dentication of novel insulin resistance related ceRNA network: LncRNA, RP11 ‐ 773H22.4; miR-3163, miR-1; mRNAs:RET , IGF1-R, m-TOR, GLUT-4, AKT2 in T2DM and its potential editing by CRISPR/Cas9

Background: In this study, we aimed to construct Insilco, a competing endogenous RNAs (ceRNAs) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients’, matched control and cell line samples. And also, to evaluate the ecacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatic tools, we identied and veried a ceRNA network panel of lncRNA, miRNAs and mRNAs related to insulin resistance and then we validated its expression in 123 patients’ and 106 matched controls and cell line samples using real time PCR. Results: LncRNA-RP11 ‐ 773H22.4, together with RET , IGF1-R and m-TOR mRNAs showed signicant upregulation in T2DM compared with matched controls while miRNAs: miR-3163, miR-1 and mRNAs: GLUT-4 and AKT2 expression displayed marked downregulation in diabetic samples.Two guide RNAs were designed to target the sequence anking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool ecacy was assessed by measurement of the network downstream proteins, GLUT4 and mTOR by immunouorescence. CRISPR/Cas9 successfully knockout LncRNA-RP11-773H22.4 as evidenced by the reversal of the gene expression of the identied network at RNA and protein levels to the normal expression pattern after gene editing. Conclusion: The presented study provides the signicance 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. we constructed a competing endogenous RNA network and identied thereby, several potential key molecules. We described the important role played by non-coding RNAs in the pathogenesis of IR. Our study highlighted specic lncRNA, miRNAs and mRNAs related to the pathogenesis of IR and obtained from public databases, which might be used as novel therapeutic targets for type T2DM. Secondly, we evaluated the expression of this identied genetic network in clinical samples. Finally, we evaluated the therapeutic ecacy of the gene editing tool CRISPR/Cas9 in the improvement of IR in type 2DM in lymphocyte cell line. Our results demonstrated that there was signicant up-regulation in the expression of LncRNA-RP11 ‐ 773H22.4, RET, m-TOR and IGF1-R mRNAs, together with signicant down-regulation in the expression of miR-3163, miR-1, GLUT-4 and AKT2 mRNAs in the diabetic group compared to the healthy control in both patients’ clinical samples and lymphocyte cell line. the function of miR-1 through AKT activation in H9c2 cells 32 . These results are similar to our study results that revealed the deregulation of miR-1 level in case of T2DM. Levels of circulating miR-1 have been associated with occurrence of myocardial infarction 33 . Also, it has been reported to suppress the growth of hepatocellular carcinoma cells by modulating ET-1 34 and to be down-regulated in primary human lung cancers 35 , gastric 36 and breast cancers 37 . approach based on computational method together with clinical validation to provide novel insights into the molecular mechanisms of IR. We implemented combined bioinformatics analysis to retrieve a set of ceRNA network (RET mRNA, LncRNA-RP11-773H22.4 and miRNA-3163 miRNA) related to insulin resistance and their targeting signaling pathway genes (IGF1-R, GLUT-4, AKT2 and m-TOR mRNAs) and (miR-1 miRNA), retrieved from public databases. Afterwards, we investigated the identied ceRNA network expression in patients’ clinical samples and lymphocyte cell line by qPCR and IF. Then, we did CRISPR/Cas9 knockout of LncRNA-RP11 ‐ 773H22.4 at the sequence of lncRNA-miRNA interaction in the cultured lymphocytes by designing more than one gRNA anking the area of interaction. This approach resulted in the restoration of normal expression of the identied genes in comparison to the healthy controls as conrmed by qPCR and IF. We thereby, suggest the CRISPR/Cas9 knockout of LncRNA ‐ RP11 ‐ 773H22.4 as a potential therapeutic target for treatment of T2DM. Taken together, our study offers valuable insights that help to decipher our understanding of diabetes and insulin resistance. More invitro functional studies are needed to validate our result and consider the safety issues related to CRISPR/Cas9 system; as genotoxicity and off target mutations with possibility of using more accurate nucleases to reduce off-target effects.


