Loss Of PLCE1 Effects The Invasion And Metastasis Of Esophageal Carcinoma Cells

BackgroundEsophageal squamous cell carcinoma(ESCC)is one of the most malignancies diseases with high morbidity,mortality and a poor prognosis.It’s incidence is a complex process involving many factors,variation accumulation and interaction of multiple genes.With the recent large clinic-based cohorts analysis and genome-wide association studies confirmed the phospholipase C epsilon 1(Phospholipase C Epsilon-1,PLCE1)gene as a susceptibility gene of esophageal cancer affects the occurrence and development of esophageal cancer.However,current studies of this gene do not fully explain the relationship between PLCE1 and invasion or metastasis of esophageal cancer.Therefore,it is of great significance to further study the function of PLCE1 in order to demonstrate the molecular mechanism of development of esophageal cancer.ObjectiveOur aim is to investigate the effect of PLCE1 on cell invasion and metastasis of esophageal cancer.This study can also provide new ideas and methods to the diagnosis of esophageal cancer and gene therapy.Methods1.we designed two single-guide RNAs(sgRNAs)targeting conserved exons,exon2 and exon3 respectively of the transcript to perform genetic inactivation of the gene in independent cell lines named EC-9706 which is derived from esophageal squamous cell carcinoma via CRISPR/Cas9 system.2.Using real-time quantitative PCR(qPCR)and Western blotting to detect the expression level of mRNA and protein between the two groups of cells(knockout and un-knockout).3.Combined with wound healing and transwell in vivo,the two groups of cells were observed in the migration and invasion.4.Observing the difference tumor graft growth kinetics about the two groups of cells in nude mice xenograft.5.Using flow cytometry to Detect the Mean fluorescent intensity between the cells of control group and the mutant group,PLCE1 gene was observed with epithelial mesenchymal transition(EMT)correlation related transcription factors(Snail、β-catenin、Slug、ZO-1).6.Detecting the expression level of protein in the mutant group and control group cells by Western blot firstly;Observing the expression level of protein about related transcription factors of EMT in cells and transplantation tumors,further understanding the functional association between the EMT and PLCE1 in esophageal squamous cell carcinoma.Results1.The sequence of target sites was knocked out successfully by using CRISPR/Cas9system;2.The results of qPCR and Western blotting showed that the expression level of mRNA in control-type cells was significantly higher than that in mutant-type cells(P <0.01);The results of Immunobloting indicated that during the mutant-type cells,which protein expression was deprived completely.3.The results of wound healing showed that the wound healing capacity of the mutant cells were significantly crippled(P < 0.01);For the migration assay,the cells that have migrated through the permeable filter were stained(P < 0.01);Transwell migration assay showed that the cell migration ability of the mutant-type cells was significantly lower than that of the control-type cells(P < 0.01);The PLCE1 deprived cells significantly decreased their invasion ability through the basement membrane.4.The results of nude mice xenograft indicated that the volume and weight of tumorin mutant group were significantly lower than the control group,and the growth capacity was decreased(P < 0.01).5.The Mean fluorescent intensity results of flow cytometry showed that Snail,ZO-1and other transcription factors in EMT were expressed in mutant-type cells,whereas it was highly expressed in wild-type cells(P < 0.001).6.The results of Immunobloting indicated that Snail,one of transcription factors about EMT,which was high expression in the control-type cells compared to the mutant-type cells;The protein expression of Snail was significantly decreased in the mutant group of nude mice xenograft,these results further illustrates the flow cytometry results from the level of protein.ConclusionsLoss of PLCE1 can reduce the migration and invasion of esophageal squamous cell carcinoma cells.This gene can affect the functional changing of esophageal squamous cell carcinoma by EMT.The interaction between PLCE1 and EMT might be a new direction for future research and gene therapy.