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.
ABSTRACT
Objective: To identify key transcriptional regulatory regions of ezrin gene in HeLa cells, thus this work maked a preliminary study for revealing its transcriptional regulatory mechanism. Methods: The recombinant pGLB plasmids containing different lengths of DNA fragments upstream of translation initiation site of ezrin gene as reporter promoters were constructed using nested-deletion method, and the promoter activities were detected using dual-luciferase reporter assay system. The potential transcription factor binding sites at key transcriptional regulatory region of ezrin gene were predicted using on-line analyzing programme. Results: The recombinant pGLB plasmids containing different lengths of 5′-flanking region of ezrin gene were obtained. When the lengths of ezrin 5′-flanking region were reduced from - 1 324 to - 890, the transcriptional activity decreased by about 80%. If the length of 5′-flanking region were deleted from - 146 to - 32, the transcriptional activities were nearly abolished. Sp 1 transcriptional factor binding sites were ubiquitous at the - 1 324/ - 890 and - 146/ - 32 regions. Conclusion: The - 1 324/ - 890 and - 146/ - 32 regions were two key transcriptional regulatory regions of ezrin gene in HeLa cells. Sp 1 may be an important factor for regulating the transcription of ezrin gene.