| Summary: | 博士 === 國防醫學院 === 醫學科學研究所 === 102 === Cervix infected by human papillomavirus (HPV) is an important factor for cervical cancer. HPV 16 and 18 are the most common high-risk types. The viral DNA is observed about 90% of cervical cancers and responsible for cervical cancer progression. Furthermore, E6 induces DNMT1 through repression of tumor suppressor p53. EZH2 is induced by E2F through E7-mediated release of E2F from pRB-E2F complex. Methylation of histone H3K27 acts mainly through EZH2, which is thought to be a potent repressive marker associated with gene silencing. The viral oncogene can cause aberrant gene expression through influencing epigenetic modifications. Our previous studies have demonstrated that inactivation of LMX1A (LIM homeobox transcription factor 1) gene is through promoter hypermethylation and LMX1A functions as a metastasis suppressor in cervical cancer with partially inhibition of EMT downstream genes.
Previous data have shown that LMX1A expression pattern is similar to Sp1 in cervical cancer progression. We found that Sp1 directly binds to the LMX1A promoter and activates endogenous LMX1A expression in cells. It implies that Sp1 is a transactivator for LMX1A regulation. Our data have shown that DNA methylation and histone modification are involved in the LMX1A regulation. Knockdown of EZH2 decreased H3K27me3 histone modification but was insufficient to restore LMX1A expression. Therefore, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A and then depleted drugs for 3 days. H3K14ac was enriched at the LMX1A promoter in EZH2-knockdown cells and LMX1A mRNA was still expressed. compared to control cells. Chromatin precipitation (ChIP) assay also showed that H3K27me3 and DNMT1 was decreased at the LMX1A promoter in EZH2-knockdown cells.
In this study, we found that overexpression of LMX1A increases the expression of inhibitor of the DNA binding 2 (ID2) but decreases the SNAIL expression. Previous data have shown that ID2 and SNAIL participates in epithelial mesenchymal transition (EMT) process. Overexpression of ID2 inhibited cellular proliferation and invasion abilities in cervical cancer. On the other hand, inhibition of ID2 enhanced the invasion and anchorage-independent growth (AIG) abilities. Using reporter assay, we found that LMX1A indirectly mediates the ID2 regulation. Furthermore, we performed co-immunoprecipitation (Co-IP) assay and observed that the LMX1A interacts with transcription factor 3 (TCF3, E47) or histone deacetylase 1(HDAC1).
We have demonstrated that LMX1A is positively regulated by Sp1. LMX1A may encounter epigenetic silencing during tumor precession by DNMT and EZH2. Suppression of EZH2 may delay resilencing of LMX1A after the removal of 5-aza-dC and trichostatin A in cervical cancer cells.
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