PLCE1 Is Essential For Invasion And Proliferation Of Esophageal Carcinoma Cells

BackgroundEsophageal cancer,occurs in the esophageal epithelium is one of the least study and the largest tumor,which is one of the most common gastrointestinal cancer,accounting for 2% of all malignancies.In recent years through large clinical specimen collection and genome-wide association analysis PLCE1 genes are closely related to the susceptibility of esophageal squamous cell carcinoma,but biochemical characteristics of PLCE1 are not fully understood in the development of esophageal cancer,which needed to be further clarified.So we knock out PLCE1 gene in esophageal carcinoma cell lines to study the specific role in tumor development.ObjectiveTo investigate the invasion and metastasis of esophageal cancer cells and the effect of proliferation and its related mechanism after PLCE1 gene is knocked out.MethodsCrisper / Cas9 gene editing technology for PLCE1 gene esophageal cancer cell lines was knocked out and application of gene sequencing,qRT-PCR and Western blot on the knockdown cell lines for testing to ensure that the protein is not expressed in PLCE1 knockout cell lines.The scratch experiment,Migration and Invasion assay experiment was applied to test the ability of Invasion and metastasis of esophageal cancer cells after knock out;CCK-8 method and assay of tumor-bearing nude mice detect the change of esophageal cancer cell proliferation before and after knockout;the change of cell cycle was test by flow cytometry before and after knockout;whole-genome RNA sequencing and qRT-PCR test the mRNA expression levels which related to the invasion and proliferation before and after knockout;Western blot check the EMT-related protein expression and snail distribution changes in the nucleoplasm before and after knockout.ResultsBy using gene sequencing we found that Crisper / Cas9 gene editing technology for esophageal cancer cell lines in PLCE1 gene knockout,the number of exon bases to increase or decrease is not a multiple of 3,realizing the frame shift mutation of PLCE1 gene.And by qRT-PCR and Western blot,esophageal cancer cell lines were detected after PLCE1 knockout,PLCE1 mRNA decreased obviously and PLCE1 protein is not expressed.Scratch test,Migration and Invasion experimental results showed that the invasion and metastasis decreased dramatically when knockout of esophageal cancer cell lines compared with the control group.After the adoption of CCK-8,esophageal cancer cell proliferation decreased after PLCE1 knockout;tumor-bearing nude mice experiments showed that after PLCE1 knockout,block tumor growth was significantly reduced.Whole-genome RNA sequencing and qRT-PCR experiments detected Invasion and proliferation-related mRNA expression levels were substantially reduced after PLCE1 gene deletion.After using Western blot we further found snail,?-catenin,zo-1 and Vimentin protein levels decreased significantly after PLCE1 gene deletion.After PLCE1 knockout,the distribution of Snail in nuclear plasma varied by slurry separation technology and nuclear and confocal microscopy,and snail decreased in the nucleus distribution.ConclusionCrisper / Cas9 gene editing technology can achieve the knockout of PLCE1 gene in esophageal cancer cell line.PLCE1 gene deletion can considerably reduce the proliferation of esophageal cancer cells,suggesting that the gene may be involved in regulating the proliferation of esophageal cancer cells;PLCE1 knockout deletions may significantly reduce the ability of invasion and metastasis of esophageal cancer cells,probably by regulating the snail which participates in the process of EMT to affect the invasion and metastasis of esophageal cancer.