| Object:According to the World Health Organization,there were approximately 257 million chronic hepatitis B virus(HBV)carriers worldwide.HBV can escape the host’s natural immunity and is difficult to cure.Repeated inflammatory necrosis and fibrosis of liver cells caused by persistent HBV infection is an important pathological basis for liver diseases such as cirrhosis and hepatocellular carcinoma(HCC).Approximately 887,000 people die each year of HBV-related diseases,such as cirrhosis,HCC,and liver failure.In our country,about 85% of patients with primary hepatocellular carcinoma have chronic hepatitis B virus infection.These patients usually experience the classical liver disease progression model that is hepatitis-fibrosis(cirrhosis)-hepatocellular carcinoma,but the mechanism is complex and has not been fully elucidated.Large tumor suppressor kinase 1(LATS1)is a core component of the Hippo pathway,which can inhibit the biological function of Yes associated protein(YAP)as a transcription coactivator,then regulate the dynamics balance between cell proliferation and apoptosis,inhibit tissue and organ hyperproliferation.Inactivation of Hippo pathway and excessive activation of YAP have been shown to be closely related to the formation of a variety of tumors including hepatocellular carcinoma.There are also studies showing that Hippo-YAP pathway can affect the progression or regression of liver fibrosis by regulating the activation of hepatic stellate cell activation.These all suggest that LATS1 as an upstream factor of YAP may be a key factor linking liver inflammation repair,liver fibrosis progression,and HBV-related hepatocellular carcinoma after chronic HBV infection,but there is still no relevant research.DNA methylation is one of the important epigenetic methods,which is related to the progression of diseases such as tumors.Recent studies have shown that HBV can induce high methylation and low expression of tumor suppressor genes by increasing DNA-methyltransferase,related to the formation of Hepatitis B virus-related hepatocellular carcinoma.Previous studies have shown that LATS1 is highly methylated and lowexpression in some liver tumor cells.However,whether the expression level and promoter methylation status of LATS1 has changed or play roles in progression model of hepatitis-fibrosis(cirrhosis)-hepatocellular carcinoma is unclear.The object of this study: 1.To study the expression level and change pattern in different disease states during chronic HBV infection.2.To study the clinical significance of the expression level of LATS1 in the disease progression during chronic HBV infection.3.To investigate whether the promoter methylation status of LATS1 is a regulatory mechanism of LATS1 expression in different states during chronic HBV infection.Methods: 1.Study subjects and groups: A total 113 chronic HBV patients were enrolled from the department of infectious diseases at Shengjing Hospital of China Medical University,who all underwent liver biopsies,and liver inflammation and fibrosis were scored according to the Metavir scoring system.we divided the 113 cases into three groups as follows: low fibrosis group(stages F0 and F1),medium fibrosis group(stage F2),and high fibrosis group(stages F3 and F4).Another liver biopsy was performed in22 of the 113 cases several years after the first liver biopsy,and the degree of fibrosis progressed or regressed.Another 73 patients with HBV-related HCC who underwent surgical resection at Shengjing Hospital were enrolled,5 patients of which had liver biopsies of cirrhosis stage before progressing to hepatocellular carcinoma.2.The expression of LATS1,nucleus YAP and α-smooth muscle actin(α-SMA)in liver fibrosis tissue was detected by immunohistochemistry.Analyze the correlation between the expression of LATS1 and the clinicopathological parameters of chronic HBV patients with different degrees of liver fibrosis,Analyze the correlation between nuclear YAP expression and the degree of fibrosis.The relative expression of LATS1 m RNA was detected by real-time quantitative PCR.Western blot was used to quantitatively detect the expression of LATS1,phosphorylated YAP(p-YAP),and α-SMA in liver fibrotic tissues and analyze their correlation.3.The expression of LATS1 in HBV-associated HCC,adjacent tissues,and liver cirrhosis before HCC was detected by immunohistochemistry.The correlation between the expression of LATS1 in tumor tissues and the clinicopathological parameters of patients with HBV-related HCC was analyzed.Real-time quantitative PCR was used to detect the relative expression of LATS1 m RNA.The receiver operating characteristic curve was used to evaluate the diagnostic value of LATS1 in cirrhosis tissue of HCC.4.DNA was extracted from 25 cases of liver fibrosis tissue,30 cases of hepatocellular carcinoma and adjacent tissues to the tumor,and methylation specific PCR(MSPCR)and bisulfite sequencing PCR(BSP)were used to detect the methylation level of LATS1 promoter.Then we analyzed the correlation between the promoter methylation status and the expression level of LATS1.5.We used MSP and western blot to screen the cell line with high methylation level and low expression of LATS1 between Hep G2,Hep3 B,Huh7,PLC / PRF / 5 liver cancer cell line and HL-7702 liver cell line.We added 5-azacytidine to the cell.Then we detected the methylation status of LATS1 promoter by MSP,and detected the expression of LATS1,YAP,and p-YAP by western blot to investigate the effect of LATS1 promoter methylation level on LATS1 expression and the activity of Hippo pathway.Results: 1.Immunohistochemical staining score of LATS1 in F0 stage was 10.01 ± 1.71,F1 was 9.34 ± 1.97,F2 was 8.01 ± 1.82,F3 was 6.51 ± 1.74,F4(including liver