胰腺癌细胞ezrin基因转录调控区及其gRNA靶位点鉴定  

Identification of ezrin gene transcriptional regulatory region and its gRNA target site in pancreatic cancer cells

作　　者：代基强 野庆松 白圣凯 张青峰 高书颖 DAI Ji-qiang;YE Qing-song;BAI Sheng-kai;ZHANG Qing-feng;GAO Shu-ying(Department of Bioengineering,Zhuhai Campus of Zunyi Medical University,Zhuhai 519041,Guangdong,China;School of Life Sciences,Nanjing University,Nanjing 210093,Jiangsu,China)

机构地区：[1]遵义医科大学珠海校区生物工程系,珠海519041 [2]南京大学生命科学学院,南京210093

出　　处：《医学研究生学报》2020年第2期133-138,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金(31360212);贵州省科学技术基金(黔科合J字[2014]2180号)。

摘　　要：目的ezrin基因在胰腺癌中过表达,其上游序列对于基因表达具有重要作用。文章拟敲除胰腺癌细胞ezrin基因转录调控区,鉴定其基因编辑gRNA靶位点。方法将携带ezrin基因上游片段的报告基因表达载体瞬时转染胰腺癌细胞Panc-1,采用双荧光素酶报告基因检测系统,鉴定ezrin转录调控区。使用在线软件预测ezrin转录调控区上下游gRNA靶位点,构建基因编辑重组质粒pX459-sgRNA-L和pX458-sgRNA-R。将重组质粒共转染Panc-1细胞,针对gRNA靶位点进行基因组DNA的PCR扩增和亚克隆测序分析,鉴定ezrin转录调控区的靶向敲除。向转染重组质粒的Panc-1细胞中加入嘌呤霉素初步筛选,采用水溶性四氮唑盐法检测细胞增殖能力。结果荧光素酶报告基因检测结果显示,Panc-1细胞中ezrin基因-1297/-1186片段对SV40启动子和ezrin启动子具有转录增强作用。亚克隆测序结果显示,携带ezrin转录调控区gRNA靶位点序列的重组质粒共转染至Panc-1细胞,细胞基因组DNA出现片段缺失,位于gRNA-L和gRNA-R靶位点之间。细胞增殖实验结果显示,转染重组质粒的Panc-1细胞,其增殖能力显著受到抑制。胰腺癌Panc-1细胞ezrin基因-1297/-1186为转录调控区,其上游gRNA-L和下游gRNA-R序列可作为基因编辑靶点。结论实现了ezrin转录调控区的靶向敲除,Panc-1细胞增殖能力的抑制有可能与ezrin转录调控区的敲除有关。Objective ezrin gene is overexpressed in pancreatic cancer,and its upstream sequence plays an important role in gene expression.This study intends to knock out ezrin transcriptional regulatory region and identify its gRNA target sites for gene editing in pancreatic cancer cells.Methods The reporter gene expression vectors carrying the upstream segment of ezrin gene were transiently transfected into Panc-1 cells.The ezrin transcriptional regulatory regions were identified by double luciferase reporter gene detection system.Then,the online software was utilized to predict the gRNA target sites located at the upstream and downstream of ezrin transcriptional regulatory region.Two recombinant plasmids pX459-sgRNA-L and pX458-sgRNA-R contained these two sequences were constructed for gene editing.Moreover,in order to identify the targeted knockout of ezrin transcriptional regulatory region,the recombinant plasmids were co-transfected into Panc-1 cells,and the genome DNA contained gRNA target sites were amplified,subcloned and sequenced.Finally,Panc-1 cells transfected with recombinant plasmids were preliminary sorted using puromycin treatment.The cell proliferation was detected by water-soluble tetrazolium salt method.ResultsLuciferase data showed that ezrin gene fragment-1297/-1186 enhanced the transcriptional activity of SV40 promoter and ezrin promoter in Panc-1 cells.Subclonal sequencing data revealed that the recombinant plasmids carrying the gRNA target sequence of ezrin transcriptional regulatory region were co-transfected into Panc-1 cells could trigger the genomic DNA fragments,which located between gRNA-L and gRNA-R target sites.Cell proliferation assay showed that the proliferation was significantly inhibited after transfection.Conclusion The targeted knockout of ezrin transcriptional regulatory region was achieved and the inhibition of Panc-1 cell proliferation may be related to this knockout.

关 键 词：EZRIN基因 胰腺癌 基因编辑 靶向敲除 

分 类 号：R735.1[医药卫生—肿瘤] R730.23[医药卫生—临床医学]