5′CpG Methylation Of CD147 Promoter

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in Asia and Africa. The ratio of morbidity rate to mortality rate is 1:0.98. In China, there are about 120,000 people dead of HCC, about 44% of deadth of HCC in the world. The mortality rate of HCC ranks the second in all kinds of malignant tumors, so it is very important for prevention and treatment of HCC. Aberrant methylation of CpG islands within promoter region usually happened earlier than cell hyperplasia; the global hypomethylation occurs with the cancer genesis and becomes more obviously with cancer development. So changes in DNA methylation patterns can be detected for diagnosis and prognosis evaluation. According to our long-term study on HCC, CD147 is a membrane protein highly expressed on HCC. CD147 can promote tumor cells invasion and metastasis by stimulating tumor cells and neighboring fibroblasts secreting the matrix metalloproteinases (MMPs), and also can by other pathways such as urokinase-type plasminogen activator system, vascular endothelial growth factor (VEGF) and resistance of anoikis, etc.. Based on the proof of CpG islands having existed in CD147 promoter region, research in the association of methylation in HCC and its role in transcription regulation has significance in theory and application which helps understanding HCC genesis, development, early diagnosis and biotherapy of cancer.The first purpose of our study was to identify whether there was methylation involving in CD147 transcription. The second purpose was to analyze CpG islands within CD147 promoter, identify its potential methylated CG dinucleotide and methylation states in every site in normal and HCC cell lines. The third purpose was to investigate the association of methylation and HCC through detecting methylation states in generous HCC specimen, evaluate the feasibility of detecting methylation states as a new HCC diagnosis method.There were three parts in our study: (1) We identified the treatment condition of 5-Aza-DC. CD147 expression was detected in different HCC cell lines and normal liver cell line through FCS and realtime RT-PCR. The changes after 5-Aza-DC treatment were also detected by realtime RT-PCR; (2) The bisulfite procedure was optimized using pBluescript SK plasmid. Than we extracted genome DNA, amplified bisulfite promoter region and identified CD147 methylated sites in promoter region by cloning and sequencing; (3) We characterized CD147 methylated sites in paraffin block specimen using the same method as before.In this study, we optimized 5μm as the best 5-Aza-DC treatment concentration and reaction parameters of realtime RT-PCR. Quantitative results showed that the expressions of CD147 in HCC cell lines were obviously higher than that in normal liver cell and 5-Aza-DC treated cells higher than control respectively, which indicated that inhibiting methylation of CD147 promoter region could up-regulate the expression of CD147. In the second, the methylation state was observed by bisulfite DNA sequencing. There were totally 47 CG dinucleotides in the fragment of CD147 promoter from -200bp to transcript start site, and global hypormethylation was found in all HCC cell lines but not in normal cells. Identical results were also observerd in HCC tissues and normal liver and blood WBC. The methylation level was decreased from HCC tissues to normal tissues. We also found low degree of methylation in fetal liver, which suggested that there was methylation regulation in embryonic development.In conclusion, we demonstrated methylation indeed presented in CpG island of CD147 promoter and regulated transcription of CD147. These conclusions could help us improve our understanding the molecule machanism of HCC. Detecting the methylation state of CD147 promoter could be used for diagnosis and prognosis. Furthermore, interventing the methylation state of CD147 may become a new strategy for cancer therapy.