Introduction
Type 2 diabetes is a prevalent chronic metabolic disorder associated with resistance to insulin action and insu cient insulin secretion and represents a global continuously growing healthcare burden that is reported by the International Diabetes Federation (IDF) diabetes atlas ninth edition in 2019, as approximately 463 million adult (20-79 years old) are living with diabetes mellitus and this number is expected to increase to 700 million by 2045 1,2 . Egypt, most notably, has topped the scale compared to other countries when it comes to the prevalence of DM. According to IDF, Egypt's diabetes prevalence (15.56% in adults of 20-79 years of age with annual mortality of 86,478 deaths) has placed it on the top 10 list of countries in terms of the number of diabetics 3 .
A major focus of research nowadays is the different signaling pathways implicated in T2DM in order to develop novel therapeutic strategies for diabetes and its complications. Although T2DM is a multi-faceted disease with contributing genetic, epigenetic and environmental factors, the reduced sensitivity to insulin in target cells constitutes the hallmark of this disease 4 . Insulin resistance represents the major pathological process underlying the disease and involves a complex network of defects in signal transduction, such defects in insulin Receptor Substrate 1/2 (IRS-1/2), phosphatidylinositol 3-Kinase (PI3K)/Serine/Threonine Kinase (AKT) and Glucose Transporter 4 (GLUT4) 5 .
Epigenetics has been shown to be involved in the regulation of in ammation and cellular senescence, both of which are associated with type 2 diabetes. Abnormal epigenetic modi cations including changes in non-coding RNA levels have been implicated in the pathogenesis of a myriad of diseases, notably diabetes. Studies postulate that RNAs affect the level of one another by competing for a small pool of micro RNAs, this is described as the competing endogenous RNA theory (ceRNA) and is supported by growing evidence in a number of diseases 6 . Long non-coding RNA (lncRNA) can act as a ceRNA in uencing the post-transcriptional regulation. That's because they have miRNA binding sites and can affect miRNAs levels available for binding to their target mRNAs. Consequently, they abolish the repression of these mRNAs explaining their negative correlation with miRNA's expression 7 . There's a list of long noncoding RNAs linked to the development of T2DM, including HOTAIR, MEG3, LET and MALAT1 8 . In addition to lncRNAs, many miRNAs are also implicated in the development of T2DM namely as miR-135, 202 and 214 along with their targets (Rock-1, Akt2, and Vamp2). In particular, miR-135 is involved in insulin resistance through its effect on insulin receptor substrate 1 (IRS-1) while miR-214 overexpression leads to Akt2 down regulation, resulting in subsequent defects in insulin stimulation of glucose transport and ultimately insulin resistance. This has been investigated in C2C12 and L6 cell line models of insulin resistance 9 .
Gene editing is a recent breakthrough with a considerable potential in understanding and treatment of several diseases. By the virtue of this technique, researchers could target predetermined parts of the DNA sequence and produce speci c alterations including insertions, deletions, point mutations or translocations through the production of double-stranded breaks at target sites and as a result many DNA repair systems are activated. Gene editing e ciency signi cantly improved after the discovery of a novel tool extracted from Streptococcus pyogenes; CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein-9 nuclease (Cas9), which led to a potentially low cost, time-saving of whole human genome editing (including non-coding RNAs) that are consistent with high throughput screening protocols 10 .
The present paper aimed to determine potential ceRNA panel related to insulin resistance from databases followed by the validation of its expression in both patients' clinical samples and lymphocyte cell lines. Besides, we called in question the role of this identi ed insulin resistance (IR) related panel in the pathogenesis of type 2 DM. Finally, we evaluated the e cacy of the gene editing tool CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network in T2DM.
Inclusion criteria of the study; Proved diagnosis of T2DM according to ADA practice guidelines, age of > 18 years at the time of consent giving and the ability to provide a written, informed consent, Exclusion criteria of the study; History of malignant disease, patients on steroids for the last 6 months, end stage organ disease as chronic liver disease ,pregnant or lactating women.
Whole Blood samples were collected at Ain Shams University hospitals during the period March 2018 to May 2019. Sera were obtained by centrifugation while Peripheral Blood Mononuclear Cells (PBMCs) were isolated using lymphoprep (Axis-Shield PoC AS, Oslo, Norway). All participants signed an informed consent and the study was approved by the ethical committee of the Faculty of Medicine at ASU no. FWA 000017585. Sera were obtained by centrifugation while Peripheral Blood Mononuclear Cells (PBMCs) were isolated using lymphoprep (Axis-Shield PoC AS, Oslo, Norway).
