Identification of ezrin gene transcriptional regulatory region and its gRNA target site in pancreatic cancer cells
10.16571/j.cnki.1008-8199.2020.02.005
- VernacularTitle: 胰腺癌细胞ezrin基因转录调控区及其gRNA靶位点鉴定
- Author:
Ji-qiang DAI
1
;
Qing-song YE
2
;
Sheng-kai BAI
1
;
Qing-feng ZHANG
1
;
Shu-ying GAO
1
Author Information
1. Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, Guangdong, China
2. School of Life Sciences, Nanjing University, Nanjing 210093, Jiangsu, China
- Publication Type:Journal Article
- Keywords:
ezrin gene;
pancreatic cancer;
gene editing;
target knockout
- From:
Journal of Medical Postgraduates
2020;33(2):133-138
- CountryChina
- Language:Chinese
-
Abstract:
Objectiveezrin 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.MethodsThe 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.ConclusionThe targeted knockout of ezrin transcriptional regulatory region was achieved and the inhibition of Panc-1 cell proliferation may be related to this knockout.
