Identification of Novel Insulin Resistance Related ceRNA Network in T2DM and Its Potential Editing by CRISPR/Cas9.

Marwa Matboli,Meram Mohamed Bekhit,Nada Essawy,Sanaa Eissa,Basant Abdulrahman,Marwa Mostafa Kamel,Ghada F Mohamed

doi:10.3390/ijms22158129

Abstract

Background: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients’, matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence. Results: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing. Conclusions: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.

Highlights

type 2 diabetes mellitus (T2DM) is defined by both insulin resistance and pancreatic beta cell malfunction, leading to hyperglycemia [13]
According to the screened insulin resistance candidate genes, their interacting miRNAs and associated long non-coding RNAs (lncRNAs), a competing endogenous RNA (ceRNA) network was constructed based on their biological interactions
Each step might involve a large number of candidate mRNAs, miRNAs or lncRNAs, only several of them were used to construct the ceRNA network after a step-by-step screening and validation process

Summary

Introduction

Type 2 diabetes is a prevalent chronic metabolic disorder that represents a global growing healthcare burden, characterized mainly by resistance to insulin action along with insufficient secretion. The International Diabetes Federation (IDF) reported in 2019 that approximately 463 million adults (20–79 years old) are living with diabetes mellitus, and this number is expected to increase to 700 million by 2045 [1,2]. Egypt has topped the scale compared to other countries when it comes to the prevalence of DM. According to the IDF, Egypt’s diabetes prevalence (15.56% in adults of 20–79 years of age, with an annual mortality of 86,478 deaths) has placed it in the top 10 countries in terms of the number of diabetics [3]

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