Cell-free long non-coding RNA-H19 and miRNA-29a, miRNA-29b expression in Type 2 Diabetes Mellitus patients.

Background: Type 2 diabetes mellitus [T2DM] has been one of the common diseases and characterized by increased blood glucose levels. T2DM participates in several organ damages as well revealed that cell-free non-coding RNAs and microRNAs (miRNAs) have been demonstrated to serve as important diagnostic/prognostic biomarkers in the pathophysiology of diabetes. Materials/Methods: The present study included clinically conrmed untreated newly diagnosed 200 cases of T2DM and 200 healthy subjects. Blood samples collected in uoride vials were used to check blood glucose for fasting and after 2 hours of breakfast (postprandial), blood samples collected in EDTA vials used for the Hba1c level. Blood samples from all the participants were collected in plain vials used for cell-free total RNA extraction. Total extracted RNA was quantied and 100ng was used to synthesized the cDNA for cell-free lncRNA H19, miRNA-29a, and miRNA-29b expression using specic primers/ probes using quantitative real-time PCR method. Serum Biochemical parameters were analyzed after collection of the sample to observe the changes among T2DM cases and healthy controls. Subjective data were recorded to evaluate the association with lncRNA H19, miRNA-29a, and miRNA-29b expression among T2DM cases. Results: It was observed that type 2 diabetic patients had decreased [0.59 fold] lncRNA H19 expression while increased miRNA-29a [5.62 fold] and miRNA-29b [5.58 fold] expression. Decreased expression of lncRNA H19 was observed to be associated with gender [p=0.004], hypertension [p<0.0001], weight loss [p=0.02] and fatigue [p=0.02]. Increased miRNA29a expression was linked with hypertension [p<0.0001], alcoholism [p=0.04], and smoking [p<0.0001] as well as miRNA-29b expression was associated with hypertension [p=0.0001], weight loss [p=0.002], smoking [p=0.0002], alcoholism [p<0.0001]. Low [<1 fold] and high [>1 fold] expression of lncRNA H19 expression was linked with miRNA-29a [p=0.005] and miRNA-29b [p<0.0001] expression. lncRNA H19 expression showed negative correlation with miRNA-29a expression [r= -27, p<0.0001] and miRNA-29b [r= -47, p<0.0001]. Conclusion: The present study concluded that lower lncRNA H19 expression, and increased miRNA-29b, miRNA-29b expression associated with the severity of T2DM patients. Decreased lncRNA H19 expression, and increased miRNA-29b, miRNA-29b expression observed to be interrelated with clinicopathological ndings of T2DM patients could involve in pathogenesis disease. H19 and increased expression of miRNA-29a as well as miRNA-29b in T2DM patients, Decreased expression was linked with hypertension, weight loss, and fatigue. Increased cell-free miRNA-29a expression was linked with hypertension, smoking, and alcoholism while miRNA-29b expression was linked with hypertension, weight loss, smoking, and alcoholism. This suggested that decreased expression of cell-free lncRNA H19, increased expression of miRNA-29a and miRNA-29b may be associated with the worseness of disease and involved in the pathogenesis of the disease. MiRNA-29a and miRNA-29b expression alteration observed to link with mainly smoking, alcoholism and this would be a factor for the severity of the disease. This data shed light on this gene interaction may be an essential factor for disease occurrence and worseness of disease. LncRNA H19, miRNA-29a, and miRNA-29b expression pattern information for the diagnosis/prognosis and management of T2DM cases. Further investigations are warranted to derive the difference in gene expression among T2DM cases.


Introduction
Type 2 diabetes mellitus [T2DM] is strongly associated with impaired glucose level, which developed due to the inability of the pancreas to secret enough insulin as well as insulin resistance. Hyperglycemia causes injury to major organs such as blood vessels, liver, brain, heart, kidneys, eyes, and can heavily contribute to the health and nancial burden associated with the disease. The liver is particularly involved in maintaining blood glucose levels and excessive hepatic glucose production [HGP] contributing hyperglycemia in T2DM patients [1,2]. Numerous long non-coding RNAs [lncRNAs] have been identi ed, and projects like ENCODE have focused on the identi cation and characterization of lncRNAs. ENCODE project revealed that there are at least 9,640 human loci lncRNAs, and the number grows continuously [3].
LncRNAs play a signi cant role in regulation at transcriptional and, post-transcriptional levels, and the organization of chromatin [4]. Studies suggested that LncRNAs also play a vital role in tumorigenesis [5].
LncRNAs and microRNAs [miRNAs] have different functions and participate in translational repression by targeting the genes, thereby shifting the nature of disease states. LncRNAs have involved in multiple cellular processes and emerged as potent genetic regulators. LncRNAs serve as a negative regulator of miRNAs and contributes to inhibits the level of expression [6]. Inc RNA H19 is positioned on chromosome number 11p15.5, about 100 kb distal of insulin-like growth factor 2 [IGF2], and simultaneously H19 and IGF2 are transcribed from a conserved embossed gene cluster [7]. In T2DM patients, approximately 5 times lower H19 levels were observed in skeletal muscle compared to healthy individuals [8].
Considerable attention received on multiple genes linked to epigenetic regulation of path-physiology of diabetes. MiRNAs are a group of short sequence about 22 nucleotides and target the multiple mRNAs that regulate protein-coding genes expression [9] and have been predicted that miRNAs regulate over 60% of all coding genes in mammals [10]. Several miRNAs have been associated with impairment of glycemic homeostasis and the diabetes progression [11,12].

