The Relationship Between G9a And Malignant Biology Of Hepatocellular Carcinoma And Its Clinical Significance

Background:Hepatocellular carcinoma is a worldwide disease that seriously affects human health.It is still unsatisfactory to the efficacy of liver cancer,using surgery combined with transcatheter arterial chemoembolization,molecular targeted therapy,immunotherapy and other comprehensive treatment,since early metastasis and postoperative recurrence is a common and fatal causes.The recurrence and metastasis of liver cancer presents a complex multi-factor process,with rapid progress,multiple steps,a variety of factors together.Therefore,it is urgent to find a gene that plays a central role in the recurrence and metastasis in the process of liver cancer.We can produce more effective drugs so that the survival and prognosis of patients can be improved.Euchromatic histone-lysine N-methyltransferase 2(EHMT2)is also known as G9 a,and its important feature is that the C-terminal contains the classical SET domain and plays histone methylation transferase(HMT)activity,which is the methylation of the protein.G9 a consists of two parts.The function of the longer part is to complete the amino acid sequence and the function of the shorter part is to identify the methylation site.G9 a expression levels in breast cancer,bone marrow cancer,bladder cancer,prostate cancer and colon cancer and other tumors were significantly increased.G9 a was highly expressed in non-small cell lung cancer tissues and could promote the invasion and metastasis of lung cancer at the cellular level.G9 a also showed high expression in endometrial carcinoma and promoted the growth of tumor cells.At the cellular level,the expression of G9 a was silenced by Small interfering RNA(Si RNA).The experiments in vivo and in vitro showed that the infiltration and metastasis of tumor cell were inhibited.G9 a was involved in proliferation,apoptosis and metastasis in many kinds of tumors.It was the focus of epigenetic research in recent years.However,G9 a had little research in hepatocellular carcinoma,and the role that G9 a plays in the progress and evolution of hepatocellular carcinoma was not reported.Objectives:To investigate the expression of G9 a in hepatocellular carcinoma and hepatocarcinoma cells,to investigate the relationship between G9 a and clinical pathology and prognosis of patients,to study the changes of cell viability and migration and invasion ability of hepatoma cells after silencing G9 a by cell experiment and nude mice experiment,to study the possible mechanism of G9 a in hepatocellular metastasis.Methods:To study the expression of G9 a in hepatocellular carcinoma and corresponding adjacent tissues by real-time fluorescence quantitative PCR(RT-qPCR)and immunohistochemistry,to investigate the expression of G9 a correlated with the clinicopathological features of patients and the prognosis of patients with hepatocellular carcinoma,to study the expression of G9 a down-regulated by lentivirus transfection in human hepatocellular carcinoma cell line,to observe the role of G9 a in vitro and in vivo in hepatocarcinoma cells,to screen the genes and targets that G9 a might play by PCR array,to verify the mechanism of G9 a by Western blot.Results:Part 1: 30 cases of primary hepatocellular carcinoma tissues and 30 cases of adjacent liver tissues were studied by RT-qPCR.The results showed that the mean level of G9 a m RNA in HCC tissues was 2.34 ± 1.00,and the mean levels of G9 a m RNA in normal tissues were 0.99 ± 0.29,liver cancer and adjacent tissues were significantly different expression(P <0.05)?The expression levels of G9 a in 350 patients with hepatocellular carcinoma were studied by immunohistochemistry.The results showed that there were 176 cases(50.3%)of G9 a positive expression,the express levels of G9 a were correlated with tumor TNM stage and tumor size(P <0.05),regardless of sex,age.Tumor size,TNM stage and G9 a expression levels can be used as an independent factor in monitoring the prognosis of patients with liver cancer by Cox analysis.K-M survival time curve showed that HCC patients survival time with higher expression statue of G9 a was significantly shorter(P<0.05).G9 a was negatively correlated with the prognosis of liver cancer and can be used as a molecular marker to predict the prognosis of patients with liver cancer.Part 2:We studied the expression levels of G9 a in five hepatocellular carcinoma cell lines by RT-qPCR and immunoblotting.The results showed that the expression levels of G9 a in HCCLM3 and MHCC97 H cell lines wrer the highest.Interference targets Y2713,Y2714,Y2715 have a knockout effect on G9 a expression.We established lentiviral stability strains successfully.Interference target Y2714 stable strains have a knockout effect on G9 a expression in HCCLM3 and MHCC97 H cells lines.Part 3:The ability of cell proliferation and invasion in vitro was decreased after knockouting G9 a in HCCLM3 cell lines.It had weak tumorigenicity after knockouting G9 a in vivo and tumor volume was significantly smaller and tumor growth was slower.Part 4: PCR array showed the expression levels of MMP2,MMP7,MMP9 and MMP10 in metalloproteinase(MMPs)was decreased,while the expression levels of TIMP3 wrer increased,and the expression levels of CD44,VEGF related to vascular invasion and metastasis were decreased.Western blot confirmed a decrease in H3K9me2 after downregulation of G9 a expression.The expression levels of MMP2,MMP9,CD44V6,VEGF m RNA and protein was down-regulated by RT-qPCR and Western blot,while TIMP3 was highly expressed..Conclusions: The expression levels of G9 a in hepatocellular carcinoma were significantly higher than that in adjacent tissues.The expression levels of G9 a were correlated with tumor TNM stage and tumor size,and was correlated with the prognosis of hepatocellular carcinoma and had clinical value in predicting the prognosis of hepatocellular carcinoma.In vitro cell experiments and in vivo nude mice experiments confirmed that G9 a enhanced HCC cell viability and migration and invasive ability.G9 a may enhance the expression of MMP2,MMP9,CD44v6 and VEGF through the specific molecular signaling pathway,which may influence the malignant ability of hepatocellular carcinoma cells,invasion and metastasis and other malignant biological behaviors.