| Summary: | 碩士 === 國防醫學院 === 微生物及免疫學研究所 === 100 === Hepatocellular carcinoma (HCC) is one of the common malignant tumors in the world. Our previous work showed that LMX1A is downregulated through DNA methylation in cervical cancer. Furthermore, we characterized the tumor-suppressor function of LMX1A in cervical cancer in vitro and in vivo. Here, we would like to investigate whether LMX1A posses TSG function in HCC and is downregulated through epigenetic changes.
In order to study whether LMX1A plays as a tumor-suppressor gene in HCC, we overexpressed LMX1A in HCC cells and checked its effect on HCC cells. Ectopic expression of LMX1A could significantly inhibit colony formation. However, there is no significant decrease in cell proliferation by MTS assay. These data suggest that LMX1A posses TSG function in HCC cells. We used bisulifte sequencing and methylation-specific PCR (MS-PCR) to detect the promoter methylation status of LMX1A and found that HepG2 displayed promoter hypermethylation whereas the methyaltion status of PLC5 and Tong was low. RT-PCR analysis of these HCC cell lines showed transcriptional silencing. After PLC5 cells were treated with trichostatin A (TSA), histone deacetylase inhibitor (HDACi), LMX1A expression was restored. These data suggested that histone modification might be involved in the regulation of LMX1A.
There are several predicted Sp1 binding sites in the promoter region of LMX1A and some papers demonstrated that Sp1 may interact with histone deacetylase 1 (HDAC1), then down-regulate the promoter activity of target gene. Therefore, we want to further sutdy whether Sp1 and HDAC are involved in the regulation of LMX1A. LMX1A promoter luciferase activity was enhanced after PLC5 cells treated with TSA. After two Sp1 binding sites were mutated in LMX1A promoter, LMX1A promoter activity was decreased. It implied that Sp1 might play as a transactivator of LMX1A. Then we found Sp1 could enhance LMX1A promoter activity by overexpression of Sp1. Moreover, when we knocked down endogenous Sp1 by Mithramycin(MTM, Sp1 inhibitor), LMX1A promoter activity was failed to elevate after cells treated with TSA. This result further proved Sp1 might play as a transactivator of LMX1A in PLC5 cell. Furthermore, we found the lysine 14 of histone 3 and histone 4 were acetylated after PLC5 cells treated with TSA. Taken together, these data suggest that HDAC and Sp1 might be involved in the regulation of LMX1A, and after cells treated with TSA, Sp1 might play as a transactivator of LMX1A.
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