The Mechanism Study Of Notch1 Signaling Pathway To Promote The Invasion And Metastasis Of Gastric Cancer And Colon Cancer By Regulating The Expression Of Fascin1

Part one:The mechanism study of Notchl signaling pathway to promote the invasion and metastasis of gastric cancer by regulating the expression of FascinlSection 1:Study on the regulation of Notchl gene and Fascinl gene in gastric cancerObjectives:To explore the regulation relationship between Notchl gene and Fascinl gene in gastric cancer cells.Methods:1.The expression of Notchl and Fascinl were detected by immunohistochemical method in 40 cases of gastric cancer and adjacent normal tissues,and the expression of Notchl and Fascinl was detected in 4 cases of human gastric cancer and adjacent tissues by immunoblotting.2.The human gastric cancer cell lines MGC-803 and SGC-7901 were used as experimental objects,and Control-siRNA and Notchl-siRNA were transfected into cells respectively.The expression of mRNA and protein in Notchl and Fascinl in different groups were detected by Real-time and Western blot.3.The survival of Notchl and Fascinl was analysed by database.Results:1.The positive rate of Notchl protein in gastric carcinoma cancer and adjacent tissues were 62.5%and 20%respectively,and the difference was significant(P<0.05).The positive rate of Fascinl in gastric cancer was 30%,while no expression was detected in adjacent tissues,and the difference was significant(P<0.05).Spearman correlation analysis showed that the expression of Notchl and Fascinl in gastric cancer was significantly positively correlated(r = 0.394,P = 0.012).2.The results of Western blot showed that the expression of Notchl and Fascinl in gastric cancer was significantly higher than that in the adjacent tissues in 4 cases of gastric cancer and adjacent tissues.3.In gastric cancer cells,the protein and mRNA levels of Notchl and Fascinl were significantly decreased after Notchl-siRNA transfection with MGC-803 cells and SGC-7901(P<0.05),but there was no significant change in Normal group and Control-siRNA group.4.The overall survival and progression free survival of patients with high expression of Notchl or Fascinl were lower than those with low expression in gastric cancer by Kmplot database analysis.Conclusion:1.Notch1 and Fascin1 have the role of oncogene in gastric cancer,and they are positively correlated in gastric cancer.2.Notch1 can regulate the expression of Fascinl at transcription and protein level.3.The high expression of Notch1 or Fascinl in patients with gastric cancer has poor prognosis.Section 2:Notch1 gene regulates invasion and migration of gastric cancer cells by Fascin1Objective:To investigate the effect of Notch1 on invasion and migration of gastric cancer cells by Fascin1.Methods:1.Control-siRNA,Notch1-siRNA,Notch1-siRNA+Control plasmid and Notch1-siRNA+Fascin1 plasmid were respectively transfected into MGC-803 and SGC-7901 cells and then cultured for 24 hours,the ability of migration and invasion in different groups were measured by transwell experiment.2.Control-siRNA and Fascin1-siRNA were transfected into MGC-803 and SGC-7901 cells respectively.Then the migration and invasion ability of the cells in different treatment groups were detected by transwell experiment.Results:1.In MGC-803 and SGC-7901 cells of gastric cancer,compared with Control-siRNA group,the migration and invasion ability of Notch1-siRNA cells decreased significantly;compared with Notch1-siRNA group,the migration and invasion ability had no obvious change in Notch1-siRNA+Control plasmid group,but the migration and invasion ability was improved in Notch1-siRNA+Fascin1 plasmid group.This indicated Fascin1 can rescue the decrease of migration and invasion ability of gastric cancer cells induced by down-regulation of Notch1 expression.2.In MGC-803 and SGC-7901 cells of gastric cancer,the migration and invasion ability of Fascinl-siRNA group was significantly lower than that of Normal group and Control-siRNA group.Conclusion:Both Notch1 and Fascin1 can promote the invasion and migration of gastric cancer cells,and Notch1 can regulate the invasion and migration of gastric cancer cells by targetting Fascin1.Section 3:The mechanism of Notch1 regulating the transcription of Fascin1Objective:To investigate the possible mechanism of Notch1 regulation of Fascinl gene transcription in gastric cancer