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Research Article
Sanaa Eissa
Ain Shams University
Corresponding Author
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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 efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatic tools, we identified and verified 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 significant 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 flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measurement of the network downstream proteins, GLUT4 and mTOR by immunofluorescence. CRISPR/Cas9 successfully knockout 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.
Conclusion: The presented 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.
Keywords
Insulin resistance, lncRNA, miRNA, genes, gene editing, Diabetes Mellitus
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No competing interests reported.
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A new preprint design is coming!
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
Sanaa Eissa
Ain Shams University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 23 Mar, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Epigenetics & Genomics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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 efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatic tools, we identified and verified 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 significant 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 flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measurement of the network downstream proteins, GLUT4 and mTOR by immunofluorescence. CRISPR/Cas9 successfully knockout 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.
Conclusion: The presented 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.
Figure 1
Figure 2
Figure 3
No competing interests reported.
This is a list of supplementary files associated with this preprint. Click to download.
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