All participants signed an informed consent and the study was approved by the ethical committee of the Faculty of Medicine at ASU no. FWA 000017585.

| Measurement Of HOMA-IR:
Fasting insulin level was measured in sera of diabetic patients and healthy controls using enzyme-linked immunosorbent assay ELISA (DRG® Insulin ELISA (EIA-2935), DRG International, Inc. USA). Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was calculated according to the equation: Fasting insulin (µU/L) x fasting glucose (nmol/L)/22.5 11 2.3 | Bioinformatics-based selection of ceRNA panel related to insulin resistance: The identi ed panel was obtained through the following steps: Retrieval of a set of candidate genes (mRNAs) related to insulin resistance signaling pathways from two public microarray databases available at (https://www.ebi.ac.uk/gxa/home) and (https://www.proteinatlas.org). Veri cation of the identi ed candidate genes expression in skeletal muscle and adipose tissues through gene cards (https://www.genecards.org/), so as to decrease the false discovery rate. Construction of mRNA/ miRNA/ lncRNA genetic axis linked to the identi ed candidate genes in IR in T2DM including LncRNA-RP11-773H22.4, miR-3163 and miR-1 microRNAs using a lnCeDB database, available at (http://gyanxet-beta.com/lncedb/index.php). Alignment between LncRNA-RP11-773H22.4 and retrieved miRNAs: miR-3163 and miR-1, alignment between mTOR mRNA and retrieved miRNAs, as well as, the alignment between LncRNA-RP11-773H22.4 and the 2 synthesized gRNAs, to identify sequences of lncRNA-miRNAs interactions to be targeted by CRISPR/Cas9 gene editing tool through alignment database tool available at (https://www.ebi.ac.uk/gxa/home). Detailed bioinformatics are shown in supplementary gures (1a-o). We used miRNeasy® RNA isolation kit (Cat no. 74104; Qiagen, USA) to extract total RNA from the PBMCs following the manufacturer's instructions. We assessed the RNA concentration and integrity using DeNovix DS-11 micro-volume spectrophotometer (Wilmington, USA). Then, it was reverse transcribed into cDNA using RT2 rst strand kit (Cat no. 330401; Qiagen, USA) for the target mRNAs and miScript II RT Kit (Cat no. 218161; Qiagen, USA) for the non-coding RNAs as per the manufacturer's protocol using Thermo Hybaid PCR express (Thermo Scienti c, USA).
2.4.2 Quantitative real time-PCR of the identi ed ceRNA panel related to insulin resistance: The levels of the identi ed mRNAs (RET, IGF1-R, m-TOR, GLUT-4 and AKT2) in PBMCs were measured using custom RT 2 Pro ler™ PCR Array (Cat no. 330171; Qiagen, Helman Germany, Ensembl: ENSG00000165731, ENSG00000140443, ENSG00000198793, ENSG00000181856 and ENSG00000105221, respectively) and RT² SYBR Green ROX qPCR Mastermix (Cat no: 330520 ;Qiagen, Helman Germany), using Applied Biosystems Tm 7500 Real-Time PCR system (Foster city, California, United States). Relative expression levels for LncRNA-RP11-773H22.4 were analyzed by RT² SYBR Green ROX qPCR Master mix (Cat no: 330520; Qiagen, Helman Germany) and lncRNA qPCR Primer Assay for Human RP11-773H22.4 (ENST00000588211) supplied by Qiagen. GAPDH (Ensembl: ENSG00000111640) was used as a reference gene. miR-3163 and miR-1 miRNAs relative expression levels in PBMCs were investigated by a miScript SYBR Green PCR Kit (Cat no. 218073; Qiagen, Helman Germany), a miScript universal primer and a miRNA-speci c forward primer (Hs_miR-3163_1 miScript Primer Assay) (Accession: MIMAT0015037) (Cat no. MS00020769; Qiagen, Helman Germany) for miR-3163 and (Hs_mir-1-1_PR_1 miScript Precursor Assay) (Accession: MI0000651) (Cat no. MP00000175; Qiagen, Helman Germany) for miR-1, and RNU-6 was used as an internal control. All PCR primers were obtained from Qiagen. The PCR program cycling conditions were adjusted according to the type of the measured RNA according to the manufacturer's protocol. The 2 −ΔΔCt technique was used to measure the expression of the IR speci c RNA-based candidate genes panel using Applied Biosystems 7500 software v2.3. Reference genes were used as an internal control to normalize the raw data of the samples and compare these results to a reference sample. In this study, appropriate standardization strategies were carried out to recognize any experimental error introduced at any stage during extraction and processing of the RNA according to MIQE guidelines 12 .
