TXNDC9 Promotes The Invasion And Metastasis Of Hepatocellular Carcinoma By Upregulation Of MMP2 And MMP9

Objective:The aim of this research was to investigate the expression of TXNDC9 in HCC and its relationship with clinical pathological features,to explore the mechanism by which TXNDC9 promotes the invasion and metastasis of HCC,and to confirm the scientific hypothesis that TXNDC9 promotes the invasion and metastasis of HCC by upregulating MMP2 and MMP9.Methods:1.Hepatocellular carcinoma tissues and corresponding adjacent liver tissues were collected from 80 patients with hepatocellular carcinoma surgically removed in the First Affiliated Hospital of Gannan Medical University from January 2021 to June2022.The expression levels of TXNDC9 m RNA and protein in hepatocellular carcinoma tissues and adjacent liver tissues were detected by q RT-PCR and Western blot,respectively.Meanwhile,the expression levels of TXNDC9 protein and MMP2,MMP9 proteins in hepatocellular carcinoma tissues and adjacent liver tissues were detected by immunohistochemistry.The correlation between the expression of TXNDC9 protein and MMP2,MMP9 protein was analyzed.2.The clinicopathological information of patients was tallied,and the relationship between the expression levels of TXNDC9 and clinicopathological features of patients was analyzed.3.The expression levels of TXNDC9 m RNA and protein in human immortal hepatic cell lines THLE-3 and hepatocellular carcinoma cell lines Hep G2,Hep3B,Huh7 and HCC-LM3 were detected by q RT-PCR and Western blot,respectively,and to screen the hepatocellular carcinoma cell lines with high expression of the TXNDC9.4.The expression of the TXNDC9 gene was interfered by siRNA,and the expression levels of TXNDC9 m RNA and protein in hepatocellular carcinoma cells before and after the interference were detected by q RT-PCR and Western blot,so as to detect the transfection effect.5.The expression of the TXNDC9 gene has been interfered by siRNA,and the expression levels of TXNDC9,MMP2 and MMP9 proteins in HCC cells were detected by Western blot before and after the interference,and to confirm that TXNDC9 could enhance the expression of MMP2 and MMP9.6.The Transwell assay were used to detect the changes in the invasion and migration ability of HCC cells before and after siRNA interference of TXNDC9 gene expression,and to determine whether there was a certain correlation between the TXNDC9 expression and the invasion and migration of hepatocellular carcinoma cells.7.The statistical data were analyzed using SPSS 22.0 statistical software,and Graph Pad Prism 8.0 was used for statistical graphing.The t-test orχ~2-test was used for comparison of differences between groups,and Spearman’s correlation analysis was used for correlation analysis.The differences were considered statistically significant at P<0.05.Results:1.The results of q RT-PCR and Western blot showed that the m RNA and protein expression levels of TXNDC9 in HCC tissues ware significantly higher than those in adjacent liver tissues,and the difference was statistically significant(P<0.05).The Immunohistochemical results showed that the positive rate of TXNDC9 protein expression in HCC tissues was 70.00%(56/80),which was significantly higher than that in adjacent liver tissues(37.50%);The positive rate of MMP2 protein expression in HCC tissues was 71.25%(57/80),which was significantly higher than that in adjacent liver tissues(31.25%);The positive rate of MMP9 protein expression in HCC tissues was 71.25%(57/80),which was significantly higher than that in adjacent liver tissues(28.75%),and the difference was statistically significant(P<0.01).Correlation analysis showed that the protein expression level of TXNDC9 and MMP2in HCC tissues had a positive correlation(r=0.605,P<0.001),and the protein expression level of TXNDC9 and MMP9 was positively correlated(r=0.643,P<0.001).2.The results of the correlation analysis between TXNDC9 expression level and clinicopathological features in hepatocellular carcinoma revealed that tumor number,vascular invasion,tumor capsule integrity,Edmondson-Steiner grade,and tumor TNM stage significantly correlate with the expression of TXNDC9(P<0.05).However,there was not significantly correlated with the gender,age,HBs Ag status,presence of liver cirrhosis,AFP level and tumor size(P>0.05).3.The results of q RT-PCR and Western blot showed that compared with the expression of TXNDC9 m RNA and protein in human immortal hepatic cell lines THLE-3,the expression of TXNDC9 m RNA and protein in all hepatocellular carcinoma cell lines up-regulated significantly,and the difference was statistically significant(P<0.05).In addition,the TXNDC9 m RNA and protein expression levels in HCC-LM3 cells were significantly higher than those in Hep G2,Hep3B and Huh7cells,and the difference was statistically significant(P<0.05).4.The results of the q RT-PCR and Western blot showed that the expression levels of TXNDC9 m RNA and protein in the HCC-LM3 cells that were transfected with TXNDC9-siRNA were significantly decreased compared with those in HCC-LM3 cells transfected with Control-siRNA,and the difference was statistically significant(P<0.01).5.The results of Western blot revealed that compared with the expression levels of TXNDC9,MMP2 and MMP9 proteins in HCC-LM3 cells transfected with Control-siRNA,there was significantly decreased in HCC-LM3 cells transfected with TXNDC9-siRNA,and the difference was statistically significant(P<0.01).6.The results of Transwell assay revealed that the invasion and migration abilities of HCC-LM3 cells transfected with TXNDC9-siRNA were significantly reduced,and the difference was statistically significant(P<0.01).Conclusion:1.The expression level of TXNDC9 in hepatocellular carcinoma tissues was significantly higher than that in adjacent liver tissues,And the expression levels of TXNDC9 protein in hepatocellular carcinoma tissues was correlated with the expression of MMP2 and MMP9 protein positively.2.The expression level of TXNDC9 was related to tumor number,presence of vascular invasion,integrity of tumor capsule,Edmondson-Steiner grade,and tumor TNM stage.However,there was no significant correlation with gender,age,HBs Ag status,presence of liver cirrhosis,AFP level,and tumor size.3.The TXNDC9 was discovered to be highly expressed in HCC cell lines Hep G2,Hep3B,Huh7 and HCC-LM3 when compared to human immortal hepatic cell line THLE-3,with HCC-LM3 cells having the highest expression.4.Downregulation of TXNDC9 expression significantly reduced the expression of MMP2 and MMP9 in HCC-LM3 cells.5.Downregulation of TXNDC9 expression significantly reduced the invasion and migration abilities of HCC-LM3 cells.