Corresponding Author: Mandana AmeliMojarad
Department of Biotechnology, Tehran University of Medical Science, Tehran (Iran)
Simvastatin Therapy Increased miR-150-5p Expression in the Patients with Type 2 Diabetes and COVID-19
Mandana AmeliMojarad Melika AmeliMojarad Alireza Pourmadian
Department of Biotechnology, Tehran University of Medical Science, Tehran, Iran
The corona virus disease 2019 (COVID-19) pandemic was caused by severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) infections which have globally spread since 2019 and have been considered the greatest major threat worldwide . The mortality rate of COVID-19 is higher, particularly in the elderly population and patients with other malignancy such as hypertension, diabetes mellitus, and cardiovascular disease, among them, Type 2 diabetes mellitus (T2DM) have a higher risk of development in COVID-19 patient increasing the risk of severe outcome and mortality [2, 3]. T2DM is one of the common chronic conditions and a public health problem, with increasing prevalence over the past decades . Although the exact mechanism of Insulin resistance (IR) in T2DM is not clearly understood, dyslipidemia is closely considered to relate to IR in diabetes. In addition, serum lipid profiles were found to deregulated in diabetics more than in non-diabetic individuals from different ethnic groups . mentioning the association between hyperlipidemia and increased risk of developing abnormal glucose metabolism in patients [5, 6]. Statins are lipid-lowering drugs, that reduce circulating lipids, and most specifically cholesterol packaged in low-density lipoprotein (LDL) particles while increasing the HDL cholesterol levels [6, 7]. Statins are also a candidate for treatment of cancer with their inhibitory effects on pro-inflammatory cytokines [8, 9]. Moreover, statins disturb viral binding by decreasing cholesterol as an essential component of lipid rafts in cellular plasma membranes and disrupting virus entry. There is a previous study that statins therapy reduced mortality in influenza viral infections . It has also been proved that statins can modulate expressions of microRNAs previously . For instance, statins could inhibit miR-133a expression which was robustly induced by cytokines/oxidants, and prevent endothelial dysfunction . MicroRNAs (miRNAs) which are classified as short non-coding RNAs and they can regulate gene expression, metabolism, inflammation, and inhibit the viral translation with the ability to suppress the expression of targeted genes through post-transcriptional mechanisms [13–15]. Therefore, determining miRNAs as crucial factor in a diverse biological processes that are modified in COVID-19 patients significantly and targeting them might be an effective and highly promising tool for patients who are susceptible for T2DM and COVID-19 . miR-150 is a regulator that suppresses inflammation and is considered as a novel biomarker based on its high expression in different main tissues, such as liver, adipose tissue and skeletal muscle [14, 17, 18]. More interestingly, the higher risk of T2DM was associated with lower levels of miR-150 and its down regulated observed in cases with obesity plus T1D, or T2D [18, 19]. Therefore, in this study, we study the statins effects on circulating levels of miR-150 in patients with T2DM and COVID-19 and evaluating its status as a biomarker to estimate the risk of developing the diseases.
Materials and Methods
In the present observational, pilot study, a group of 30 plasma samples of subjects with positive COVID-19 and T2DM compared with patients only with COVID-19 before and after treatment with 10 mg/day of statins (simvastatin) for 2 months. Between (March 1st and Feb 30th, 2021) were compared with COVID-19 patients who did not exhibit T2DM or other respiratory problems was obtained. We age-matched the groups by choosing ages between 30–66 years of age. The criteria for inclusion were PCR confirmation of SARS-CoV-2 and less than 48 hours hospitalization of each case. For exclusion were: suspected to any other infections besides COVID-19; patients with neoplastic or autoimmune diseases. Written consent was received from all patients. Our study was based on Helsinki Declaration and the ethical committee of Tehran University and the registry code (IR.IUMS.REC.1399.9223497212) The patient details are provided in Table 1.
Lipid profile before and after a 2-month treatment with simvastatin. TC: total cholesterol. LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; VLDL-C: very low-density lipoprotein cholesterol; TG: Triglycerides; ApoAI: apolipoprotein AI; ApoB: apolipoprotein B (*p < 0.05, **p < 0.01, ***p < 0. 001)
Measurements of Biochemical parameters
Venous blood was collected in EDTA tubes after a 12-h overnight fast. Plasma were separated immediately afterward in a refrigerated centrifuge to avoid any loss of analytics. Modular analyzer DDPPII Hitachi (Roche, Switzerland) was used for lipid variables and colorimetric enzymatic methods for assessing total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) and triglyceride (TG) and nephelometry was used for measuring Apolipoproteins AI (apoAI) and B (apo B).
Extraction of RNA and cDNA synthesis
Anticoagulant EDTA tubes were used for blood samples collection and peripheral blood mononuclear cells (PBMCs) were obtained with centrifugation.RNA was extracted using RNX™-plus reagent (SinaClon, Iran). The cDNA was synthesized by the advanced miRNA cDNA Synthesis Kit (TaqMan). RT-qPCR with the SYBR green was used to evaluate the expression levels of selected miRNA, the reaction was set as 5 min at 95 °C, 10 s at 95 °C for 45 cycles, and 5 min at 95 °C, with specific forward primer and the universal adaptor reverse primer. The relative expression was calculated with 2-∆∆Ct method, and U6 was used as internal controls, respectively .
Target Prediction of miR-150-5p and Functional Enrichment Analysis
We analyzed the potential miR-150-5p target genes with MiRWalk 3.0 (http://mirwalk.umm.uni-heidelberg.de) and evaluated the other overlapping genes with three different highly recognizable miRNA-target prediction tools miRDB: (http://mirdb.org), and Targetscan7.2: http://www.targetscan.org/vert_72/) and Miranda (http://www.microrna.org) for accurate results. 11 selected overlapping genes were obtained. Then we used Metascape (metascape.org/gp/index.html) for further GO annotation analysis. P < 0.05 was considered as significant. Data was analysied with Graph Pad Prism 6 (Graph Pad Software, USA) and are shown as mean ± standard deviation (SD). To examine the normal distribution of the variables Kolmogorov–Smirnov test was used and Mann–Whitney U test. Student’s t-test was used to evaluate the differences between the two groups. P < 0.05 was considered as statistically significant.
Effects of Statins supplementation on lipid levels and miR-150 expression
Lipid levels before and after simvastatin treatments are presented as mean ± SD in Table 1. Treatments significantly improved the lipid profile in both groups. Simvastatin treatments significantly improved the LDL-C and Apo B. There were no significant differences in weight, BMI TG, total cholesterol, HDL-C, or ApoA1. Next to verifying the statin effects on miR-150-5p, miR-150 expression levels were measured before and after simvastatin treatments. our data revealed that plasma levels of miR-150-5p were lower in both groups and the expression level of miR-150-5p, was significantly increased after two months of treatment with simvastatin, especially in patients with COVID-19 and T2DM (Fig. 1).
M.A and M.A contributed equally to writing and drafting. A.P worked on editing all authors and approved the final submitted version.
Statement of Ethics
This research was confirmed by the Tehran College of Therapeutic Sciences with the code of ethics IR.IUMS.REC.1399.9223497212. Written informed consent was obtained from all patients.
The authors declare that no conflict of interests exists.
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