Molecular Mechanisms Of PTK7 Regulating Invasion,Metastasis And Apoptosis Of Esophageal Squamous Cell Carcinoma

BackgroundPTK7 is Receptor protein tyrosine kinase without Catalytic activity.PTK7 binds to the corresponding ligands and dimerizes,and its own phosphorylation activates a series of downstream cascades and regulates cell proliferation,differentiation,and apoptosis.In recentt studies,PTK7 is highly expressed in acute myeloid leukemia,colon cancer,non-small cell lung cancer,breast cancer and ovarian cancer,and antibody therapy targeting PTK7 can significantly reduce tumor-initiating cells and continue to promote tumor improvement.In general,PTK7 is closely related to tumorigenesis.Overexpression of PTK7 can promote the invasion and metastasis of esophageal squamous carcinoma cells,and the high expression of PTK7 inhibits the radiosensitivity of tumors.In the previous study,we verified the expression of PTK7 in esophageal squamous cell carcinoma and paracancerous tissues by immunochemical staining.The results showed that PTK7 was highly expressed in the cancer cell membrane,but not in the adjacent tissues.However,the mechanism of PTK7 in esophagealsquamous cell carcinoma is not well understood,so it is necessary to further explore the biological role and molecular mechanism of PTK7 in esophageal squamous cell carcinoma.This study intends to investigate the biological effects of PTK7 on proliferation,apoptosis,invasion and migration of esophageal squamous carcinoma cells by up/silently expression PTK7 gene,and to study its specific signaling mechanism regulating cell proliferation and apoptosis,finally,to provide experimental basis for the pathogenesis and biological treatment of esophageal squamous cell carcinoma.ObjectiveTo explore the molecular mechanism of PTK7 on invasion and metastasis of ESCC,reveal the theoretical basis of the proliferation,invasion and metastasis of ESCC,and to explore the specific mechanism of PTK7 regulating the invasion,migration and proliferation of esophageal cancer cells.Methods1.Immunohistochemical staining was used to detect the expression of PTK7 in ESCC tissues and normal tissues.PTK7 gene was firstly obtained from TE-1 Cells by reverse transcription polymerase chain reaction(RT-PCR),then Recombinant pcDNA3.1-PTK7 plasmad was constructed using the pcDNA3.1 vector system.2.Effect of PTK7 overexpression on proliferation,invasion andmigration,cell cycle and epithelial-mesenchymal transition of esophageal squamous carcinoma cells.(1)The human esophageal cancer cell line TE-1 was transfected with the PTK7 overexpression recombinant plasmid to construct a cell line stably expressing PTK7.(2)The expression of PTK7 protein in TE-1 was detected by Western blot.(3)The role of PTK7 in cell invasion was clarified by scratch test,colony formation experiment,Transwell experiment,flow cytometry.(4)Western Blot assayed the effect of PTK7 overexpression on epithelial-mesenchymal transition-related proteins E-cadherin,N-cadherin and Vimentin.3.Effect of silencing PTK7 expression on proliferation,invasion,migration,apoptosis and epithelial-mesenchymal transition of esophageal squamous carcinoma cells(1)siRNA was used to interfere with the expression of PTK7 in TE-1cells.And western blot analysis of PTK7 protein expression in TE-1after siRNA interference.(2)CCK8 and scratch test were used to detect the effect of siRNA interference on the growth and proliferation of TE-1 cells.The effect of plate clone formation assay on the clonality of TE-1 cells was detected by flow cytometry.Effects on the apoptosis of TE-1 cells,Transwell migration and invasion assays on invasion and migration ability.(3)Western Blot detects the effect of PTK7 silencing expression on E-cadherin,N-cadherin and Vimentin.4.Molecular mechanism of PTK7 regulating proliferation and apoptosis of esophageal squamous carcinoma cells.(1)Fluorescence quantitative PCR was used to detect the expressionof apoptosis-related factors in TE-1 cells after siRNA silencing PTK7 expression,and PTK7 was involved in the regulation of apoptosis in esophageal cancer cells by regulating the expression of TP53 gene.(2)Western Blotting detection of the expression of P38 and JNK proteins after overexpression and silencing expression on PTK7.(3)Fluorescence quantitative PCR and Western Blotting were used to detect the effect of PTK7 on the expression of MKK3 and MKK6.