cells.Methods:1.It was predicted whether the transcription factor RBP-J k of Notchl signaling pathway has binding sites in the Fascinl promoter by the database.2.MGC-803 cells as experimental object,whether the RBP-J k had binding sites in the promoter of Fascinl was verified by chromatin immunoprecipitation(ChIP)assay.Gastric cancer cell line MGC-803 was cultured for 24 hours without any treatment,transfected with Control-siRNA and transfected with Notchl-siRNA,and the effects of different treatment factors on the binding of RBP-J k.and Fascinl promoter were detected by ChIP assay.Results:1.In the PROMO database,we predict that the transcription factor RBP-J k of Notch1 pathway has two potential binding sites on the Fascinl promoter.2.ChIP experiment:(1)When protein complexes was precipitated by specific antibody RBP-J k,the bands can be amplified in the sample,but the bands was not amplified by IgG antibody.The results showed that RBP-J k had binding sites in the promoter of Fascinl;(2)Compared with the Normal group and Control-siRNA,the band signal was weakened in Notch1-siRNA group,and the result showed that Notchl can regulate the binding ability of RBP-Jk on the Fascinl promoter.Conclusion:In gastric cancer cells,the transcription factor RBP-J k has binding sites on Fascinl promoter,and Notchl regulates Fascinl transcription by RBP-J k.Part Two:The mechanism study of Notchl signaling pathway to promote the invasion and metastasis of colon cancer by regulating the expression of FascinlSection 1:Study on the regulation of Notchl gene and Fascinl gene in colon cancerObjectives:To explore the regulation relationship between Notchl gene and Fascinl gene in colon cancer cells.Methods:1.The expression of Notchl and Fascinl were detected by immunohistochemical method in 60 cases of colon cancer tissues and paracancerous tissues,and the expression of Notchl and Fascinl was detected in 6 cases of human colon cancer tissues and paracancerous tissues by immunoblotting.2.The human Colon cancer cell lines HCT-116 and LoVo were used as experimental objects,and Control-siRNA and Notchl-siRNA were transfected into cells respectively.The expression of mRNA and protein in Notchl and Fascinl in different groups were detected by Real-time and Western blot.3.Control plasmid and NICD1 plsamid were transfected into cells respectively.The expression of protein in Notchl and Fascinl in different groups were detected by Western blot.Results:1.The positive rate of Notchl protein in colon cancer and paracancerous tissues were 73.3%(44/60)and 26.7%(16/60)respectively,and the difference was significant(P<0.05),and the expression of Notchl in colon cancer was positively correlated with TNM stage and lymph node metastasis.The positive rate of Fascinl in colon cancer was 55%(33/60),while no expression was detected in paracancerous tissues,and the difference was significant(P<0.05),and the expression of Fascinl in colon cancer was positively correlated with TNM stage,lymph node metastasis and tumor size.Spearman correlation analysis showed that the expression of Notchl and Fascinl in colon cancer was significantly positively correlated(r=0.288,P=0.026).2.The results of Western blot showed that the expression of Notchl and Fascinl in colon cancer was significantly higher than that in the paracancerous tissues in 6 cases of colon cancer and paracancerous tissues.3.In colon cancer cells,the protein and mRNA levels of Notchl and Fascinl were significantly decreased after Notch1-siRNA transfection into HCT-116 and LoVo cells(P<0.05),but there was no significant change in Normal group and Control-siRNA group.4.In colon cancer cells,the protein levels of Notchl and Fascinl were significantly increased after NICD1 plasmid transfection into HCT-116 and LoVo cells(P<0.05),but there was no significant change in Normal group and Control plasmid group.Conclusion:1.Notchl and Fascinl have the role of oncogene in colon cancer,and they are positively correlated in colon cancer.2.Notchl can regulate the expression of Fascinl at transcription and protein level.Section 2:Notchl regulates invasion and migration of colon cancer cells by FascinlObjective:To investigate the effect of Notchl on invasion and migration of colon cancer cells by Fascinl.Methods:1.Control-siRNA,Notchl-siRNA,Notchl-siRNA+Control plasmid and Notch1-siRNA+Fascinl plasmid were