Piezo1 Protein Affects The Proliferation,cycle And Apoptosis Of Esophageal Squamous Cell Carcinoma Through The P53 Signaling Pathway

BackgroundEsophageal cancer ranks seventh in terms of incidence(604,000 new cases)and sixth in mortality overall(544,000 deaths),the latter signifying that esophageal cancer is responsible for one in every 18 cancer deaths in 2020.EC contains two types according to histological type:Adenocarcinoma(EAC)and Squamous cell carcinoma(ESCC).More than 80%of esophageal cancers are ESCC.ESCC is a malignant epithelial carcinoma of the esophageal epithelium.It has a poor prognosis,with an overall 5-year survival rate less than 20%.The formation of ESCC is a multi-step process,which involves a series of genetic and epigenetic changes.Therefore,it is urgent to seek new therapeutic targets for ESCC.Piezol is a mechanosensitive ion channel protein in mammals.This protein is a large transmembrane protein because it contains more than 2500 amino acids and 2432 transmembrane helices.Studies have found that cells could squeeze against each other to prevent the formation of tumors.However,high expression of Piezo1 can prevent living cells from squeezing and cause epithelial cells to cluster,leading to the formation of tumors.In epithelial cells,Piezol protein is a major factor in sensing mechanical stimuli to control cell behavior and is believed to be a key protein in tumor formation.ESCC belongs to epithelial cell carcinoma,and the esophagus is a site that receives more mechanical stimulation.Therefore,research on the role of Piezol in ESCC can provide the basis for its clinical application.P53 signaling pathway is a classic apoptotic pathway.As a transcription factor,P53 can be induced by a variety of factors,leading to cell cycle arrest and apoptosis.Many studies have shown that p53-mediated cell cycle and apoptosis play an important role in preventing tumor progression.Piezol has been reported to cause apoptosis in breast cancer,prostate cancer and other cancer cells,but how Piezo1 affects THE apoptosis of ESCC cells is rarely reported.In addition,the relationship between Piezol protein causing apoptosis and the P53 signaling pathway has not been found yet and needs further exploration.ObjectiveThrough immunohistochemistry and bioinformatics,Piezo 1 protein was highly expressed in ESCC.By constructing a esophageal squamous cell carcinoma cell line with stable knockdown of piezo 1 protein,the effect and mechanism of knockdown of piezo 1 protein on ESCC were verified at the cellular and animal levels,so as to provide a new target for the treatment of ESCC.Methods1.Immunohistochemistry and TCGA database data were used to validate the expression of Piezo1 in ESCC tissues.2.qRT-PCR(Real-time Fluorescent Quantitative Polymerase Chain Reaction)was used to detect the expression of Piezo1 protein in EC 109,EC9706,TE-1,KYSE30 and KYSE510,and selected for subsequent experiments.3.Lentiviral transfection was used to construct ESCC lines with stable knockdown of Piezo 1 protein.qRT-PCR and Western Blot were used to detect the mRNA and protein levels of Piezo 1 in the selected ESCC lines.Flou-4-Ca2+ fluorescent probe was used to investigate the effect of knockdown Piezo1 protein on Ca2+flux before and after adding Yoda1.4.CCK-8,scratch and transwell assay were used to detect the proliferation,migration and invasion ability of knockdown Piezol protein in ESCC.Flow cytometry was used to detect the effect of knockdown Piezo1 on the cell cycle of ESCC.TUNEL assay was used to detect the effect of knockdown Piezol on apoptosis of ESCC cells.5.Western Blot was used to detect the expression of knockdown Piezol on EMT pathway factors E-cadherin and N-cadherin.The protein expression of p53 pathway related factors p53,Bax and apoptosis factors Caspase3 and Cleaved-Caspase3 were detected by Western Blot.qRT-PCR was used to detect the p53,Bax mRNA expression in ESCC.Immunoprecipitation assay detected the interation between Piezo1 and p53 in ESCC.6.The cell lines of the ESCC knockdown group and the control