Sample collection
This study included a total of 400 study subjects, 200 were newly diagnosed untreated type 2 diabetes mellitus patients and, 200 were healthy subjects. 3 ml of blood samples were withdrawn from all the study subjects and collected in plain vials. Fasting as well as postprandial samples collected in uoride vials from all the 200 T2DM subjects and 1 ml blood in EDTA vials for Hba1c. All the criteria were followed, such as fasting blood glucose [glucose level ≥ 126 mg/dL] and postprandial glucose [2-hour blood glucose ≥ 200 mg/dL] were monitored for diagnosis of T2DM disease. T2DM patients with Hypertension (BP ≥ 140 or ≥ 90 mm Hg), increased urination (polyurea by physical examination as well as 8, 12 & 24hrs urine test), weight loss (> 4 kg weight loosed in last 3 to 6 months), fatigue, wound healing, blur vision (Fundus/ uorescein angiography), loss of appetite, smoking, alcoholism were subjective data recorded for analysis.
Blood samples centrifuged at 1500 rpm to collect the serum and stored at -80 0 C for further process. The ethical committee board ethically approved this research study and informed written consent was obtained from all the participants before the study commenced. The present study was ethically approved and the study was conducted at the Department of Zoology, Gurukula Kangri University, Haridwar, India.
Total RNA extraction Stored serum samples of T2DM cases and healthy controls were thawed, and total cell-free RNA extraction was done by kit [mirVana total RNA isolation kit, Thermo Fisher Scienti c]. All samples are then stored at -70 0 C in 2 ml nuclease-free Eppendorf tubes. The concentration and quality of cell-free RNA were checked by the A260/280 ratio using Nano-drop.
cDNA synthesis and QRT-PCR for lncH19 expression study 100 ng of the total cell-free extracted RNA from T2DM patients and healthy controls were used to synthesized cDNA using kit [Verso, Thermo scienti c, USA] following kit provided protocol. Expression of cell-free lncRNA H19 was done by quantitative real-time PCR using SYBR green dye using forward primer sequence 5'-ATCGGTGCCTCAGCGTTCGG-3' and reverse primer sequence 5'-CTGTCCTCGCCGTCACACCG-3' and β-actin were used as internal control, and the forward primer sequence 5'-CGACAACGGCTCCGGCATGTGC-3', reverse primer sequence 5'-GTCACCGGAGTCCATCACGATGC-3'. The program followed as qRT-PCR for lncRNA H19 and β-actin was performed for 40 cycles; initial denaturation was at 94 0 C for 40 seconds, annealing temperature was at 60 0 C for 40 seconds, extension at 72 0 C for 40 seconds and 20 micro litters reaction volume was used. Ending additional step at 72 0 C for 5 minutes to end up reaction and melting curve examined between the ranges 35 0 C to 90 0 C to con rm the target ampli cation. A control without cDNA was included in each experiment, and every reaction was done in duplicate. The relative quanti cation by 2 − [ΔΔCT] method was used to compute the lncRNA H19 level expression.
Polyadenylation, cDNA synthesis for miRNA-29a, and miRNA-29b expression study Total 100 ng RNA was taken to polyadenylate and cDNA synthesis by miRNA Reverse transcription kit [TaqMan, Thermo Fisher Scienti c] by following kit protocol. Essential reagents such as reverse transcriptase enzyme and other reagents were added to switch in poly [A] -tail and then into cDNA using a universal RT primer provided with the kit.
QRT-PCR for miRNA-29a, and miRNA-29b expression Quantitative real-time PCR [qPCR] was performed to compute the expression level of cell-free miRNA-29a and miRNA-29b. qPCR was done by advanced Taqman master mix [4444556], Taqman probes for miRNA-29a [478587_mir], and miRNA-29b [478369_mir] for quanti cation and miRNA- 16 [477860_mir] were used as internal control as normalizer to calculate the level of expression.

