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

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.

Evaluation of schistosomicidal activity of myrrh extract: parasitological and histological study.

Ninety mice were divided into three main groups: G. I (non-infected control), G. II (infected non treated control) and G. III (infected treated), which was further subdivided into 4 subgroups: IIIA, IIIB, IIIC & IIID, where the drug was administered in a dose of 500 mg/kg body weight for five days before infection for subgroup IIIA, on the 1st day post infection (PI) for subgroup IIIB, 21 days PI for subgroup IIIC and 45 days post infection for subgroup IIID. All animals were sacrificed 80 days after the start of the experiment. Anti-schistosomal activity was assessed parasitologically by estimating the percentage reducetion of worm burden, egg count/gm tissues (liver & intestine), and the oogram pattern; histologically & histopathologically by examination of jejunum using different stains. The percentage reduction of worm burden was 30.35%, 64.54%, 76.92% and 98.46% respectively when compared to the infected non-treated control G. The maximum effect of the drug was observed in subgroups IIIC & IIID. Also, there was marked reduction in the egg count in tissues (liver & intestine). In addition the oogram pattern showed that myrrh had high antischistosomal activity. The histologically and histopathologically infected untreated (GII), when compared to non-infected non-treated control G. showed apparent shortening and flattening of the jejunal villi with focal loss of the epithelial covering. Loss of PAS positive brush border of many enterocytes with goblet cells hyperplasia was seen. Bilharzial granulomas were frequently encountered in the submucosa and the musculosa with numerous eosinophils content. In subgroups IIIA & IIIB, there was mild amelioration of the mucosal structural abnormalities. The granulomas were less frequently seen with decrease of their eosinophils. In subgroups IIIC & IIID there was restoration of the jejunal mucosal continuity, marked decrease in the granulomas and paucity of eosinophils. The present data proved that myrrh has a valuable schistosomicidal effect against different maturation stages of S. mansoni. The chemotherapeutic effect was more evident when the drug was given to the infected mice on the 21st as well as on the 45th day PI. The drug proved a promising chemoprophylactic agent when used five days before exposure to infection.