Study on the possible role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer

• Main Authors: Hsu, Chia-Lin, 許佳琳
• Other Authors: Lin, Ya-Wen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/89510494379121369071

碩士 === 國防醫學院 === 微生物及免疫學研究所 === 99 === 英文摘要（Abstract） Cancer has become the leading cause of death in Taiwan, and cervical cancer is the one with the highest incidence and mortality in women. In addition to genetic deletions and mutations, many studies suggested that epigenetic alterations including DNA methylation and histone modification are also involved in inactivation of tumor suppressor genes (TSG). An epigenomic approach used in our previous work showed that LMX1A is methylation-silenced in cervical cancer. LMX1A, a LIM-homeobox gene, is known to participate in developmental event. Furthermore, we characterized the function of LMX1A by examining cell lines, animal models and human cervical neoplastic tissues, and found that tumor-suppressor function of LMX1A in cervical cancer, especially in invasion and metastasis. Hence, we would like to further elucidate what mechanisms are involved in epigenetic silencing of LMX1A in cervical cancers. According to the preliminary data, LMX1A was re-expressed in cervical cancer cells which were treated either with 5-aza-2'-deoxycytidine (DAC) only or Trichostatin A (TSA) in combination, we speculated that histone modification might be involved in regulation of LMX1A. Further, we identified the promoter region of LMX1A. After bioinformatic analysis, several transcription factor binding sites, including Sp1 are found in the promoter region of LMX1A. One report demonstrated that HPV-16 E6 and E7 oncoproteins activate Sp1 expression through TGF-beta pathway in cervical cancer. Dysregulation of p53/Sp1 control lead to DNA methytransferase-1 (DNMT1) over-expression has been reported in lung cancer. In addition, Sp1 may interact with histone deacetylase 1 (HDAC1), then down-regulate target gene promoter activity in glioma. Moreover, another report indicated enhancer of zeste homolog 2 (EZH2), which is a highly conserved histone methyltransferase, may be an important epigenetic regulator in cervical cancer carcinogenesis. Therefore, we propose that Sp1 or EZH2 might be involved in the regulation of LMX1A expression. At first, we found Sp1 could enhance LMX1A promoter activity by overexpression of Sp1. Then we use luciferase reporter assay and site-directed mutagenesis to prove Sp1 might be involved in the regulation of LMX1A. Secondly, we used RNA interfere (RNAi) to knock down human EZH2. We have selected three stable clones, and confirmed the knockdown efficiency by QPCR and western blot. However, the LMX1A expression level was not significantly restored. We also found that the methylation level of LMX1A promoter in siEZH2 stable clones was slightly lower than control. Taken together, these data suggest that Sp1 and EZH2 might be involved in the regulation of LMX1A. In addition to histone modification, our preliminary data showed that DNA methylation may take part in epigenetic silencing of LMX1A in cervical cancer.