LncRNAs appear to be a likely biomarker for the prediction and diagnosis of disorders, because some complicated diseases, such as diabetes mellitus, demonstrate a dysregulated expression. It has been revealed that type 2 diabetes is 12 times more likely in people with a lower expression of lncRNA GAS5 [22, 23]. Moreover, the relative expression of lncRNA KCNQ1OT1 was significantly higher in the T2D group than in the control group [24]. Ji et al., also reported an elevated expression profile of lncRNA NR2F1-AS1 in uncomplicated T2D patients [25]. On the other hand, glucose homeostasis maintained by pancreatic β-cells through insulin secretion indicates the pivotal role of β-cells. Research shows that NFATs can regulate the development and proliferation of β-cells in human and mouse islets. Moreover, among all isoforms of NFATc, NFATc3 has the highest expression in islets, which reflects the underlying role of this marker in β-cells function and blood sugar balance [20, 26].
In the current study, lncRNA NRON and its target genes, including NFATc3 were selected to evaluate their association with the development of T2DM. As far as we are concerned, there is a paucity of research on expression regulation of lncRNA NRON and NFATc3 in Type 2 diabetes mellitus. Herein, the mRNA transcript level of lncRNA NRON was higher in the patient group than in the healthy group. In contrast, qRT-PCR and Western blotting displayed a decreased expression of NFATc3 gene and protein in T2DM. Hu et al., induced diabetes in mice fed with a high-fat diet and then examined changes in NFATc3 expression. The results revealed that NFATc3 expression levels were downregulated in pancreatic β-cells of HFD-fed mice, which may be associated with insulin secretion impairments and diabetes [26]. Li et al., studied the association between lncRNA NRON and NFATc3 in atrial fibrosis. They found that the overexpression of lncRNA NRON, by inhibiting NFATc3 nuclear transport, can suppress the expression of miR-23a, leading to the alleviation of myocardial fibrosis [27]. Previous research demonstrated that NRON, by blocking the nucleation of NFATc3, inhibited interleukin (IL)-12 expression and transcription in myocardial cells, which contributes to the weakening of atrial fibrosis [28]. Furthermore, the results of correlation found in our study showed that lncRNA NRON upregulation was associated with NFATc3 downregulation, and positively correlated with HbA1c, FPG, HDL-C. Wang et al., implicated a correlation between three upregulated lncRNAs and metabolic variables, including FPG, HbA1c, BMI, 2hPG, and HOMA-IR [29]. Jiang et al., reported an association between two candidate lncRNAs (lnc-HIST1H2AG-6 and lnc-AIM1-3) and the levels of FIN, FPG, HOMA-B and HbA1c-B [30]. In addition, a negatively correlation was observed between NFATc3 expression and HbA1c, FPG. Another study has demonstrated the significant association of NFATc1 with metabolic indices [31].
We used bioinformatic tools to further validate the present study in terms of screening the target genes of lncRNA NRON involved in T2DM development. One of the NRON targeted genes predicted to be associated with this process, was NFATc3. The results of bioinformatic analysis showed that the target genes of lncRNA NRON can play a significant role in T2DM incidence and development through different pathways and mechanisms (Fig. 5a). One of the noticeable pathways is the Wnt signaling pathway, which is vital for the activation of JNK, TCF7L2 variants, and gene expression caused by β-catenin/TCF7L2, as well as calcineurin/NFAT. As such, it is significantly correlated with T2DM and related syndromes [32]. Other roles identified for NRON target genes are Th2 and Th1 cell differentiation. The balance between Th1 and Th2 cell differentiation is important for the development and progression of type 2 diabetes, and while an imbalance in favor of Th1 cells promotes inflammation and insulin resistance, an imbalance in favor of Th2 cells provokes adipose tissue dysfunction and insulin resistance [33]. AGE-RAGE signaling pathway is also one of the predicted pathways for NRON target genes. This pathway contributes to the incidence and progression of type 2 diabetes by accumulating advanced glycation end products, which can activate the receptor for AGEs and trigger damaging cellular responses that are associated with impaired glucose tolerance, insulin resistance, inflammation, and microvascular complications [34]. Another primary function is tumor necrosis factor (TNF) signaling pathway, which has a major role in type 2 diabetes pathogenesis. A cytokine released by macrophages and adipocytes, TNF is linked to the growth of insulin resistance and glucose intolerance. TNF signaling pathway paves the way for the development of type 2 diabetes by provoking inflammation, insulin resistance, oxidative stress, and pancreatic β-cell dysfunction [35]. Circadian rhythm was also identified as a central related mechanism. Gubin et al., reported that the disruptions in the normal circadian rhythm can be linked to the development of type 2 diabetes as it regulates the timing of biological processes, including insulin sensitivity and glucose metabolism [36]. Mitophagy is also one of the predicted functions for the targeted genes. Hippo signaling pathway is another function that involves NRON target genes. It has been shown that the Hippo signaling pathway regulates pancreatic beta-cell growth, insulin secretion, and glucose homeostasis. Hence, the dysregulation of this pathway can lead to reduced beta-cell proliferation, insulin secretion, impaired glucose tolerance, and therefore, incidence of type 2 diabetes. This pathway is also involved in regulation of insulin signaling, and its dysregulation can prompt inflammation, oxidative stress, and apoptosis, all of which are contributing factors of Type 2 diabetes [37]. Our data displayed the role of NRON target genes in the estrogen and prolactin signaling pathways. These pathways are crucial in regulating glucose metabolism and insulin sensitivity and influence the risk of type 2 diabetes. Estrogen signaling improves glucose uptake and lipid metabolism, while prolactin signaling stimulates insulin secretion and improves glucose uptake. However, the dysregulation of these pathways, due to diminished estrogen levels and consistently elevated levels of prolactin, can give rise to insulin resistance and development of type 2 diabetes [38, 39]. In this regard, thyroid hormone signaling pathway has also been observed. TH levels have been shown to play a crucial role in the development and progression of insulin resistance. Research shows that the TH signaling pathway can directly affect insulin signaling pathways and glucose metabolism. Suboptimal thyroid hormone levels have been linked to insulin resistance and may contribute to the incidence of type 2 diabetes [40].
Informed by our results and findings of previous studies, it can be concluded that lncRNA NRON and its target biomarkers including NFATc3, are fundamentally involved in the incidence and progression of type 2 diabetes, as well as glucose regulation in patients with T2DM, and are therefore potential therapeutic targets for this disorder.
The present study had a number of limitations. Firstly, further research with a larger sample size is warranted. Secondly, long-term complications in T2DM, such as diabetic foot ulcer, retinopathy, nephropathy, and coronary artery disease, were not investigated. Finally, a comprehensive evaluation of other regulatory targets of lncRNA NRON and their differentially expressed levels in patients with T2DM is recommended.