2.5 | Validation of the identi ed ceRNA panel related to insulin resistance and CRISPR/CAS9 editing of LncRNA-RP11-773H22.4 in lymphocyte cell line:

Culture Of Human Lymphocytes
Human cell culture was conducted as previously described 13 . The PBMCs were transferred in 20 mL RPMI 1640 media to a T-75 culture ask containing: 10% fetal bovine serum, 1% penicillin/streptomycin, 1µg/mL phytohemagglutinin (PHA) and incubated at 37°C with 5% CO 2 for 24 hours. Next day, all the media was removed from the ask, and the cell pellet was added to a 50 mL conical tube and centrifuged at 500 Xg for 5 minutes. The pellet which contained mainly lymphocytes was resuspended. The cells were transferred to a new T-75 culture ask in 25 mL RPMI 1640 media containing: 10% fetal bovine serum, 1% penicillin/streptomycin, 1µg/mL PHA and incubated at 37°C for 3 days. After 24 hours of growth, 20 mL of fresh media was added and transferred to a larger T-175 culture ask. After 3 days, media and suspended cells were removed from the culture ask and transferred to a 50 mL conical tube then centrifuged at 500 Xg for 5 minutes. The pellet was resuspended and transferred to a new T-75 culture ask containing 25 mL RPMI 1640 with 10% fetal bovine serum, 1% penicillin/streptomycin. Lymphocytes were grown for 4 days.

Synthesis Of Grnas
DNA oligonucleotides used for gRNA synthesis were designed with the GeneArt™ CRISPR gRNA Design Tool (available at: www.thermo sher.com). The two gRNAs were then synthesized using the GeneArt™ Precision gRNA Synthesis Kit according to the manufacture's protocol and each was combined with GeneArt™ Platinum™ Cas9 Nuclease to form the Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs), Fig. 2s and table 1s.
2.5.3 Lymphocyte transfection in a 12-well plate using Lipofectamine CRISPRMAX Reagent: 50 µl Opti-MEM medium was mixed with 1.25 µg GeneArt Platinum Cas9 nuclease and 0.25 µg gRNA, then vortexed with 2.5 µl Cas9 Plus and incubated at 25 for 5 min to form RNPs. 3 µl of Lipofectamine CRISPRMAX with 50 ul Opti-MEM were mixed and incubated at 25 for 5 min before being added to RNPs solution. The mixture was incubated at 25 for 15 min, then added to cells that were plated onto 12-well plates at a density of 8.5 × 10 5 cells/well in 1 ml growth medium. 72 h post-transfection, lymphocytes were harvested for cell count and viability using trypan blue exclusion method and gene expression was analyzed before and after editing 14 . The genomic cleavage e ciency was measured by GeneArt® Genomic Cleavage Detection kit.
Fluorescence was examined by immuno uorescence microscope (Labmed; USA), using Optika ISview image acquisition and processing software.

| Statistics
Statistical Package for the Social Sciences (SPSS, Chicago, IL) version 20 was used to perform all statistical analyses. Kruskal-Wallis test, Mann-Whitney rank sum U test, independent samples t-test, ANOVA and chi square test were used for comparison, as appropriate. (ROC) curves were used to explore the predictive value of investigated panel. The relationships between the investigated parameters were assessed using Spearman rank correlations. A 2-tailed p value of ≤ 0.05 was considered a statistically signi cant.

