The Mechanisms Of CD147 Gene Transcriptional Regulation And Its Epigenetic Modification

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with an annual incidence rate of about 600,000 cases, among which 55% are in China. HCC is characterized by vascular invasion, rapid progression and poor prognosis. The 5-year overall survival rate of individuals with HCC is only 8.9%, and this has barely improved over the past two decades. New prognostic markers are needed to help identify patients who are likely to have a poor prognosis and benefit from more aggressive treatment approaches.CD147 is a transmembrane protein with highly glycosylated modifications. It contains two extracellular immunoglobulin domains (C2 and I set), exhibiting the characteristics of the immunoglobulin superfamily members. CD147 expression significantly contributes to tumor progression by inducing the expression of MMPs (mainly MMP-2 and MMP-9) and vascular endothelial growth factor, activating the urokinase -type plasminogen activator system, conferring the resistance of tumor cells to anoikis and thus promoting tumor invasion and metastasis. Additionally, CD147 overexpression in tumor cells was significantly associated with poor prognosis. Nonetheless, although CD147 plays a critical role in tumor progression and prognosis, the mechanisms underlying the up-regulation of this molecule in cancer cells remain largely unknown.The gene expression has been shown to be dynamically regulated by many factors at both the transcriptional and posttranscriptional level. Among these mechanisms, the transcription factors seem to play the most crucial roles in gene regulation. Up to date, the cis-acting elements and transcription factors involved in CD147 expression was not been descripted. 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.The most well-defined mechanism involved in the regulation of methylation-mediated gene expression is the direct binding blocking of transcription factors to their CpG-containing binding sites. Sp1 is a well-characterized sequence-specific transcriptional factor that regulates a large number of house-keeping and tissue-specific genes by binding to GC-rich DNA sequences in the promoter region of many human genes. Numerous previous studies have shown that methylation status of the Sp1 consensus binding site influences the Sp1 binding and thus regulates the gene transcription. The 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 cancer biotherapy. The first purpose of our study was to identify the minimal promoter region of CD147 and the critical transcriptional factors. 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 liver and HCC cell lines, and investigate the transcription factor binding status. 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 five parts in our study: (1) We detected the expression of cancer-associated molecule CD147 in HCC tissues and cell lines by immunohistochemical staining, real time RT-PCR and western blot; (2) We identified the minimal promoter region of CD147 by luciferase reporter assay and nested deletions of the CD147 promoter region; (3) With the assistance of the TRANSFAC database, the Sp1 binding sites were found in the critical promoter of CD147. Whether Sp1 could bind to the CD147 promoter was confirmed by point mutation experiment, in vivo and in vitro binding assays. These results were proven by blocking Sp1 using RNAi or mithramycin A treatment and upregulating Sp1 using transfection with eukaryotic expression vector; (4) CD147 promoter methylation statuses and related expression levels in normal and HCC cell lines and 54 paired HCC and adjacent non-tumor (ANT) tissues were respectively examined by bisulfite genomic sequencing, methylation-specific PCR, real-time RT-PCR, western blot and immunohistochemistry. Whether the binding of Sp1 to CD147 promoter could be influenced by methylation status was also investigated; (5) We characterized CD147 methylated sites in paraffin block specimen using methylation-specific PCR and BGS. For all HCC patients, follow-up analysis was carried out and the correlations of promoter methylation statuses with CD147 expression level, the clinicopathologic features and prognosis of patients were statistically analyzed.In this study, we explored the regulation of CD147 in HCC. The expression of CD147 was significant higher in HCC tissues than adjacent normal liver tissues. Over 80% of the HCC tissues expressed differential high levels of CD147. Then, we cloned the 5 -flanking region of human CD147 gene and identified a critical promoter region at -108 to -42 which contained one binding site for Sp1 (+1 as relative to the translation start site), which was essential in upregulating CD147 promoter activity. EMSA and ChIP assays demonstrated that Sp1 could bind to the CD147 promoter. These results were proven by blocking Sp1 using RNAi or mithramycin A treatment and upregulating Sp1 using transfection with eukaryotic expression vector. Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines. Demethylation with 5-Aza-2 -deoxycytidine led to increased CD147 expression through enhancing Sp1 binding affinity and in vitro methylation of CD147 promoter reduced its transcriptional activity. CD147 promoter methylation level in HCC tissues (22.22%) was lower than that in ANT tissues (46.30%; P<0.05). Within HCC tissues, a significant inverse correlation was observed between CD147 expression and methylation level (r = -0.615). Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1% vs. 58.3%; P<0.05) and death rate (83.3% vs. 50.0%; P <0.05) than patients with methylated CD147 promoter.In conclusion, our results suggest that Sp1 is essential for regulating the CD147 gene expression. We demonstrated methylation indeed presented in CpG island of CD147 promoter and regulated transcription of CD147. Promoter hypomethylation upregulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients. 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, intervening the methylation state of CD147 may become a new strategy for cancer therapy.