AbstractBackgroundHepatocellular Carcinoma (HCC) is the leading cause of cancer deaths worldwide as well as in Egypt. We aimed to use Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) gene editing technique to induce forced down-regulation of the circRNA which consequently modified miRNA expression in HepG2 cell line to prove the regulatory relationship between the RNA parts of an in silico-detected competing endogenous RNA network in HCCMethodWe first retrieved hsa_circ_0000064-miR-1285-TRIM2 mRNA from public microarray databases followed by in silico modelling to mimic the regulation kinetics of cirRNA associated ceRNA network. Secondly, we performed polymerase chain reaction (PCR)-based amplification of synthetic fragments, Gibson assembly of both CRISPR and non CRISPR based circuits, E-coli transformation, plasmid purification, HePG2 cell line transfection. Finally Expression levels of the chosen RNAs in hepatocellular carcinoma (HCC) cell line, HepG2, were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the cytotoxic effect was validated by viability assay.TRIM2 protein expression was proved by immunohistochemistry and flowcytometry.ResultsInduction of hsa_circ_0000064 into HepG2 cell line via CRISPR-and non-CRISPR mediated synthetic circuit resulted in statistically significant decrease in cell number and, then, cellular viability with marked increase in hsa_circ_0000064 and TRIM2 mRNA levels and concomitant decrease in miR-1285 expression in HepG2 cell line compared with control (p<0.0). Moreover exogenous expression of hsa_circ_0000064 in HepG2 cell line showed increased expression of the tumor suppressor protein, TRIM2.ConclusionsOur integrative approach, including in silico data analysis and experimental validation proved that CRISPR-mediated synthetic circuit-based overexpression of hsa_circ_0000064 was more efficient than conventional transient transfection, representing a promising therapeutic strategy for HCC.Data AvailabilityOur Data was made available online on the IGEM wiki of team AFCM-EGYPT:http://2017.igem.org/Team:AFCM-Egypt. Synthetic parts have been submitted to IGEM Parts Registry.Financial DisclosureThe project was funded by Armed Forces College of Medicine AFCM, Zewail City of Science and Technology, National Research Center NRC, VitaBiotics, PHARCO Pharmaceuticals, Sim Era and DANUB Paintings. IDT provided 20 kb of DNA synthesis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Integrated analysis of competing endogenous RNA network revealing potential prognostic biomarkers of hepatocellular carcinoma