| Data And Resource Availability
The datasets and RESOURCE generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Results
3.1 | Demographic, clinical data and diabetes laboratory pro le of the diabetic and healthy groups Demographic data regarding age, sex and smoking showed no signi cant difference (p > 0.05). There were signi cant differences in fasting and 2 hour post prandial blood glucose level, BMI, blood pressure, lipid pro le, albumin and creatinine ratio (alb./creat)., fasting insulin, glycosylated hemoglobin A1c (HbA1c), HOMA-IR between the diabetic and healthy groups (p < 0.05) ( Table 1). We calculated the sensitivity and speci city of each partner in the genetic network by ROC curve analyses (Fig. 2s). The overall positivity rates of these candidate genes in the diabetic and healthy groups are shown in table (2). There was a highly signi cant positive correlation between each of the following: LncRNA-RP11-773H22.4, RET, IGF1-R, m-TOR mRNAs, glycemic control and insulin resistance. Whereas, we observed a marked negative correlation between each of the abovementioned parameters and miR-3163, miR-1 miRNAs, GLUT-4 and AKT2 mRNAs in both the diabetic and healthy groups (P < 0.01) using Spearman's correlation as shown in table (3).

.1 Effect of CRISPR/Cas9 editing on cell count and viability
By applying one-way ANOVA post hoc test, we could not observe any difference in lymphocyte's count and viability in the diabetic cell line before and after CRISPR/Cas9 editing. Of note, CRISPR editing slightly decreased the cell count and viability as compared to healthy control which may be because of its potential genotoxic effect or its companion chemicals used for its transfection for further study (Table 2s).  There was a marked positive correlation between each of the following: LncRNA-RP11-773H22.4, RET, IGF1-R, m-TOR mRNAs. However, we noticed a highly signi cant negative correlation between each of the above mentioned parameters and miR-3163, miR-1 miRNAs, GLUT-4 and AKT2 mRNAs among the diabetic groups before and after CRISPR/Cas9 editing and the control group (P < 0.01) using Spearman's correlation (Tables 5 and 6).

Effect of CRISPR/Cas9 editing on GLUT-4 and m-TOR proteins (major effectors in the insulin signaling pathway).
Lymphocytes obtained from healthy donors "normal pool" showed compact lymphocytes colonies with GLUT4 high uorescence intensity and loss of mTOR expression (Figs. 2a,b). On the other hand, lymphocytes obtained from T2DM showed a merged collection "small colonies" of cells which were uniformly distributed of enlarged size, high nuclear/cytoplasmic ratio, irregular nuclear contour, with membrane vesicles and marked mTOR uorescence intensity (+++) in cytoplasm and nucleus along with faint GLUT4 expression (+). After CRISPR editing, a discrete spread out lymphocytes were uniformly distributed of medium cell size, slightly regular nuclei, moderate nuclear /cytoplasmic ratio, and few membranous vesiculation with moderate faint uorescence of mTOR (+) and dense clustering expression of GLUT-4 with high uorescence intensity (+++).
both insulin resistance and pancreatic beta cell malfunction, leading to hyperglycemia 15 .
In this study, we rst constructed a competing endogenous RNA network and identi ed thereby, several potential key molecules. We described the important role played by non-coding RNAs in the pathogenesis of IR. Our study highlighted speci c lncRNA, miRNAs and mRNAs related to the pathogenesis of IR and obtained from public databases, which might be used as novel therapeutic targets for type T2DM. Secondly, we evaluated the expression of this identi ed genetic network in clinical samples. Finally, we evaluated the therapeutic e cacy of the gene editing tool CRISPR/Cas9 in the improvement of IR in type 2DM in lymphocyte cell line.
Our results demonstrated that there was signi cant up-regulation in the expression of LncRNA-RP11-773H22.4, RET, m-TOR and IGF1-R mRNAs, together with signi cant down-regulation in the expression of miR-3163, miR-1, GLUT-4 and AKT2 mRNAs in the diabetic group compared to the healthy control in both patients' clinical samples and lymphocyte cell line.
RET is a protooncogene residing on chromosome 10q11.2 coding for a tyrosine kinase receptor.it participates in many intracellular signaling pathways including the PI3K-AKT and MAPK-ERK. The involvement of RET oncogene in the same pathway of insulin signaling may contribute to its link with the molecular pathogenesis of insulin resistance. Mutational activation of RET protooncogene results in the overproliferation of the affected cells as well as increased risk of metastasis and poor clinical outcome 16 . Consequently, RET mutation has been associated with multiple endocrine neoplasia 17 , medullary thyroid carcinoma 18 , breast cancer 19 , Non-Small Cell Lung Cancer (NSCLC) 20, and many other malignancies. However, it has never been described in T2DM.
