The Expression Of H3K9Ac, H3K14Ac And H4K20TriMe In Epithelial Ovarian Tumors And Their Clinical Significance

Background and ObjectiveThe epithelial ovarian cancer derived from the monoptychial surface epithelium of the ovary. Due to our poor understanding of the molecular pathogenesis of this disease, we lack valid strategies for early detection and therapy. The survival rate of 5-year for the epithelial ovarian cancer is 30%~40%.Most studies about its pathogenesis focused on a large number of genetic changes, but this kind of mechamism can not completely explain the ovarian oncogenesis. At the same time the majority of studies about cancer gene therapy stay on the animal experimental stage and its clinical practice faced with many difficulties. Recently a lot of studies have demonstrated that epigenetic abnormalities may play important roles in ovarian oncogenesis except genetic mechanism. Genetics is based on the changes in DNA sequence, but epigenetics is defined as heritable changes in gene expression that are not accompanied by changes in DNA sequence. Epigenetics is represented by methylation of cytosines at CpG sites and histone modification, which has a crucial role in chromatin packaging and gene expression. Recent reseach has demonstrated that global histone modification patterns predict the risk of prostate cancer recurrence. Genome-wide changes in the structure of the epigenome induced by histone modification can lead to the genomic instability and carcinogenesis. The modification change of histone acetylation is not in DNA sequence, so this modification can be reversible. Histone deacetylase inhibitors (HDACIs) have been tested as antitumor drugs through reversing histone acetylation, which are under phase I/II clinical trials. But little is currently known about the role of histone modification in epithelial ovarian tumors. Our experiments were to detect the expression of acetylation of histone H3 Lys9, Lys14 (H3K9Ac, H3K14Ac) and trimethylation of histone H4 Lys20 (H4K20TriMe) in normal ovarian epithelium, ovarian adenomas, ovarian borderline tumors and ovarian epithelial carcinomas. And the relationship between the three markers expression and the histological grade and clinlic stage was analyzed. Then we tested the expression of H3K9Ac, H3K14Ac and H4K20TriMe in the epithelial ovarian carcinoma cell line SKOV-3 under different concentration of sodium butyrate. The aim of our studies was to explore the roles of H3K9Ac, H3K14Ac, and H4K20TriMe in the diagnosis and therapy of epithelial ovarian tumors.MethodsThrough immunohistochemical staining of samples of normal ovarian epithelium, ovarian adenomas, ovarian borderline tumors and ovarian epithelial carcinomas, we determined the mean optical density of expression of H3K9Ac, H3K14Ac and H4K20TriMe. We furtherly analyzed the relationship between the three markers expression and tissue differentiation and clinical stages. Then the epithelial ovarian carcinoma cell line SKOV-3 was cultured with different concentrations of sodium butyrate(0mmol/L, 4mmol/ L, 8mmol/L, 12mmol/L, 16mmol/L)and the expression of H3K9Ac,H3K14Ac and H4K20TriMe in SKOV-3 cells was detected by western blot.Results1. The expression levels of H3K9Ac and H4K20TriMe were gradually reduced in normal ovarian epithelium, benign ovarian adenomas, borderline ovarian tumors and ovarian epithelial carcinomas (P<0.05). But the expression of H3K14Ac remained invariable (P>0.05).2. The expression of H3K9Ac,H3K14Ac and H4K20TriMe has no difference in serous cystadenomas and mucinous cystadenomas, borderline serous cystadenomas and mucinous cystadenomas(P>0.05). Compared with mucinous cystadenocarcinomas the expression of H3K9Ac and H4K20TriMe in the serous cystadenocarcinomas was lower (P<0.05). But the expression of H3K14Ac has no difference in serous and mucinous cystadenocarcinomas (P>0.05).3. The reduced expression of H3K9Ac and H4K20TriMe correlated well with histological grade and advanced tumor stage. The expression of H3K9Ac and H4K20TriMe was lower in poorly differentiated carcinomas than that in the well differentiated carcinomas (P<0.05) and it was lower in the late clinical stage than those in the earlier stage (P<0.01).4. When the concentration of sodium butyrate was 4~16mmol/L, the growth rate of SKOV-3 cells had been greatly inhibited. The cell survivals of SKOV-3 cultured with SB for 48 hours are 85.6%, 62.4%, 52.6%, 48.6%, which assumed the obvious dose-related effects. The total cell numbers had the tendency of decrease with the same dose.5. HE staining was observed under the light microscope: the number of SKOV-3 cells cultured with SB was greatly reduced with the increase of concentration and many cells were dead. The morphology of SKOV-3 cells became round or orbicular-ovate for crenation and chromatin became compact hyacinthine spheroid for concentration. But the SKOV-3 cells cultured without SB grew in clumpes and the morphology of cells was fusiform or polygonal. The cytoplasm was rich and cell nucleus situated in the center. 6. After SKOV-3 cells were cultured with different concentrations of sodium butyrate(0mmol/L, 4mmol/L, 8mmol/L, 12mmol/L)for 48 hours, the expression of H3K9Ac was greatly increased and that of H3K14Ac and H4K20TriMe remained invariable.Conclusions1. The expression of H3K9Ac and H4K20TriMe was different between normal ovarian epithelium and epithelial ovarian tumors and was gradually down-regulated with the malignancy transformation of ovarian epithelial tumors. The down-regulated expression of H3K9Ac and H4K20TriMe maybe participate in ovarian epithelial tumors initiation which will provide a new way for the diagnosis of this tumor.2. The expression of H3K9Ac and H4K20TriMe had great difference in benign ovarian adenomas, borderline ovarian tumors and ovarian epithelial carcinomas. The reduced expression of H3K9Ac and H4K20TriMe correlated well with histological grade and advanced tumor stage. H3K9Ac and H4K20TriMe may be the potential markers for differential diagnosis between benign and malignant tumor and the prognosis of patients with epithelial ovarian carcinomas.3. The growth rate of SKOV-3 cells had been greatly inhibited when they were cultured with SB, which indicated that HDACI has the role of antitumor.4. The expression of H3K9Ac was greatly increased and the expression of H3K14Ac and H4K20TriMe remained invariable when SKOV-3 cells were cultured with sodium butyrate for 48 hours. They suggested that HDACIs display antitumor fuction through reversing special histone acetylation which maybe have great relation to the up-regulation of many crucial gene expressions.