The Research Of Demethylation And Expression Of ALDH1A3 Promoter In HBV-related Hepatocellular Carcinoma

Objective: As the fifth most common malignancy in the world,hepatocellular carcinoma(HCC)is at the third place in the mortality rate of tumor.About 600,000 people die of HCC worldwide each year.At present,hepatitis B(HBV)infection is the most important risk factor for HCC.There are around 248 million people who have chronic hepatitis B in the world while around 95 million in China.Some of these patients' hepatitis B may slowly develop into cirrhosis and then HCC.Presently,the diagnosis and treatment of HCC still meet critical issues,such as difficulty in early diagnosis,deficiency of effective treatment and original new drug,high rate of recurrence and metastasis,etc.Therefore,the research on molecular pathogenesis of HCC and the development of new technologies and drugs for the diagnosis and treatment of HCC are particularly important.As a member of the ALDH family,ALDH1A3 is mainly responsible for oxidizing total trans-retinal into retinoic acid and then participates in physiological processes in cells.More and more studies have found that ALDH1A3 expression is regulated by many factors and related to the occurrence,development and prognosis of tumors.Moreover,ALDH1A3 shows cancer stem cells(CSC)characteristics in a variety of tumors,which is closely related to the colony formation,ball formation,tumorigenesis and angiogenesis activity of cancer cells.As an important way of epigenetic modification,DNA methylation plays an important role in maintaining gene imprinting of genomic DNA,stability of chromosomes and transcriptional silencing of repeated genes.In the respect of epigenetics,DNA methylation can widely occur in HCC,which has been proved to be associated with HBV infection,but the role of HBV-induced changes in DNA methylation in the development of HCC is not clear yet.In this study,we use HCC clinical specimens and two kinds of HCC cells,equipped with methylation microarray,methylation PCR,qRT-PCR and western blot,to detect methylation level and expression of ALDH1A3 gene in HBV-related HCC so as to explain the relationship between ALDH1A3 gene and the HBV-related HCC.Methods:1.Collected 6 cases of HCC tissue from patients with HBV high-copy and 6 cases of tissue from ones without HBV-infected,and extracted DNA from these tissues.Used methylation microarray technology to compare and analyze the two sample groups so as to screen differential gene and site as well as compare the whole status of methylation and the level of ALDH1A3 methylation in the two groups.2.Collected and extracted DNA from 3 cases of HCC tissue from patients with HBV high-copy and 3 cases of tissue from ones without HBV-infected.Cultured and extracted DNA from HepG2.2.15 cells that express HBV and HepG2 cells that express HBV negatively.According to the result of differential site analyzed by methylation microarray,we designed the primers in promoter region of ALDH1A3.By using methylation specific PCR(MSP),the methylation differences in the promoter region of ALDH1A3 were further verified in tissues and cells.3.Extracted mRNA and protein from the two groups of HCC tissues and two kinds of cells in the previous step.Compared the mRNA and protein expression of ALDH1A3 in the HCC tissues and cells by qRT-PCR and Western blot.All data were analyzed by one-way analysis of variance(ANOVA)using SPSS 21.0 software and checked by normal test and anova test.P < 0.05 was considered to be statistically significant.Results:1.The results of methylation microarray analysis showed that the DNA of HCC tissues with HBV high-copy presented a hypomethylation state,and the screened ALDH1A3 from those genes that have differential methylation also showed hypomethylation state.2.The results of Methylation PCR showed that the ALDH1A3 promoter region of ALDH1A3 in HCC tissues with HBV high-copy presented hypomethylation while the promoter region of ALDH1A3 in HCC tissues without HBV-infected presented hypermethylation.The difference was significant(P<0.01).The promoter region of ALDH1A3 in HepG2.2.15 cell showed hypomethylation,while the promoter region of ALDH1A3 in HepG2 cell showed hypermethylation,the difference was significant(P< 0.01).3.The results of qRT-PCR and Western blot showed that ALDH1A3 expression in HCC tissues with HBV high-copy was higher than that in HCC tissues without HBV-infected at both mRNA level and protein level and the ALDH1A3 expression in HepG2.2.15 was higher than that in HepG2.The difference was significant(P< 0.05).Conclusion:HBV-related HCC has a whole genome hypomethylation and HBV mediates the demethylation and high expression of the promoter region of ALDH1A3 gene.