Glucose transporters (GLUTs), are a group of plasma membrane transporters that help glucose translocation into mammalian cells. The most noteworthy of these GLUTs is the glucose transporter 4 (GLUT 4), the center of most studies of insulin resistance since it is the major mean of glucose transport in insulinsensitive peripheral tissues i.e. skeletal muscles and adipose tissues. GLUT4, coded by SLC2A4 gene, transports glucose by a process of alternating between the plasma membrane and intracellular storage vesicles 21 . Surprisingly, GLUT4 has proven to be involved in cancer metastasis, through its role in cellular consumption of glucose at basal levels, cancer cell proliferation and survival in breast cancer cells 22 and multiple myeloma cells 23 .
AKT-2 is an important mediator in the PI3K pathway involved in insulin signaling and in uencing numerous downstream proteins that affect metabolism, growth, and cell survival. Akt2 expression is highest in insulin-sensitive tissues and is believed to contribute largely to the role of insulin in metabolism 24 .
Therefore, derangement in its protein product may contribute to the pathogenesis of insulin resistance and/or T2DM. Multiple studies have speci ed that Akt2 is responsible for insulin-dependent glucose uptake in humans as well as in rodents and its dysfunctional is related to insulin resistance and impaired glucose tolerance which goes hand in hand with our study 25 . AKT-2 ampli cation has been evident in one quarter of the cell lines of ovarian carcinoma and 2 out of 15 primary tumors of the ovary 26 . Furthermore, it has been implicated in other types of cancer, namely hepatocellular carcinoma 27 , breast and prostatic cancers 28 .The role of this gene is not exclusive to diabetes and cancer, it has also been shown to contribute to a lot of neurological and cardiovascular diseases 29 .
The role of miRNAs has been delineated in several studies related to various critical protein cascades that share in insulin signaling pathways 30 . Among the most notable of these miRNAs is micro-RNA-1, described in several diseases and cancers. micro-RNA-1 is highly expressed in muscle cells where it suppresses proliferation of precursor cells and encourages myogenesis 31 . miR-1 downregulation shown to be related to diabetes induced oxidative stress. A study by Chen et al., revealed that under oxidative stress insulin regulates the function of miR-1 through AKT activation in H9c2 cells 32 . These results are similar to our study results that revealed the deregulation of miR-1 level in case of T2DM. Levels of circulating miR-1 have been associated with occurrence of myocardial infarction 33 . Also, it has been reported to suppress the growth of hepatocellular carcinoma cells by modulating ET-1 34 and to be down-regulated in primary human lung cancers 35 , gastric 36 and breast cancers 37 .
In this study, the newly investigated miRNA: miR-3163 in T2DM is retrieved from databases based on its association with many downstream effectors in insulin signaling pathway. It was found that its level is signi cantly deregulated in T2DM in both clinical and cell line samples. In cancer, miR-3163 has been shown to have a role in suppressing the translation of Skp2 mRNA in NSCLC cells; hence, inhibiting their growth 38 . Furthermore, miRNA-363 strongly in uences ACG2 expression affecting cell growth, apoptosis and resistance to drugs in retinoblastoma neoplastic stem cells 39 . It also has been shown to suppress tumorigenesis in ovarian cancer 40 .
In addition, recent studies reported that lncRNAs may work as competing endogenous RNAs (ceRNAs) and crosstalk with mRNAs by competitively bind their common pool of miRNAs in most of human diseases 41 .
Interestingly, our study demonstrated that Lnc-RNA-RP11-773H22.4 is expressed in patients with T2DM and it was correlated with poor glycemic control and insulin resistance, thus could then be used as a target of novel therapy These non-coding RNAs have been proven to be involved in many nodes in the development of cancer and DM through affecting the expression of the disease associated genes at the epigenetic level, as well as transcriptional and posttranscriptional levels 8,42 .
Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that is critical for insulin signaling. It is now considered a key player in the activation of the phosphatidylinositol 3-kinase-AKT. The IGF-1R is considered to be one of the important receptor proteins for insulin signaling pathway. Speci cally, IGF-1 and its receptor affect the sensitivity of muscle to insulin. Interestingly, Chakraborty et al., found that miR-1 in uences the expression of both, IGF-1 and its receptor 43 . Through modulating IGF-1 and IGF-1R, miR-320 promotes insulin resistance in the endothelium and adipocytes. Moreover, it was previously established to be involved in malignant transformation of cells and neoplasia 44 and implicated in breast 45 , colon 46 and prostate cancers 47 . Taken all together, we can conclude that the importance of IGF-1 and IGF-1R in insulin signaling and tumorigenesis is similar. More recent studies have demonstrated that cross talk between insulin resistance and IGF-1R may also occur in malignancies, as increased insulin signaling is observed during the downregulation of IGF-1R in tumor cells 48 .
A key player in the phosphatidylinositol 3-kinase (PI3K)-related kinase family is mTOR, a special type of multi-subunit serine/threonine kinase. Surprisingly, mTOR is the target of the valuable drug rapamycin, which is used to coat coronary stents and prevent organ transplant rejection. This versatility can thus be explained by the critical role of mTOR in response to miscellaneous signaling cascades when stimulated by variations intracellular and environmental conditions 49 . Being a major node in the (PI3K/AKT/mTOR) pathway, we think that its disruption will result in hyperglycemia and diabetes 50 . mTOR activation in uences multiple diseases such as cancer 51 , obesity 52 , cardiovascular diseases 53 and neurodegenerative disorders 54 . Normal cells convey a special network interaction between all the aforementioned pathways. In our study there was a highly signi cant positive correlation between the expressions of LncRNA-RP11-773H22.4, RET, m-TOR mRNAs, HbA1c and HOMA-IR. We observed a markedly negative correlation as well between them and miR-3163, miR-1 miRNAs expression in the diabetic and healthy groups (P < 0.01). These correlations have been validated in both clinical samples and in lymphocyte cell line which concede with the in-silico data analysis. We believe that LncRNA-RP11-773H22.4 has an inhibitory effect on miR-3163 miRNA, thus releasing the inhibitory effect of that miRNA on RET mRNA and other mRNAs in the panel.
Our study provides evidence that CRISPR/Cas9 genome editing system is a simple, cheap, and fast tool to manipulate genomes which is expected to have a broader therapeutic application in insulin resistance by modulating the deregulated genetic network of insulin signaling pathway (Fig. 3).
gure (3): Proof of concept of ceRNA hypothesis in IR in T2DM In this paper, we used for the rst time an integrative approach based on computational method together with clinical validation to provide novel insights into the molecular mechanisms of IR. We implemented combined bioinformatics analysis to retrieve a set of ceRNA network (RET mRNA, LncRNA-RP11-773H22.4 and miRNA-3163 miRNA) related to insulin resistance and their targeting signaling pathway genes (IGF1-R, GLUT-4, AKT2 and m-TOR mRNAs) and (miR-1 miRNA), retrieved from public databases. Afterwards, we investigated the identi ed ceRNA network expression in patients' clinical samples and lymphocyte cell line by qPCR and IF. Then, we did CRISPR/Cas9 knockout of LncRNA-RP11-773H22.4 at the sequence of lncRNA-miRNA interaction in the cultured lymphocytes by designing more than one gRNA anking the area of interaction. This approach resulted in the restoration of normal expression of the identi ed genes in comparison to the healthy controls as con rmed by qPCR and IF. We thereby, suggest the CRISPR/Cas9 knockout of LncRNA-RP11-773H22.4 as a potential therapeutic target for treatment of T2DM.
Taken together, our study offers valuable insights that help to decipher our understanding of diabetes and insulin resistance.
More invitro functional studies are needed to validate our result and consider the safety issues related to CRISPR/Cas9 system; as genotoxicity and off target mutations with possibility of using more accurate nucleases to reduce off-target effects.
The study was approved by the ASU-Faculty of Medicine ethical committee. All methods were carried out in accordance with declaration of Helsinki guidelines and regulations. Informed consent was obtained from all participants and/or their legal guardians.
*Consent for publication.
All authors have read the manuscript and agree to all its contents. All authors give their consent for publication.
*Availability of data and materials.
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

*Competing interests.
The authors declare no competing interests *Funding.  Proof of concept of ceRNA hypothesis in IR in T2DM