Statistical analysis:
All the statistical testing was done using Graph Pad Prism software version 6.05 and SPSS 20.0 version.
Mann Whitney U test used to calculate the differences in biochemical parameters among T2DM subjects and healthy controls. QRT-PCR outcome analysis was done by the relative cycle threshold [Ct] method to compute the LncRNA H19, miRNA-29a, and miRNA-29b expression levels by the relative quanti cation method using the 2 −[ΔΔCt] . The Mann -Whitney U test was done to calculate the differences in lncRNA H19, miRNA29a, miRNA29b expression level among the subjective variables. Results more than or less than one were considered to specify up-regulation or down-regulation of expression.
Spearman correlation analysis was performed to check the degree of association by calculating the rvalue of lncRNA H19 with miRNA-29a and miRNA-29b. Regression analysis was performed to calculate the R 2 and beta coe cient to check the effect of lncRNA H19 and biochemical parameters on miRNA-29a and miRNA-29b expression.
All values were normalized relative to the control values, which were depicted as a value of 1. P-value of less than 0.05 was considered statistically signi cant.

Demographic and clinical characteristics of T2DM patients and healthy controls
In brief, the present study included 200 cases of type 2 diabetes mellitus and 200 healthy control subjects [ Table 1]. Among the T2DM cases, 68% of patients were males, and 32% were females while in healthy controls, 70% were males, and 30% were females, more details mentioned in Table 1.    Table 4]. T2DM patients who were hypertensive showed 5.96-fold miRNA-29a expression, whereas nonhypertensive reported having 5.09-fold miRNA-29a expression. Besides, the differences between diabetic smoker and non-smoker patients in H19 expression were observed to be statistically signi cant [P < 0.0001]. We also studied whether cigarette smoking has an impact on miRNA-29a expression. We observed that the T2DM patients with smoking habit showed 5.98-fold miRNA-29a expression, and nonsmokers showed 5.24-fold miRNA-29a expression [P = 0.04]. T2DM subjects with the habit of alcohol consumption showed 6.17-fold miRNA-29a expression, while non-alcoholic T2DM patients showed 5.04fold miRNA-29a expression. Furthermore, a statistically signi cant correlation was observed in H19 expression between alcoholic and non-alcoholic diabetic patients [P < 0.0001]. Our observations illustrated that miRNA-29a expression in T2DM patients is directly associated with hypertension and alcohol consumptions.  the observed R 2 value was 0.13 (p = 0.003) and 0.14 (p = 0.001) ( Table 6). The beta coe cient was also calculated and the observed beta value for lncH19 to miRNA-29a was − 0.298 (p < 0.0001) and concerning miRNA-29a was-0.028 (p < 0.0001) ( Table 7).   Correlation of lncRNA H19 with miRNA-29a and miRNA-29b A negative correlation of lncRNA H19 with miRNA-29a and miRNA-29b was observed [ Figure 2]. It was found that lncRNA H19 expression showed negative correlation with miRNA-29a [r= -0.27, P < 0.0001] as well as negative correlation with miRNA-29b [r= -0.47, P < 0.0001] was also observed.

