Detection Of Aberrent GSTP1 Promoter Methylation Of In Tumor And Serum From Hepatocellular Carcinoma Patients And Cloning Of GSTP1 Methylated Promoter

Background and objective: Hepatocellular carcinoma is one of the commonest cancerous diseases worldwide, particularly in parts of Asia and Africa, rating the fifth in occurrence and the third in mortality. It presents one of the major health threats in China. In our country, most HCCs are associated with HBV infection and arise from cirrhosis. GSTs, encoded by several different gene family of α , μ, π and θ , are a family of important drug-metabolizing enzymes that catalyze the conjugation of carcinogens, natural toxins and exogenous drugs. The pi class GST is one of particular interest in investigating cancergenesis. Previous studies have proved that CpG island hypermethylation was an important molecular mechanism for the development of HCC. The epigenetic silencing GSTP1 gene expression by promoter hypermethylation was common in human HBV-related HCC. All of the reports indicated GSTP1 might serve as potential biomarker for the diagnosis and prognostication of HCC. The present study detected and analyzed GSTP1 promoter methylation in tumor, non-tumor and corresponding serum from HCC patients using MSP, in order to develop a sensitive, specific, and noninvasive blood test for cirrhosis developmentmonitoring and early detecting HCC.To further study the regulation mechanism of the GSTP1 gene methylation promoter, we would constructe the methylated and unmethylated GSTP1 gene promoter in vector pGL3-Basic, which contain the luciferase as reporter gene.Methods: 26 surgically resected cancerous, non-cancerous specimens of primary hepatocellular carcinoma patients and corresponding 32 peripheral serum samples were collected. Control subjects were obtained from peripheral samples of 12 healthy volunteers. Breast cancer cell line MCF-7 with CpG islands hypermethylation of GSTP1 was as positive control. All genomic DNA were extracted using common phenol-chloroform approach and the 5'CpG islands methylation status of GSTP1 gene were studied by methylation-specific PCR. Data of methylation and pathological data of hepatocellular carcinoma were statistically analyzed.We amplified the promoter region (-408-+36) of GSTP1 gene using PCR, and linked it to pMD 18 T-vector. After methylation treated by Sss I and untreated, the recombinant DNA were subcloned to luciferase reporter vector pGL3-Basic. The recombinants were verified by sequencing and methylation specific PCR.Results: GSTP1 promoter methylation was detected in breast cancer cell line MCF-7, while none of 12 healthy subjects was detected for GSTP1 methylation in their PBMCs. Aberrant GSTP1 promoter methylation was demonstrated in 23/26(88.5%) of cancerous tissues and 18/26 (69%) of corresponding non-cancerous liver tissues from HCC patients. The circulating tumor DNA was detected in fifth percent (16/32) of serum fromHCC patients. Significant correlation between methylation status of cancerous and non-cancerous was found (P = 0.022). The concordance of methylation in tumors and non-tumors, tumors and serum samples was statistical significance. (Kappa=0.454, P=0.006; Kappa = 0.264, P=0.047. respectively). Frequent GSTP1 CpG islands methylation in HCC is not associated with clinical parameters (HbsAg / cirrhosis history) (P>0.05), but is related to AFP of HCC significantly (P=0.022). The association of GSTP1 methylation status in non-tumors and sera is statistically significant (P=0.0003, Fisher's exact test). Additionally, metastasis was association with tumor methylation status ((Fisher's exact test, p=0.046).We obtained successfully the methylated GSTP1 promoter recombinant andunmethylatedone(pGL3- GSTP1 prom and pGL3- GSTP1 pro). Conlusion: The CpG island hypermethylation of GSTP1 gene is an important molecular event in the early stages of the multistep process of HCCs. The epigenetic silencing of GSTP1 gene expression by promoter hypermethylation play a vital role during the development of the HBV-related HCC, and may be a valuable marker for noninvasive disease monitoring and HCC early diagnosis.We gained the recombinants pGL3 - GSTP1 prom and pGL3 - GSTP1 pro. It established the foundament for further studying the regulation mechanism of the GSTP1 promoter.