Tregs suppress self-reactive T cells and inhibit their number and functions, helping to maintain peripheral tolerance and prevent the onset of autoimmune diseases [38, 39]. Tregs suppress the functions of a number of cell types, including CD4 + TH cells, B cells, CD8 + cytotoxic T lymphocytes (CTLs), and antigen-presenting cells(APC), to effectively block immune responses, inflammation, and tissue destruction [40–42].In the present study, alteration in Treg cells' frequency in SLE has been analyzed in the light of miRNAs expression, which possibly influences Tregs.
Our study observed a substantial increase in Treg cells in both active and inactive SLE patients compared to controls, with the most significant increase in active ones. This result agreed with Singla et al.'s(2017)  results, who reported a significant increase in Tregs in childhood SLE and mentioned that active lupus patients had a higher percentage than inactive lupus patients do. A previous study by Suarezet al. (2006) also observed a significant elevation in both active and inactive SLE patients with a maximum increase in inactive ones. In contrast, Kailashiya et al. (2019)found an insignificant difference in Treg cells' percentage in SLE patients. Knowing that corticosteroids (glucocorticoids and cyclophosphamide) were used as a regular treatment of lupus due to their suppressing effect on the immune response, specifically the development of pro-inflammatory cytokines, the observed rise in Tregs in our SLE patient might be returned to the impact of immunosuppressive therapies. These therapeutics have been proven to augment Tregs frequency in several conditions, including lupus [46–49].
Several miRNAs have been discovered to be essential in immune homeostasis. The role of microRNAs in immune cell lineage differentiation and their physiological functions in maintaining normal innate and adaptive responses is well known [50, 51]. Aberrations in the miRNA-mediated immune-cell development and function regulation have been related to autoimmune diseases [52–54]. Intuitively, miRNA dysregulation is one of the main contributors to the collapse of self-tolerance, leading to autoimmunity .
In the current study, there was a lowering in miR-125a expression level in both active and inactive phases of the disease compared to the healthy group. These findings agree with Zhaoet al. (2010)  and Wang et al. (2012) , who reported a reduction in miR-125 level in SLE patients. In T cells isolated from lupus patients, diminished levels of miR-125a had been reported . miR-125a promotes the up-regulation of the inflammatory chemokine RANTES, which is needed for the adverse effects of inflammatory processes. Its deficiency impairs Treg maintenance and immunoregulatory capacity, while over expression of miR-125a stabilizes Treg-mediated self-tolerance .
A significant diminution in miR-24 expression levels was observed in SLE patients, either active or inactive, compared to normal controls. No previous studies have been performed on the change in miR-24 level in lupus patients to the best of our knowledge. Murata et al. (2013) reported an increase in the expression level of miR-24 in Rheumatoid Arthritis (RA) patients. Both miR-24 and miR-125a can play a role in the inflammation's enhancement. Via direct targeting of Furin, miR-24 might control the processing of latent transforming growth factor (TGF-1) , and miR-125 targets the tumor necrosis factor-alpha-induced protein 3 (TNFAIP3) . TGF-β1 plays a suppressive role in immune system regulation. Moreover, furin expression in T-cells is also essential for maintaining peripheral immune tolerance [63, 58].
Our results recorded a significant elevation in miR-146a in active and inactive SLE patients compared to normal controls. Our data agree with Chen et al. (2017)  and Zheng et al. (2017) , who observed over expression of miR-146a in SLE patients. On the other hand, Luo et al. (2011)  reported a reduction in miR-146a expression in lupus patients. The TLR4/NFB signaling pathway is negatively regulated by miR146a, and its down regulation causes inflammatory responses to be activated. Over expression of miR-146a reduced TRAF6 and consequently inhibited the activity of NF‐κB, resulting in simultaneous inhibition of TNF‐α, IL‐1β, and IL‐8 synthesis .
Similar to previously published studies of Chen et al. (2017)  and Shumnalieva et al. (2018)  who found over expression of miR-155 in SLE patients; our data showed a significant increase miR-155 in the peripheral blood of SLE patients with a maximum elevation in inactive patients. This data disagrees with Wang et al. (2012) , who reported an unexpected reduction in the expression level of miR-155 in SLE patients. Over expression of miR-155 contributes to the development of antibodies, irregular T cell differentiation, kidney failure, and lupus-like symptoms [69, 70]. Some miRNAs, such as miR-155, commonly associated with a compromised immune response and increases disease activity, were differentially expressed in multiple autoimmune diseases .
We demonstrated a significant increase in miR-21 was observed in both active and inactive status of the disease compared to healthy controls. In accordance with this data, the study of Wanget al. (2012) on SLE patients pointed to the up-regulation of miR-21 expression in SLE patients. The same observation was previously mentioned by Pan et al. (2010) , who observed a significant increase in miR-21.In accordance, patients with active disease have substantially higher levels of miR-21 in their PBMC than normal subjects and patients with inactive disease . Elevated miR-21 levels promoted CD4 + T cell activation, B cell hyper-responsiveness, and over expression of autoimmune-associated methylation-sensitive genes through repression of DNMT1, PDCD4, or PTEN expression [72, 74, 75]. Besides, the inhibition of miR-21 in CD4 + T cells from SLE patients might reverse T cells' activation [74, 76].
A significant elevation in miR-148a expression levels in SLE patients was observed in the present study, with the maximum increase in the active group. This finding was in agreement with Wang et al. (2012)  and Chen et al. (2017), who observed an increase in miR-148alevel in SLE patients. Moreover, our finding was consistent with Pan et al. (2010), who observed that miR-148a was up-regulated in SLE patients. miR-148a expression was up-regulated in CD4+ T cells from patients with SLE patient. miR-21, miR-126, and miR-148a over-expression resulted in DNA hypomethylation in CD4 + T cells by direct inhibition of DNMT1 protein expression, thus inducing CD4 + T cell activation and secretion of autoimmune-related proteins, such as CD70, CD11a, and LFA-1[72, 77, 76].In females, DNA methylation serves as a housekeeping mechanism for physiological X-chromosome inactivation [78–81].It might be estimated that increased circulating miR-21 and miR-148a, in turn, might also accelerate disease progression through the cell-cell communication processes between these apoptotic bodies, exosomes, and target cells, such as quiescent lymphocytes. Zhang et al. (2020) pointed to the elevation of miR-148a, which is generally associated with the immune response and increases the disease's activity.
In conclusion, we approved the numeric rising in Treg cells' frequency in SLE patients, especially those in an active state. Although, we stressed the idea that these elevated cells might be malfunctioning. Studying the expression of some miRNAs associated with Treg cells pointed to the increase in miR146a, miR155 miR148a, and miR-21, coinciding with the reduction of miR-24.We hypothesized that the increase in miR-21, miR-148a, and miR-155 (Treg positive regulators) accompanied by a decrease of miR-24 (Treg negative regulators) favors the elevation of Treg cells, leading to this observed increase of Treg cell frequency. There is a lack of consensus in the research on the relationship between Treg and rheumatic diseases. The majority of evidence proposes Treg cells' impairment, quantitatively and/or qualitatively. Herein, our results provide a novel insight into Treg-miRNA's role in lupus patients' regulation network.
However, our study has some potential limitations, such as the lack of previous studies on some miRNAs in SLE patients (such as miR-24) and using a sorter to test the function of detected Treg cells. Thus, further studies are needed to confirm our findings. We performed the same research on another important autoimmune disease (RA) to examine our assumption's strength in view of this hope.