The Experimental Research On The Mrna Expression Of ASPP1, ASPP2 And The CpG Island Methylation Of Their Promoters In Hepatocellular Carcinoma

Objective: The p53 protein is one of the best-known tumor suppressors, and losing apoptosis function of p53 contributes to tumorigenesis. About 30～40% of hepatocellular carcinoma (HCC) had mutated p53 gene which led to lose apoptotic function of p53. Losing of apoptosis function of wild type p53 might be relate to abnormal expression of ASPP (apoptosis-stimulating protein of p53) gene family. The ASPP gene family consisted of ASPP1, ASPP2 act as potent activators which stimulate the p53's apoptotic function. To understand how important the ASPP1, ASPP2 in hepatocarcinogenesis, we investigated the mRNA expression levels of ASPP1, ASPP2 and detected the methylation status of gene promoter in seven kinds of HCC cell lines and surgical specimens from 52 patients with HCC and its adjacent no tumor tissues.Methods:1. ASPP1, ASPP2 mRNA level were detected by RT-PCR and their promoter methylation were analysed by MSP-PCR in seven HCC cell lines and also observed the changes of ASPP1, ASPP2 mRNA and promoter mehtylation in Huh7 HCC cell deal with DNA methyltransferases (DNMT) inhibitiors (5'-aza-2'CdR) and/or histone deacetylase (HDAC) inhibitors (TSA). To analyses the relation of abnormal expression of ASPP1, ASPP2 and their promoter hypermethylation in HCC cell lines.2. Surgical specimens from 52 patients with HCC and its adjacent no tumor tissues were investigated for ASPP1, ASPP2 promoter methylation by MSP-PCR, in which 41 patients with HCC and its adjacent no tumor tissues was investigated p53 mutated protein by immunohistochemistry. We analyses correlation with ASPP1, ASPP2 promoter methylation, p53 mutated protein and clinicopathological factors.Result:1. By RT-PCR, ASPP1 and ASPP2 mRNA expression level was prominent different in each cell line, but the expression level in all HCC lines was lower than in HL7702 liver cell. It was almost none in Huh7 HCC cell.2. By MSP-PCR, ASPP1 and ASPP2 promoter had hemimethylation in most HCC cell lines, but had unmethylation in HL7702 liver cell and HepG2 HCC cell and had hypermethylation in Huh7 HCC cell. The ASPP1 and ASPP2 promoter methylation level were different in each cell line and correlated with mRNA expression level.3. DNA methyltransferases (DNMT) inhibition (5'-aza-2'CdR) and/or histone deacetylase (HDAC) inhibitors (TSA) revered the ASPP1, ASPP2 promoter hypermethylation to unmethylation and up-regulated mRNA expression level in Huh7 HCC cell. Both had synergistic effect and dose-dependent.4. By MSP-PCR ASPP1 promoter methylation was 21.1%(11/52), ASPP2 was 34.6%(18/52), both of ASPP1, ASPP2 were 5.7%(3/52), each of ASPP1, ASPP2 was 50%(26/52) in 52 HCC tissues; in adjacent tissues ASPP1 was 15.3%(8/52), ASPP2 was 23%(12/52), both of ASPP1, ASPP2 were 5.7%(3/52), each of ASPP1, ASPP2 was 32.6%(17/52). By immunohistochemistry p53 protein positive (mutated type p53) was 34.1% (14/41) in HCC tissues and none in adjacent tissues. In HCC tissues it was negative correlation with ASPP1, ASPP2 promoter methylation and mutated p53 protein positive (p=0.042); in adjacent tissues it also was negative correlation with ASPP1, ASPP2 promoter methylation and tumor size (p=0.022), clinical classification (p=0.046).Conclusion:1. Downexpression of ASPP1, ASPP2 is generally presence in HCC cell lines and related to theirs promoter hypermethylation.2. ASPP1, ASPP2 promoter hypermethylation was negative correlation with p53 protein positive, tumor size and clinical classification.It suggested the abnormal expression of ASPP1, ASPP2 might be involved in hepatocarcinogenesis.