(4)Flow cytometry was used to detect the effects of cell apoptosis with simultaneous silencing PTK7 and MKK3.5.The SPSS22.0 software was conducted for statistical analysis.The mean ± standard deviation was used to represent the measuring data..The T test and variance analysis were used to Inter-group difference analysis,Statistical significance was defined as P < 0.05.Result1.PTK7 expression was positvie in 57 of 68 cases with esophageal squamous cell carcinoma,only 2 of 32 adjacent tissues(including the normal tissue and atypical cell proliferation)was positive for PTK7.After total RNA was extracted from TE-1 Cells,the target gene was obtained by reverse transcription,and then the recombinant plasmid PcDNA3.1-PTK7 was obtained by double digestion reaction.2.The expression of PTK7 protein in TE-1 cells transfected with recombinant plasmid was significantly higher than that of the control group,indicating that the PTK7 stable overexpressing cell line was successfully constructed.3.Overexpression of PTK7 has an obvious effect on proliferation,invasion and migration,cell cycle and epithelial-mesenchymal transition of esophageal squamous carcinoma cells.(1)Colony formation experiments and scratch experiments showed that the proliferation of PTK7 overexpressing TE-1 cells was significantly higher than that of control cells.(2)Transwell experiments showed that the number of TE-1 cells overexpressing PTK7 through the Matrigel polycarbonate membrane was significantly higher than that of the control cells,demonstrating that PTK7 overexpression can promote the invasion and metastasis of esophageal immortalized epithelial cells and cancer cells.(3)Flow cytometry showed that the apoptosis ability of PTK7 was significantly decreased after overexpression.It is proved that PTK7 promotes immortalization and inhibits apoptosis in cells.(4)Western Blot results showed that overexpression of PTK7 increased the expression of N-cadherin and Vimentin,and decreased the expression of E-cadherin.The results suggest that PTK7 may promote EMT in esophageal epithelial cells.4.Silencing expression of PTK7 has an important effect on proliferation,invasion,migration,apoptosis and EMT-related proteins ofesophageal squamous carcinoma cells.(1)CCK8 experiments,colony formation experiments,and scratch experiments showed that the proliferation of TE-1 cells was significantly lower than that of control cells after siRNA interference.(2)Transwell experiments showed that the invasion and metastasis ability of TE-1 cells was weakened by siRNA interference,and the reverse effect of PTK7 was promoted.(3)Flow cytometry showed that the apoptosis ability of TE-1 cells was significantly enhanced after siRNA interference.Reverse proof that PTK7 has an inhibitory effect on apoptosis.(4)Western Blot results showed that the expression of PTK7 could decrease the expression of N-cadherin and Vimentin,but increased the expression of E-cadherin.The reverse suggests that PTK7 protein may be involved in the EMT process of esophageal cancer.5.PTK7 regulates apoptosis of esophageal cancer cells through the MKK3/P38/P53 signaling pathway.(1)The results of real-time PCR showed that the expression of TP53 gene was significantly increased after silencing PTK7 expression in esophageal squamous carcinoma cells.(2)Western Blot results showed that silencing PTK7 can cause an increase in P38 phosphorylation,while overexpression of PTK7 can cause a significant decrease in P38 phosphorylation,but PTK7 expressionchanges to non-phosphorylated P38 and JNK and phosphoric acid.JNK has basically no effect.(3)Real-time PCR and Western Blot showed that the relative expression of MKK3 mRNA was significantly increased and significantly decreased in esophageal squamous carcinoma cells over-and overexpressing PTK7,while the expression of MKK6 was not changed.(4)Western Blot results showed that in TE-1 cells,silencing PTK7 significantly increased the expression of P53-Ser15 protein,while overexpression of PTK7 significantly inhibited the expression of P53-Ser15 protein.(5)Western Blot experiments confirmed that the expression of p53-ser15 was regulated by MKK3 and PTK7.(6)Flow cytometry suggests that the effect of PTK7 on apoptosis may be through MKK3.Conclusion1.The expression level of PTK7 in esophageal squamous carcinoma was significantly higher than the normal Tissue,and it is shown that the high expression of PTK7 is closely related to esophageal squamous carcinoma.2.PTK7 plays an important role in promoting the metastasis and invasion of ESCC,and has an important influence on the biological behaviors such as proliferation,apoptosis,invasion and migration of esophageal squamous carcinoma cells.3.PTK7 promotes the epithelial-mesenchymal transition pathway of esophageal squamous cell carcinoma,but its specific mechanism still needs further study.4.PTK7 inhibits apoptosis in esophageal squamous cell carcinoma may be through MKK3/P38/P53 signaling pathway.