cirrhosis)was 5.83 ± 1.52,analysis of variance result was p <0.001.The q RT-PCR method was used to detect the m RNA level of LATS1 gene in hepatocyte of low,medium and high fibrosis groups and the same conclusion was obtained(p=0.001).2.Multiple logistic regression analysis showed that the expression of LATS1 in liver tissue was independently negatively correlated with the degree of liver fibrosis(OR=0.501;p= 0.006;95%Cl 0.307-0.820),and independently positively correlated with HBV-DNA(OR=3.823;p= 0.004;95%Cl 1.540-9.492).Low expression of LATS1 in liver tissue was independently associated with HBe Ag loss(OR=0.162;p= 0.009;95%Cl 0.041 0.632).3.Immunohistochemical staining score of nuclear YAP expression in low fibrosis group(F0-1)was 0.8±0.67,in medium fibrosis group(F2)was 1.99±0.67,in high fibrosis group(F3-4)was 2.81±0.57,and the results of analysis of variance showed that with liver fibrosis progression,nuclear YAP expression was also increased,p <0.001.4.The expression of LATS1 in hepatocytes decreased in 9 out of 10 cases with progressed liver fibrosis,and the expression of active YAP increased in all of the 10 cases.The expression of LATS1 in hepatocytes increased in 7 out of 12 cases with liver fibrosis remission after antiviral treatment,whereas the expression of active YAP decreased in 9of the 12 cases.These observations suggested that the progression of liver fibrosis was related to the activation of the Hippo pathway in hepatocytes.5.In the same degree of liver fibrosis,the expression of PIIIP and hyaluronic acid were higher in serum of patients with low LATS1 expression.Immunohistochemical results showed that α-SMA expression was higher in patients with lower LATS1 expression.Western blot method also demonstrated that p-YAP expression was low andα-SMA expression was high in live with low LATS1 expression.6.Immunohistochemistry staining of LATS1 in 5 patients who suffered HBV-related hepatocellular carcinoma with liver cirrhosis showed that the LATS1 in liver cirrhosis tissue before HCC was significantly lower than that of adjacent cirrhosis tissue(p=0.003)and higher than that of tumor tissue(p=0.016).7.LATS1 expression level of liver cirrhosis tissue combined with serum AFP level may have better predicting value of HCC: AUC=0.884(0.810,0.958),diagnostic sensitivity 77.8%,specificity 96.2%.8.The immunohistochemistry staining score of LATS1 in 73 cases of HBV-related hepatocellular carcinoma tissue was 4.90 ± 2.31 and the adjacent tissues was 8.68 ± 2.37.The t-test showed that the expression of LATS1 in tumor tissues was significantly lower than adjacent tissues(p<0.001).The relative expression of LATS1 m RNA in tumor tissues was also significantly lower than that in adjacent tissues.The expression of LATS1 in tumor tissue was directly proportional to the degree of differentiation of hepatocellular carcinoma and inversely proportional to the serum AFP level.9.MSPCR method was used to detect the promoter methylation status of the LATS1.22(73.33%)of 30 cases of HBV-associated hepatocellular carcinoma were hypermethylation,while only 11 of the 30 adjacent tissues(36.67%)were hypermethylation.The level of methylation of LATS1 promoter in liver tumor tissues was significantly higher than in adjacent tissues(p = 0.009).The promoter methylation level of LATS1 in liver fibrosis tissues of different degrees was not significantly different.10.In 33 hypermethylation cases,25 cases were low expression of LATS1 and 8were high expression of LATS1,whereas in 27 unmethylated cases,only 3 cases were low expression of LATS1 and 24 were high expression of LATS1.The promoter methylation status of LATS1 region was closely related to the expression of LATS1.11.In 11 cases with early recurrence of hepatocellular carcinoma,promoter methylation status of LATS1 in 8 adjacent tissues(72.73%)were hypermethylation.And in 19 cases without early recurrence of hepatocellular carcinoma,only 3 cases of adjacent tissues(15.79%)were hypermethylation.This result suggest that the promoter methylation status of adjacent tissues was associated with early recurrence of hepatocellular carcinoma(p = 0.004).12.The result of BSP quantitative analysis showed that the promoter methylation level of adjacent tissue was significantly lower than that of tumor tissue,and the promoter methylation level of adjacent tissue without early recurrence was significantly lower than that of adjacent tissue with early recurrence.13.Cell lines with high methylation and low expression of LATS1 were treated with5-azacytidine.LATS1 expression and p-YAP expression was up-regulated after demethylation.It was confirmed that LATS1 methylation status regulates LATS1 expression and up-regulates Hippo pathway activity.Conclusion: 1.The expression level of LATS1 in hepatocyte decreased with liver fibrosis progression.During the progression from cirrhosis to hepatocellular carcinoma,the expression of LATS1 in tumor tissue decreased,while the expression of LATS1 in adjacent tissue increased.The expression of LATS1 in tumor tissue was significantly lower than that in adjacent tissue.2.During chronic HBV infection,LATS1 downregulation in the liver leading to inactivation of Hippo pathway is related to the progression of liver fibrosis.3.Abnormal high expression of LATS1 in cirrhosis tissue may be valuable in the prediction of hepatocellular carcinoma.The hypermethylation status of LATS1 promoter in adjacent tissue may be a predictor marker of early recurrence of hepatocellular carcinoma.4.The methylation status of LATS1 promoter is negatively correlated with the expression of LATS1 in hepatocellular carcinoma tissues and adjacent tissue.The expression of LATS1 is regulated by the methylation state of LATS1 promoter. |
