The Molecular Mechanism Of NDRG1 Regulates Invasion And Metastasis Of Hepatocellur Carcinoma Via Integrin ?3

Background and objective:Liver cancer is one of the most common digestive tract malignancies,which has high morbidity and mortality.China is one of the countries with high incidence of liver cancer,the new and the number of deaths account for about half of the world.Liver cancer is a malignant tumor with difficulty in early diagnosis and poor prognosis.Recurrence and metastasis are the main factors affecting the prognosis of liver cancer.Thus it is a hot spot to search for biomarkers of liver cancer,including tumor susceptibility genes and metastasis-related genes.Our previous experiments confirmed that NDRG1 could inhibit the invasion and migration abilities of BEL7402 cell line.Therefore,the study of the molecular mechanism of how NDRG1 plays a role in tumor suppression was the focus of this study.The purpose of our study was to search for the changed metastasis-related genes after NDRG1 gene was knocked down,and to investigate the molecular mechanism of NDRG1 regulating expression of integrin ?3 through Smad2/3 pathway,which may provide new clues for invasion and metastasis of hepatocellular carcinoma.Methods:1.The changed metastasis-related genes after NDRG1 gene was knocked down in BEL7402 and SMMC7721 cells were detected by PCR Array assay.2.The expression of the changed metastasis-related genes after knockdown of NDRG1 in BEL7402 and SMMC7721 cells was detected by Western Blot,ITGB3 gene continued in-depth study.3.Real-Time PCR was used in detection of ITGB3 gene expression in different cell lines.SiRNA technology was used to interfere the expression of ITGB3 gene in BEL7402 cells,and the interference effect was verified by Real-Time PCR and Western Blot.4.After sh-NDRG1 and si-ITGB3 were co-transfected into BEL7402 cells,the effects of integrin ?3 on NDRG1 function were examined by Transwell migration and Matrigel invasion assay.5.Western Blot was used to test the expression of TGF-?/Smad signaling key molecule Smad2,Smad3,p-Smad2 and p-Smad3.6.BEL7402 cells were treated by sh-NDRG1 and SB431542,Western Blot was used to test the effect of TGF-?/Smad pathway inhibitor SB431542 on the expression of integrin ?3 after knockdown of NDRG1 in BEL7402 cells,and Transwell migration and Matrigel invasion assay were used to exam the effect of SB431542 inhibiting migration and invasion of BEL7402 cells induced by sh-NDRG1.7.BEL7402 cells were transfected with si-Smad2,si-Smad3 or common pathway regulated protein si-Smad4 by respectively,then Real-Time PCR and Western Blot were used to test the interference effects and to further verify the effect of the key proteins of TGF-?/Smad pathway on the expression of integrin ?3 regulated by NDRG1.Results:1.PCR Array demonstrated that NDRGI knockdown upregulated CTSL1,IL-1?,ITGB3,MMP10,SERPINE1 and downregulated MMP13,TNFSF10 in BEL7402 cells.The genes ETV4,ITGB3,MMP10 and SERPINE1 were upregulated in SMMC7721 cells,while CDH6 and TNFSF10 were downregulated.2.The results of Western Blot showed that the expression of integrin ?3 was up-regulated in BEL7402 cells and SMMC7721 cells after NDRG1 knockdown of 72h.3.The ITGB3 mRNA endogenous expression in various cells differed;BEL7402 cell was the most,followed by HepG2 cell,and the least was in SMMC7721 cell.The results indicated that si-ITGB3 could effectively interfere the expression of integrin?3 in BEL7402 cell.4.Transwell migration and Matrigel invasion experiment results indicated that si-ITGB3 could inhibit the migration and invasion of BEL7402 cells induced by sh-NDRG1,in other word,NDRG1 can inhibit the migration and invasion of BEL7402 cells by interfering integrin ?3 expression.5.NDRG1 knockdown could cause the expression of Smad2,p-Smad2 and p-Smad3 up-regulated in BEL7402 cells.6.The pathway inhibitor SB431542 could block the expression of integrin ?3 induced by sh-NDRG1 and block the migration and invasion of BEL7402 cells induced by sh-NDRG1.This result suggested that NDRG1 could inhibit the migration and invasion of BEL7402 cells via TGF-?/Smad pathway.7.The results showed that si-Smad2,si-Smad3 and si-Smad4 could effectively knockdown the expression of corresponding protein respectively;and the knockdown of Smad2,Smad3 or Smad4 could block the expression of integrin ?3 regulated by NDRG1,which further confirmed that NDRG1 inhibited integrin ?3 expression in BEL7402 cells by Smad2/3 pathway.Conclusion:1.NDRG1 knockdown upregulated the expression of ITGB3.2.NDRG1 could inhibit the invasion and migration ability of BEL7402 cells by interfering with the expression of integrin ?3.3.NDRG1 could inhibit the expression of integrin ?3 by blocking the Smad2/3 pathway in BEL7402 cells.