this miR in ENL, where the disseminated deposits of M. leprae antigens resulting in a systemic disease. Coherent with this, we also observed that in patients with RR and ENL there was a positive correlation between the IB and the expression of miR-125a-3p. ENL is a deployment of leprosy that has a distinct immune response in relation to other clinical forms. The role of miR-125a-3p in this case might be unlike PB considering that different triggers could be driving the scene. Therefore, there are two possible explanations: a) The increase in miR-125a-3p expression in ENL would inhibit mechanisms of autophagy, promoting metastasis that in turn mirrors the correlation with a high bacillary index; b) Alternatively, this correlation would re ect an increase in miR-125a-3p as an "attempt" to regulate apoptosis and contain the spread of the bacillus. Members of the miR-125 family have been validated to act either as diseasesuppressing or disease-promoting functions in certain contexts such as tumor metastasis and infections.
It seems that the role played by this miRNA in PB and ENL are in fact distinct. The information provided in this article opens the perspective for a functional study that might help elucidate mechanisms to validate the miR-125a-3p as a possible therapeutic target for leprosy treatment.

Conclusion:
This work underpins the role of miRNAs with immunological and clinical parameters of leprosy, reinforcing that epigenetic mechanisms underlying the disease regulates its pathogenesis. Functional studies will be important to validate the ndings described here.

Declarations
Ethics approval and consent to participate: The study has been approved by the Ethical Committee of the Hospital Universitário Prof. Edgar Santos, Federal University of Bahia, number 2.149.677. All patients read and signed an informed consent form.

Consent for publication:
Not applicable.
Availability of data and material: The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests:
The authors declare that they have no competing interests.  Tables   TABLE 1 miR-125a-3p differential expression in leprosy skin lesions Values expressed in logfc, analyzed by Welch's t test. Figure 1 Normalized log2 expression of miR-125a-3p in leprosy patients. The logFC = log fold change, corresponds to a direct measure of how many times a gene is more or less expressed between groups.

Figures
Data were analyzed using the Welch t test, through the R program, and considered signi cant if P < 0.05.

Figure 2
Positive correlation analysis between the bacillary index and the relative expression of miR-125a-3p in biopsy samples of reactional patients (RR + ENH). The data were analyzed by the Spearman Correlation test, using the GraphPad Prism software, where r ≥ 0.7 was considered. Values of p <0.05 was considered statistically signi cant.