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Abstract Type 2 DM is a chronic metabolic disease characterized by insulin resistance and beta cell dysfunction. It is an extremely heterogeneous disease in which the insulin resistance and beta cell dysfunction resulted from environmental and genetic factor. Type 2 diabetes is the most prevalent form of diabetes worldwide and account for 90% of cases globally. It represents a major public health threat and considered as a principal cause of morbidity and mortality affecting almost 6% of the world’s population. Patients with type 2 diabetes usually have insulin resistance and relative insulin deficiency and are often associated with a strong genetic predisposition. It’s one of the fast growing healthcare problems in Egypt. According to international diabetes federation, Egypt is the ninth leading country in the world for the number of patients with type 2 DM. The prevalence of T2DM in Egypt was tripled over the last two decades with a significant impact on morbidity, mortality, and health care resources. Insulin resistance is an enormous health care problem and the central pathogenic factor for the development of type 2 DM; in addition, it is also strongly associated with several metabolic risk factors as obesity, hypertension and dyslipidemia.Several noncoding RNAs were reported linked to insulin resistance. Therefore, Therapeutic targeting of deregulated genetic network responsible for insulin resistance may open a new hope for management of type 2 DM. Competing endogenous RNAs (ceRNAs) are RNA transcripts that make communication with each other by decreasing targeting concentration of micro-RNA (miRNA) with the derepression of other messenger RNAs (mRNAs) having the common miRNA response elements (MREs). The ceRNA theory has been applied to elucidate the pathogenesis of numerous diseases such as cancer, muscular dystrophy neurodegenerative disease and diabetes. However, little is known about potential role of ceRNAs in metabolic disease, especially diabetes mellitus. Due to previous stated information, we used an integrative approach based on bioinformatics analysis together with clinical validation to provide great insights into the molecular mechanisms of IR in the development of T2DM. This study was done at Medical Biochemistry Department, Faculty of Medicine, Ain Shams University during the period from March 2016 – October 2018 and included 123 T2DM patients and 106 normal volunteers. The aim of the present study was to retrieve potential LncRNA-RP11-773H22.4 associated ceRNA network from atabases followed by validation of this network in patients’ and cell line samples by quantitative Real Time-PCR and to evaluate the efficacy of CRISPR/Cas9 gene editing tool as a potential therapeutic strategy to modulate the expression of deregulated network in type 2 DM. The study included 229 subjects classified into two groups: group 1: 123 cases of type 2 diabetes mellitus patients, T2DM diagnosis was proved according to the American Diabetes Association practice guidelines. Their ages ranged from 35 to 83 years, with a mean of 54.69± 8.855 years, the median of ages was 55 years. group 2: 106 healthy normal individuals. Their ages ranged from 36 to 69 years, with a mean age 53.29± 7.04 years, the median was 53 years. All patients were subjected to complete medical history taking, full clinical, radiological examination and routine laboratory investigations including: Complete blood picture (CBC), Fasting Blood Sugar (FBS), HbA1c and HOMA-IR. The expression of the identified genetic network was measured by quantitative real time PCR (qPCR) in the peripheral blood mononuclear cells obtained from patients’ whole blood samples and in lymphocytes cell line. The data were normalized using the endogenous GAP-dh mRNA and RNU-6 miRNA as reference controls. Then the results were calculated and statistically analyzed by the SPSS software version 20. A significant difference was observed in the positivity rates of LncRNA-RP11-773H22.4, miR-3163, miR-1 miRNAs RET and m-TOR mRNAs, which were 73.2%, 85.4%, 70.7%, 80.5% and 99.2% respectively, in the diabetic group. However, among the normal control, the positivity rates of LncRNARP11- 773H22.4, miR-3163, miR-1 miRNAs RET, and m-TOR mRNAs were 14.2%, 17%, 4.7%, 20.8% and 17% respectively (p<0.01). Using ROC curve analysis, ≥ 2.123, ≤ 0.626, ≤ 0.995, ≥ 2.99 and ≥1.6) were set as cut off values for LncRNA-RP11- 773H22.4, miR-3163, miR-1 miRNAs RET and m-TOR mRNAs respectively. They showed sensitivity of (73.2%, 85.4%, 70.7%, 80.5% and 99.2% respectively), specificity of (85.8%, 83%, 95.3%, 79.2% and 83% respectively), and accuracy of (79.04%, 84.28%, 82.1%, 79.91% and 91.7% respectively). There was a highly significant positive correlation between expressions of LncRNA-RP11-773H22.4, RET, m-TOR mRNAs, HbA1c and HOMA-IR, and highly significant negative correlation between them and miR-3163, miR-1 miRNAs expression among the two studied groups (P<0.01). Human PBMCs isolated from whole blood samples of diabetic and healthy individuals. The PBMCs were cultured and lymphocytes grew for 3-5 days, then Cas 9 knockout of LncRNA-RP11-773H22.4 at the lncRNA-miRNA interaction site was done in the cultured cells. Cell pellets were collected from the following 3 groups: diabetics before Cas9 editing, diabetics after 72 hours post-transfection by Cas9 targeting LncRNARP11- 773H22.4 and healthy group, cells were assessed for both cell count, cell viability and for molecular assays. In conclusion, the results of our study Support our previous hypothesis that LncRNA-RP11-773H22.4, miR-3163 miRNA and RET mRNA act as ceRNA network and might play an important role in the pathogenesis of insulin resistance and T2DM. And could be potential therapeutic target by gene editing tools as CRISPR/Cas9 technique to modulate the expression of the deregulated network in type 2 DM. |