Alteration Of The Hypomethylation Status Of Hoxa10 Gene In Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC) is the most common tumor of genital system, and also the fifth leading cause of cancer death in women as well as the first lethal gynecological malignancy. Moreover, although ovarian cancer constitutes approximately 25% of the cancers arising from the female genital organs, it accounts for approximately 50% of all deaths from gynecological cancers. To help improve the development of better diagnosis and better treatment, it is critical to understand molecular events involved in the development of ovarian tumors.HOX genes, a subset of the homeobox gene family, are well conserved at the genomic level during evolution. In addition to their roles as master transcriptional factors critical in the regulation of embryonic development, their stringently regulated expression patterns in various tissues and organs in adulthood indicate fundamental roles in maintaining homeostasis. A growing body of literature has emerged in the past decade on the involvement of HOX genes in the pathogenesis of cancer. Several studies have reported aberrant expression of a number of HOX genes in cancers. Aberrations of DNA methylation are now believed to be an important epigenetic alteration occurring early in many cancers including ovarian ones. In general, DNA methylation is one of the best-understood epigenetic changes in human cancers and may play important roles in carcinogenesis. Carcinogenesis is associated with changes in the epigenetic phenomenon, including two distinct and seemingly opposing trends: global decrease in cytosine methylation (hypomethylation) and methylation of cytocine in CpG islands (hypermethylation).In our previous study, Cheng et al. found that HOXA10 was strongly expressed in endometrioid and mucinous, but not in serous EOCs. It is already known that the high expression of HOXA10 gene is relative to the ovarian carcinogenesis, however the underling mechanism is unclear. This research is aiming at revealing a possible mechanism that regulates HOXA10 expression as it relates to hyomthylation status in epithelial ovarian cancers. Better understanding of the mechanism of HOXA10 expression might shed light on developing therapeutic strategies targeting HOXA10.Objective The purpose of this study was to determine the relationship between hypomethylation of HOXA10 gene's promoter and high expression in malignant ovarian tissues, and to confirm the level of hypomethylation in ovarian cell lines.Methods We performed the methylation status of 29 samples from ovarian carcinomas and 16 from normal tissues by methylation-specific polymerase chain reaction (MSP). Then, we evaluated the expression of mRNA and protein of HOXA10 in all samples to work out the relationship between the methylation status of HOXA10 and its expression in transcriptional and translational levels.We then confrmed our present study using SKOV-3 and HEY ovarian cancer cell lines treated with the demethylating agent 5-aza-2-deoxycytidine (5-Aza-dC) to detect whether the expression of HOXA10 in the two cell lines was altered. And the cell proliferation was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay.Results 1. HOXA10 is highly expressing in ovarian cancer tissues. HOXA10 expression was examined by qRT–PCR in 29 malignant ovarian tissues and 16 normal ovarian tissues. Western Blot analysis of the tissues confirmed that HOXA10 protein was present in malignant ovarian tissues. The real-time PCR assay further confirmed the quantitative relationship between the hypomethyaltion status and HOXA10 expression.2. The promoter of HOXA10 gene is hypomethylated in epithelial ovarian carcinomas. We determined the methylation status of the region in normal and malignant ovarian tissues using MSP, which showed that the promoter of HOXA10 gene in 17 out of 29 (58.62%) malignant cancers and in 4 out of 16 (25%) normal tissues was hypomethylated, and the result shows significant difference (p < 0.05). Combining the research data above, we arrived at three conclusions: (1) The promoter of HOXA10 gene is methylated in most normal ovarian samples, whereas hypomethylated in abundant epithelial ovarian cancers. (2) HOXA10 is highly expressing in epithelial ovarian cancers; nevertheless, the expression level is low in normal tissues. (3) Comparing the methylated and hypomethylated status of HOXA10 gene in different tissues (including normal and malignant ones), the HOXA10 expression is lower in former status and higher in latter status.3. Rescue of HOXA10 expression by 5-Aza-dC Treatment. The results showed that after the treatment of the SKOV-3 and HEY cell line with 5-Aza-dC, demethylation of HOXA10 and concomitant increasing HOXA10 expression occurred. And MTT test showed that the cell proliferation conditon varied on different cell lines.