| Objective: Hepatocellular carcinoma(HCC)is one of the most malignant tumors in digestive tract,with poor prognosis and high mortality.More and more evidences show that B7 protein family plays an important role in all kinds of cancers,including HCC.However,little is known about the role of human endogenous retrovirus-H longterminal repetitive associated protein 2(HHLA2,also known as B7-H5)in HCC.The change of malignant biological behavior of hepatocellular carcinoma cells is of great significance to the occurrence and development of HCC.Therefore,the purpose of this study is to explore the mechanism of HHLA2 on the proliferation and invasion of hepatocellular carcinoma cells.Methods: 1.Using TCGA and GTEx database to analyze the difference of HHLA2 expression between HCC tissue and normal tissue.Immunohistochemical method was used to observe the expression of HHLA2 between HCC tissue and normal tissue,and to analyze the relationship between the expression level of HHLA2 and the clinicopathological features of patients.The Kaplan-Meier plotter database and clinical follow-up data were used to analyze the relationship between the expression level of HHLA2 and the overall survival of HCC patients.2.Hepatocellular carcinoma cell lines overexpressing HHLA2,silencing HHLA2 and negative control HHLA2 were constructed,and the overexpression and silencing efficiency were detected by Western Blot.Scratch healing experiment,Transwell invasion experiment,CCK8 experiment,EdU experiment and plate cloning experiment were used to explore the effect of HHLA2 on the fine proliferation,migration and invasion of hepatocellular carcinoma.The effect of HHLA2 on the proliferation,tumor size,weight and metastasis of hepatocellular carcinoma cells in vivo was investigated by subcutaneous tumorigenesis experiment in female nude mice.3.Using R software v4.2.2 to statistically analyze the tumor signal pathways related to HHLA2 in TCGA database and draw the thermal map and volcanic map of differentially expressed genes according to the expression level of HHLA2.4.The changes of SPP1 protein,PI3K/AKT/mTOR signaling pathway protein and EMT-related protein were detected by Western Blot.Interfere with the expression of SPP1 in hepatoma cell lines that overexpress HHLA2,that is,restore experiment,and observe the changes of proliferation,migration and invasion ability of hepatoma cells in different groups by scratch healing experiment,Transwell invasion experiment,CCK8 experiment and EdU experiment again.Western Blot was used to detect the effect of interfering SPP1 expression on PI3K/AKT/mTOR signaling pathway proteins.Results: The results of TCGA and GTEx database analysis show that the expression of HHLA2 in HCC tissue is significantly higher than that in normal liver tissue.2.Immunohistochemical results showed that the expression of HHLA2 in HCC tissue was significantly higher than that in normal tissue.The clinicopathological features of HCC patients showed that the high expression of HHLA2 was related to the low degree of tumor differentiation.Analysis of Kaplan-Meier plotter database and clinical follow-up data showed that the overall survival time of patients with high expression of HHLA2 was significantly shortened.2.Compared with normal hepatocytes,the expression of HHLA2 in hepatocellular carcinoma cells was significantly increased.Compared with the control group,the proliferation,migration and invasion of overexpressed HHLA2 hepatoma cells and their subcutaneous tumorigenesis in nude mice were significantly enhanced,while the proliferation,migration and invasion of silent HHLA2 hepatoma cells and their subcutaneous tumorigenesis in nude mice were significantly weakened.3.The analysis of HCC data in TCGA database shows that HHLA2 has a significant positive correlation with tumor proliferation,EMT and PI3K/AKT/mTOR signaling pathways,and the expression of SPP1 in the high expression group of HHLA2 is also significantly increased.The Western Blot results showed that the expression level of SPP1 and the phosphorylation level of PI3K/AKT/mTOR signaling pathway related proteins in overexpressed HHLA2 hepatoma cells were significantly increased,but the results in silent HHLA2 hepatoma cells were the opposite.The expressions of epithelial marker-related proteins E-cadherin and α-catenin were significantly decreased in hepatoma cells overexpressing HHLA2,while the expressions of interstitial markers Ncadherin and Vimentin were significantly increased,while the results of hepatoma cells silencing HHLA2 were the opposite.4.Interfering with the expression of SPP1 in the hepatoma cell line that overexpresses HHLA2 will not affect the expression of HHLA2,but can inhibit the promotion of proliferation,migration and invasion of hepatoma cells by overexpression of HHLA2.Interference with SPP1 expression will inhibit PI3 K phosphorylation in PI3K/AKT/mTOR signaling pathway,and then inhibit the proliferation,migration and invasion of hepatocellular carcinoma cells.Conclusion: HHLA2 gene is highly expressed in HCC tissues,and its high expression is related to the low differentiation of HCC patients and the shortening of overall survival.HHLA2 can promote the expression of SPP1 in hepatocellular carcinoma cells,and SPP1 can phosphorylate PI3 K downstream of it,thus activating PI3K/AKT/mTOR signaling pathway,which ultimately leads to the enhancement of the proliferation,migration and invasion of hepatocellular carcinoma cells. |
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