Construction of a stable centromere protein F overexpression cell model of hepatocellular carcinoma using CRISPR activation system

Abstract:

Current studies have shown that centromere protein F (CENPF) was overexpressed in hepatocellular carcinoma (HCC) and might be involved in the pathogenesis of HCC. Specifically, due to the very large molecular weight (358 kDa) of CENPF full length protein, only CENPF knock-down, but not overexpression models, were applied currently to explore the carcinogenicity of CENPF in HCC. Whether CENPF overexpression is a cause or an effect in HCC remains to be illustrated. We aimed to establish a CENPF overexpression cell model using CRISPR/dCas9 synergistic activation mediator (SAM) system with lentiMPHv2 and lentiSAMv2 vectors to explore the role of CENPF overexpression in HCC. Single guide RNAs (sgRNAs) that specifically identify the transcription initiation site of CENPF gene were synthesized and inserted into the lentiSAMv2 plasmid. Huh-7 and HCCLM3 cells were first transduced with lentiMPHv2 and then selected with hygromycin B. The cells were then transduced with lentiSAMv2 carrying specific sgRNA for CENPF gene, followed by blasticidin S selection. The mRNA and protein detection results of Huh-7 and HCCLM3 cells screened by hygromycin B and blasticidin S showed that the endogenous overexpression of CENPF can be induced by sgRNA1 and sgRNA4, especially by sgRNA4. By using the CRISPR/dCas9 technique, stable cell models with overexpressed CENPF were successfully constructed to explore the role of CENPF in tumorigenesis, which provides a reference for the construction of cell models overexpressing large molecular weight protein.