Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma.
Bottom Line: Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines and tissues control.Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1%versus 58.3%; P < 0.05) and death rate (83.3%versus 50.0%; P < 0.05) than patients with methylated CD147 promoter.In conclusions, promoter hypomethylation up-regulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients.
Affiliation: State Key Laboratory of Cancer Biology, Cell Engineering Research Centre & Department of Cell Biology, Fourth Military Medical University, Xi'an, China.Show MeSH
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Mentions: To directly examine if Sp1 binds to the putative binding site within the critical CD147 promoter region, EMSA was performed with biotin-labelled oligonucleotides spanning the Sp1-binding sites as probe. Shifted complexes were observed when the labelled wild-type probe was used (Fig. 5A, lane 2), while these complexes disappeared when 100-fold molar excess of unlabelled wild-type probe was added (Fig. 5A, lane 3). EMSA was also performed by using mutant probes in order to understand the contribution of the Sp1-binding sites to the observed binding patterns. The mutation in the Sp1-binding sites dramatically decreased the level of complex formation (Fig. 5A, lanes 4). To further confirm the specific binding of Sp1 to the CD147 minimal promoter, supershift assay was performed with anti-Sp1 antibody. The protein–DNA complex of interest was supershifted by anti-Sp1 antibody (Fig. 5A, lane 6). However, no supershift was observed when control IgG was used (Fig. 5A, lane 5). We also observed that Sp1 could not bind to the methylated CD147 gene promoter probe (Fig. 5A, lane 7). The EMSA results demonstrated that Sp1 specifically bound to the Sp1-binding sites in the CD147 gene promoter and this binding could be interfered by methylation status.
Affiliation: State Key Laboratory of Cancer Biology, Cell Engineering Research Centre & Department of Cell Biology, Fourth Military Medical University, Xi'an, China.