respectively transfected into HCT-116 and LoVo cells and then cultured for 24 hours,the ability of migration and invasion in different groups were measured by transwell experiment.2.Control-siRNA and Fascinl-siRNA were transfected into HCT-116 and LoVo cells respectively.Then the migration and invasion ability of the cells in different treatment groups were detected by transwell experiment.Results:1.In HCT-116 and LoVo cells of colon cancer,compared with Control-siRNA group,the migration and invasion ability of cells in Notchl-siRNA group decreased significantly;Compared with Notchl-siRNA group,the migration and invasion ability of cells had no obvious change in Notch1-siRNA+Control plasmid group;but the migration and invasion ability of cells was improved in Notchl-siRNA+Fascinl plasmid group.This indicated Fascinl can rescue the decrease of migration and invasion ability of colon cancer cells induced by down-regulation of Notchl expression.2.In HCT-116 and LoVo cells of colon cancer,the migration and invasion ability of Fascinl-siRNA group was significantly lower than that of Control-siRNA group.Conclusion:Both Notchl and Fascinl can promote the invasion and migration of colon cancer cells,and Notchl can regulate the invasion and migration of colon cancer cells by targetting Fascinl.Section 3:The mechanism of Notchl regulating the transcription of FascinlObjective:To investigate the possible mechanism of Notchl regulation of Fascinl gene transcription in colon cancer cells.Methods:1.HCT-116 cells as experimental object,whether the RBP-J k had binding sites in the promoter of Fascinl was verified by chromatin immunoprecipitation(ChIP)assay.HCT-116 cells was cultured for 24 hours without any treatment,transfected with Control-siRNA and transfected with Notchl-siRNA,and the effects of different treatment factors on the binding of RBP-J K and Fascinl promoter were detected by ChIP assay.2.Control-siRNA and Notchl-siRNA were transfected into HCT-116 cells respectively,and EMSA assay was used to detect the transcription factor RBP-J k DNA binding activity under different treatments.3.According to the position of the transcription factor RBP-J K binding sites,two Fascinl promoter fragments-1466～114(P1)and-973～+114(P2)were constructed,and the dual luciferase reporter was used to detect the activity of Fascinl promoter.4.Fascinl-P2 as the core promoter,base sequence of 2 RBP-J K binding sites separately and simultaneously mutated,and mutant plasmid(Fascin1-P2-M1,Fascinl-P2-M2,Fascinl-P2-M1-M2).Then mutant plasmid separately and with RBP-J k plasmid were transfected into cells and the dual luciferase reporter was used to detect the activity of Fascinl promoter.5.The Fascinl promoter activity was detected in Normal group,Control-siRNA group and Notch1-siRNA group by the dual luciferase reporter.Results:1.ChIP assay:(1)When protein complexes was precipitated by specific antibody RBP-J k,the bands can be amplified in the sample,but the bands was not amplified by IgG antibody.The results showed that RBP-Jk had binding sites in the promoter of Fascinl;(2)Compared with the Normal group and Control-siRNA,the band signal was weakened in Notch1-siRNA group,and the result showed that Notchl can regulate the binding ability of RBP-Jk on the Fascinl promoter.2.EMSA assay:Compared with Control-siRNA,Notchl down-regulation in Notch1-siRNA group can reduce the DNA binding activity of RBP-Jk.3.Dual luciferase reporter assay:(1)The fluorescence activity of Fascinl-P2 was significantly higher than that of Fascinl-Pl;(2)The Fascinl promoter activity was significantly decreased after single mutation and simultaneous mutation;After cotransfection with RBP-J k,the activity of Fascinl promoter in Ml group and M2 group could be reversed,but the activity of Fascinl promoter in the M1-M2 mutant group was not significantly changed;(3)When Notchl expression was down regulated,the activity of Fascinl promoter was significantly decreased,and RBP-J k could reverse the downward trend of Fascinl promoter activity induced by down-regulation of Notchl.Conclusion:In colon cancer cells,Notchl regulates the transcription of Fascinl by transcription factor RBP-J k;The Fascinl promoter region contains 2 RBP-J k binding sites,which can be combined with the Fascinl promoter.RBP-J k is a positive transcription factor that regulates the transcription of Fascinl.