Discussion
Diabetes mellitus is a metabolic disorder that developed due to insu cient insulin secretion from β-cells of the pancreas [18]. The frequency of T2DM has increased in a few decades and to date affects more than three hundred million lives worldwide due to increased sedentary life and easy access to fast foods [19]. The pathogenesis of T2DM is associated with a genetic disposition, environmental exposures, and multiple causes. [20]. Long non-coding RNA H19 is a multifunctional long noncoding RNA that functionally involved in the nucleus and the cytoplasm [21]. In T2DM patients, ve times lower H19 expression observed in skeletal muscle compared to healthy individuals [22]. Zhuo et al. have shown that diabetic rats had decreased expression of H19 as well as in high glucose exposed to neonatal cardiomyocytes [23].
A downregulation of cell-free lncRNA H19 observed in diabetes [24], and in the same way, the present study revealed decreased cell-free lncRNA H19 expression associated with gender, hypertension, weight loss, and fatigue. Male T2DM patients showed 0.40-fold down-regulation, hypertensive T2DM patients showed 0.47-fold down-regulation, T2DM patients who had weight-loss showed 0.38-fold downregulation, T2DM patients who had fatigue showed 0.26-fold down-regulation of lncRNA H19 expression compared to its counterpart. Increased cell-free miRNA-29a and miRNA-29b expression were observed among T2DM patients. It has revealed that the cell-free miRNA-29a expression was linked with hypertension and alcoholism. miRNA-29b expression was linked with hypertension, weight loss, smoking, and alcoholism. Increased expression of the miRNA-29 family recorded in different tissues such as the liver [25] and β cells of diabetic rodent models and humans [26].
Essentially, an increase in miRNA-29 expression level in the serum of children diagnosed with T1DM and adult T2DM patients was observed [27]. Hyperglycemia is the primary situation of diabetes mellitus and leads to up-regulates the expression of miRNA-29a and miRNA-29b [28]. Roggli et al. revealed that patients treated with proin ammatory cytokine induce the miRNA-29 family members' expression level in human beings and mouse pancreatic cells. Moreover, an increase in the expression of miRNA-29 family miRNAs is associated with the early step of diabetes in mice [29]. It has been shown that miRNA-29 suppression by using anti-miRNA-29 oligomers decreases the expression and protects the patients against diabetic nephropathy [28].
Pancreatic islets showed upregulation of miRNA-29a and miRNA-29b and involved in β-cell death, initiation of diabetes, and suggested to be the reliable diabetic marker [26]. Dahlman has revealed that the T2DM patient's skeletal muscles showed increased expression of miRNA-29 family members compared to the non-diabetic person [30]. miRNA-29b was over-expressed in diabetic rat's muscle cells and correlated with the negative GLUT4 expression, and miRNA-29a has previously been validated as repressors of GLUT4, increased miRNA-29a expression was observed in vastus lateralis muscle T2DM, and GLUT4 protein expression was observed to decrease in mice skeletal muscle over-expressing miRNA-29a [31], as well as in C2C12 muscle cells [32]. Expression of GLUT4 level has been reported to decrease in 3T3-L1 adipocytes over-expressing miRNA-29a, miRNA-29b [33].
This study observed the T2DM patients who had expression level ≤ 1 fold of lncRNA H19 showed higher expression while > 1 fold of lncRNA H19 had lower expression comparatively. It was also found that the lncRNA H19 expression had a negative correlation with miRNA-29a and miRNA-29b. This suggested that decreased lncRNA H19 expression showed to increased expression of miRNA-29a and miRNA-29b. In support of the present study, Hee et al. in 2007 and Karolina et al. in 2011 also revealed that the miRNA-29 family was increased in several tissues, skeletal muscle, derived from diverse rat models of T2DM [34,35]. It has also been revealed that miRNA-29a and miRNA-29b were up-regulated in T2DM patients' skeletal muscle tissue [36] study done by Zhang P et al in 2020, indicated that lncRNAs are important regulators of DM and are thus likely to serve as important diagnostic and therapeutic targets for this disorder [37]. Increased miRNA-29a expression was observed in T2DM and the present study provides support for the exploration of miR-29a antagonists as a potential therapeutic approach for T2DM treatment and the associated metabolic complications [38]. Huang M et al revealed that lncRNA H19 could bind to miRNA-29a competitively following miRNA response elements (MREs) of miRNA-29a and affects the expression [15]. Zhong et al said that lncH19 sponge the miRNA-29b, [16] and Mengxin Lv et al also stated that H19 could directly bind to miR-29b and reduces its expression [17]. Over-expression of miR29a levels was positively associated with metabolic disorders and demonstrated to have a potential value as biomarkers in disease prognosis [39]

Conclusions
The present study observed decreased expression of cell-free lncRNA H19 and increased expression of miRNA-29a as well as miRNA-29b in T2DM patients, Decreased expression was linked with hypertension, weight loss, and fatigue. Increased cell-free miRNA-29a expression was linked with hypertension, smoking, and alcoholism while miRNA-29b expression was linked with hypertension, weight loss, smoking, and alcoholism. This suggested that decreased expression of cell-free lncRNA H19, increased expression of miRNA-29a and miRNA-29b may be associated with the worseness of disease and involved in the pathogenesis of the disease. MiRNA-29a and miRNA-29b expression alteration observed to link with mainly smoking, alcoholism and this would be a factor for the severity of the disease. This data shed light on this gene interaction may be an essential factor for disease occurrence and worseness of disease. LncRNA H19, miRNA-29a, and miRNA-29b expression pattern information for the diagnosis/prognosis and management of T2DM cases. Further investigations are warranted to derive the difference in gene expression among T2DM cases.

Declarations
Con ict of interest: None 40. Ying-Zhi Liang, Jia-Jiang-Hui Li, Huan-Bo Xiao, Yan He, Ling Zhang, Yu-Xiang Yan, Identi cation of stress-related microRNA biomarkers in type 2 diabetes mellitus: A systematic review and meta-  Figure 1 Association of lncRNA H19 expression with miRNA29a and miRNA29b expression.