基于CRISPR转录激活系统构建内源性着丝粒蛋白F稳定过表达的肝细胞癌细胞模型

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

摘要:

临床研究表明着丝粒蛋白F (centromere protein F, CENPF)与肝细胞癌(hepatocellular carcinoma, HCC)的发生相关。由于CENPF蛋白的分子量高达358 kDa，目前有关CENPF致癌机制研究使用的体内外模型主要基于CENPF表达敲低，尚缺乏将CENPF过表达以探究其与癌变因果关系的研究，CENPF过表达改变到底是“因”还是“果”仍不清楚。本研究旨在利用包括lentiMPHv2和lentiSAMv2两个载体的CRISPR/dCas9转录激活协同激活介体(synergistic activation mediator, SAM)系统构建内源性CENPF稳定过表达的细胞模型，为阐明CENPF过表达与HCC发生发展的因果关系提供有效工具。首先设计并合成能够特异性识别CENPF基因转录起始位点的单向导核糖核酸(single guide RNAs, sgRNA)，并将其插入lentiSAMv2质粒中。利用慢病毒载体将lentiMPHv2质粒导入Huh-7和HCCLM3细胞株中，用潮霉素B筛选后再用慢病毒载体将连接好的携带sgRNA序列的lentiSAMv2质粒导入细胞中，并用杀稻瘟菌素S继续筛选。对2种抗生素筛选获得的Huh-7和HCCLM3细胞株样品进行CENPF mRNA和蛋白表达水平的检测，结果显示sgRNA1和sgRNA4序列均能够激活内源CENPF的表达，其中sgRNA4诱导转录效果更加明显。本研究利用CRISPR/dCas9系统成功构建内源性CENPF稳定过表达的HCC细胞模型，为研究CENPF过表达与肿瘤发生的因果关系奠定基础，并为构建大分子量蛋白过表达的细胞模型提供参考方案。

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.