Correlation With Promoter Hypermethylation Of TGFBI And Chemoresistance In Human Ovarian Cancer

Backgroud:Epithelial ovarian cancer is the most lethal disease among gynaecologic malignancies. Despite advances in surgical and chemotherapy management, mortality of ovarian cancer has remained virtually unchanged. Maximal surgical cytoreduction followed by systemic chemotherapy with carboplatin and paclitaxel is the current standard treatment modality for advanced ovarian cancer, and it improves the prognosis of untreated ovarian cancer.But the overall survival rate was still no significant improvement and5-year survival of advanced ovarian cancer remains <30%. The primary or acquired drug resistance is one of the main reason for treatment failure. Therefore, the discovery and reverse of chemoresistance has important clinical significance on improving the prognosis and survival rate of the ovarian patients.Chemoresistance is associated with a variety of factors, such as tumor gene amplification, translocation, deletion or mutation. In addition, epigenetics also has a close relationship with chemoresistance. The promoter methylation of CpG islands is one of the most important manifestation of epigenetics. Cancer cells exhibit significant changes in terms of DNA methylation, which can be summarized as global hypomethylation of the genome or focal hypermethylation. The silencing of suppressor gene induced by promoter hypermethylation can lead to chemoresistance.TGFBI is also called βig-H3which has been identified to regulate migration, cellular adhesion. As one of extracellular matrix(ECM), TGFBI is seen as suppressor gene because that it is upregulated in normal cells or immortalized cell lines but downregulated or undetectable phenotypes in tumor cell lines. Recently, promoter hypermethylation of TGFBI was found in lung, prostate and ovarian cancer. However, the role of TGFBI methylation in paclitaxel chemoresistance in ovarian cancer is unknown. In this study, we examined the methylation status and expression of TGFBI in epithelial ovarian cancer tissues, paclitaxel-sensitive and-resistant ovarian cancer cell lines in order to determine whether the methylation of TGFBI is asscociated with paclitaxel chemoresistance. Objective:To detect the expression and promoter hypermethylation of TGFBI in epithelial ovarian cancers.To investigate the correlation between the expression silence of TGFBI and promoter hypermethylation.Methods:RT-PCR and S-P immunohistochemistry was used to examined the expression of TGFBI in epithelial ovarian cancers and benign epithelial ovarian cysts and normal ovarian tissues. The promoter methylation of TGFBI was detected with MSP and BGS methods.Results:1. Ten of10(100%) normal ovarian tissues were positive for TGFBI mRNA, and the same status were detected in benign ovarian cysts. Among40ovarian cancer samples analyzed, only12of40(30%) were positive for TGFBI. The positive rate of TGFBI expression in ovarian cancer was significantly lower than that in normal ovarian samples and benign ovarian cysts(χ2=3.9474, p<0.05).2. Ten of10(100%) normal ovarian samples and benign cysts were positive for TGFBI protein, which was distinctly higher than that in ovarian cancer tissues(χ2=50, p<0.01).3. Twenty-nine cases were detected TGFBI promoter hypermethylation among40cases(72.5%) of ovarian cancer tissue. But only1case was found methylation status in10(10%) benign ovarian cysts and no methylation status in normal ovarian cases, which was distinctly different with the promoter hypermethylation status of ovarian cancers(χ2=24.5, p<0.01).4. The positive for TGFBI expression in ovarian cancer cases without methylation(90.91%) was significantly higher than that of ovarian cancer tissues with promoter hypermethylation(6.90%)(χ2=7.8108, p<0.01).5. In our study, the methylation rate of carcinomas with poor differentiation was higher than those with well differentiation. Meanwhile, higher methylation rate was also found in late stage patients with ovarian cancers, though no distinct correlation was found between TGFBI methylation status and clinicopathological characteristics.Conclusion:TGFBI was absent or slight expression in ovarian cancer tissues. It indicated TGFBI play a role as suppressor gene in ovarian cancer. TGFBI is frequentlymethylated and is an important mechanism for down-regulated of TGFBI expression in ovarian cancer. Its methylation may be used as a potential epigenetic biomarker for diagnosis and prognosis in epithelial ovarian cancer. Objective:To detect promoter hypermethylation and expression of TGFBI in epithelial ovarian cancer cells with MSP and BGS after5-aza-dc treatment. To investigate the correlation between TGFBI promoter methylation and chemoresistance in epithelial ovarian cancer.Methods:qPCR and Western Blotting was used to examined the expression of TGFBI in epithelial ovarian cancer cells before and after5-aza-dc treatment. The promoter methylation of TGFBI was detected with MSP and BGS methods. Cell proliferation was examined using MTT assay.Results:1. Partial TGFBI methylation detected in SKOV3and A2780cells (42.9%and35.2%of total CpG sites, respectively). In contrast, almost complete hypermethylation were found in SKOV3/TR (94.3%of total CpG sites) and A2780/TR (91.4%of total CpG sites) cells. The methylation level of TGFBI was significantly higher in paclitaxel resistant cell lines (SKOV3/TR and2780/TR) than that in the sensitive pairs (P0.001).2. After5-aza-dc treatment, the relative expression of TGFBI mRNA and protein increased significantly in SKOV3/TR and A2780/TR cells. However, no statistical differences of relative TGFBI mRNA expression and protein were found in other cells (all P>0.05).3. Our results showed that the rate of cell inhibition was significantly increased in SKOV3/TR and A2780/TR than that in control groups at several paclitaxel concentrations of0.01,0.1and1μM(P<0.05). The IC50of SKOV3/TR obviously decreased after5-aza-dc administration (0.19±0.01μM vs.0.42±0.02μM, P=0.001), which was similar with the results A2780/TR (0.012±0.0001μM vs.0.33±0.011μM;P=0.001).Conclusion:It is possible that patients with silenced TGFBI will show chemoresistance to paclitaxel and have poorer prognoses. TGFBI might be a potential therapeutic target for the enhancement of responses to chemotherapy in ovarian cancer patients.