group were inoculated subcutaneously into BALB/C nude mice to establish the mouse transplanted tumor model,the tumor weight and volume were observed and recorded.Western Blot was used to detect the expressions of p53,Bax,Caspase3,Cleaved-Caspase3,Ecadherin and N-cadherin protein of tumor tissue.7.The p53 inhibitor PFTa was added into the knockdown ESCC cell lines of Piezo1,and the protein expression levels of p53 and Bax were detected by Western Blot.Flow cytometry,TUNEL and subcutaneous tumor mouse model were used to detect the changes of cell cycle,apoptosis and proliferation of ESCC after knockdown of Piezol and inhibition of p53.The tumor tissues were collected and the expression levels of p53 and Bax proteins in tumor tissues were detected by Western Blot.Results1.The expression of Piezo1 protein was significantly higher in ESCC tissues than in paracancerous tissues via immunohistochemistry(P=0.004).The data of Piezol expression in 81 ESCC tissue samples and 11 normal esophageal tissue samples were collected from the TCGA database.The results showed that Piezo1 was highly expressed in ESCC compared with normal esophageal tissue(P<0.001).2.The mRNA expression of Piezol in EC109,EC9706,TE-1,KYSE30 and KYSE510 cells were verified by qRT-PCR,and it was found that the expression of Piezo1 in EC109(P<0.05)and EC9706(P<0.05)cells were higher than in other ESCC cells.Therefore,these two cell lines were selected for knockdown Piezol.3.Knockdown Piezo1(sh-Piezo1)and control(sh-NC)lentivirus were constructed and transfected into EC 109 and EC9706 cells.qPCR and Western Blot results showed that the mRNA and protein levels of Piezo1 were significantly reduced(EC109,P<0.05;EC9706,P<0.001).Before and after addition of Yodal,an agonist of Piezo1,the Ca2+ f1ow in ESCC cells was decreased compared with the control group(EC 109,P<0.001;EC9706,P<0.05).The results showed that the cell line of knockdown Piezo1 was successfully constructed.4.Compared with the control group,the migration(EC 109,P<0.001;EC9706,P<0.01)and invasion(EC109,P<0.01;EC9706,P<0.01)ability of ESCC were significantly reduced after knockdown of Piezo1.In EC 109 and EC9706 cells,compared with the control group,knockdown of Piezo1 increased the expression of the epithelial factors E-cadherin and decreased the expression of the mesenchymal factors N-cadherin.5.Compared with the control group,knockdown down Piezo1 could inhibited cell proligeration,blocked the number of G0/G1 phase cells to S phase and promoted cell apoptosis(EC109,P<0.001;EC9706,P<0.001);Knockdown of Piezo1 significantly increased the mRNA and protein expression of p53(EC109,P<0.01;EC9706;P<0.01)and its downstream factor Bax(EC109,P<0.01;EC9706,P<0.01),The protein expression levels of apoptotic factors Caspase3 and Cleaved Caspase3 were also significantly increased.Immunoprecipitation results showed that Piezo1 interacted with p53 in ESCC.6.In vivo experiments showed that knockdown Piezo1 inhibited tumor growth compared with the control group(P<0.01).The tumor tissues in mice were extracted,Western Blot results showed that compared with the control group,the expression of p53,Bax,Caspase3,Cleaved-Caspase3,and E-cadherin in the knockdown Piezol group were up-regulated,while the expression of N-cadherin was down-regulated.7.In the rescue experiment,the expression of p53 and Bax was significantly down-regulated after the addition of p53 inhibitor PFTα,and consistent results were obtained in tumor tissues.Meanwhile,the effects of knockdown of Piezo1 on cell cycle and apoptosis of ESCC cells were reversed.In addition,subcutaneous tumor results showed that knockdown of Piezol and inhibition of p53 reversed the inhibitory effect of Piezol on tumor growth in ESCC.Conclusions1.Piezo1 protein was highly expressed in ESCC.2.Knockdown of Piezol inhibits the proliferation,migration and invasion of ESCC cells,promotes apoptosis,blocks the cell cycle and thus inhibits the development of ESCC.3.Piezo1 regulates the p53 pathway by interacting with p53,thereby affecting the apoptosis of ESCC cells.