Hypermethylation Of Tumor Suppressor Gene Promoter And Histone Methylation In Ovary Granulosa Cell Tumors

Purpose:Ovary granulosa cell tumors (GCTs) are malignant neoplasms, which originate from the sex cord. GCT is relatively rare and account for approximately 2-5% of all ovarian malignancies. Currently the pathogenesis is not clear. GCT exhibits few clinical symptoms and its microscopic morphology is complicated and various. The diagnosis indices of GCT are relatively limited, mainly for HE dyeing added by immunohistochemistry. So far the sensitivity and specificity of the common antibodies, such as a-inhibin?CD99?calretinin?EMA?vimentin, can't meet the need. In treatment aspects, there is insufficient evidence showed that chemotherapy can delay or prevent its recurrence. In recent years, because of the rapid development of epigenetics, the relationship between aberrant epigenetic modification and tumorigenesis has become a new hot spot. Aberrant epigenetic modification is associated with cancer development and progression. Cooperative histone modification have been considered the primary mechanisms of epigenetic modification. Both aberrant epigenetic modification and histone modification are reversible, so demethylation may activate the silent suppressor genes and then execute the tumor suppressor function. This subject intends to study the hypermethylation of tumor-suppressor gene promoter and histone methylation in GCT and to explore a new study targets on GCT mechanism, diagnosis and treatment.Methods:(1) In the present study, we recruite 30 patients with GCTs from Shandong University Qilu Hospital and Shanxian Central Hospital between 2010 and 2013. All patients were diagnosed histologically with GCT, and all tissue specimens were reconfirmed by pathologists in the Department of Pathology Qilu Hospital. The present study also includes tissues from 30 patients with follicular cysts, which are selected as a control. Clinicopathological data from each patient, including age, tumor size, Federation of Gynecology and Obstetri (FIGO) stage and postoperative recurrence are collected. (2) We use methylation-specific PCR method (MSP) to detecte the tumor suppressor genes (TSGs) promoter methylation status of CDH13, DKK3 and FOXL2 in 30 cases of ovarian granulosa cell tumor specimens and 30 cases of ovarian follicular cyst. (3) To detect the expression of histone methylation enzyme EZH2 in granulosa cell tumor and ovarian follicular cyst by immunohistochemistry methods (IHC). (4) To analyze the associations between tumor suppressor gene (CDH13, DKK3 and FOXL2) methylation, expression of histone methylation enzyme EZH2 and the clinicapathological features of ovarian granulosa cell tumor. (5) To analyze the associations between the frequency of tumor suppressor gene methy-lationand and the rate of EZH2 expression. (6) Selecting 10 cases of granulosa cell randomly for SNP detection, to detect the gene sequence of CDH13 and DKK3 exons and FOXL2-C134W.Results:(1) The frequency of CDH13, DKK3 and FOXL2 promoter methylation is significantly higher in GCTs compared to healthy control tissues (86.67% vs.23.33%, 80% vs.26.67%, and 70% vs.20%, respectively; p<0.001). (2) Immunostaining of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, showed that EZH2 protein was expressed in 11 of the 30 GCT tissues, whereas all of the 30 healthy control tissues failed to express EZH2 protein (p<0.01). (3) The associations between gene methylation and clinicopathological parameters, including age, tumor size, FIGO stage and postop-erative recurrence, were also analyzed, however, no significant associations were observed. (4) No associations were found between the expression of EZH2 and the clinicopatho-logical parameters of the patients with GCT with respect to age, tumor size, FIGO stage and metastasis. (5) No Statistical correlation is found between the expression of suppressor genemethylation and histone methyltransferase.(6) CDH13 exons have not been found to have focused mutation; There is a A-->G mutations in exons7 of DK.K3 in four of the ten cases of GCT. All ten GCTs have a missense point mutation, C-->G (C134W), in FOXL2.Conclusions:(1) Significant differences were found in the methylation of the CDH13, DKK3 and FOXL2 promoters in the GCT tissues, compared with the follicular cyst tissues (P<0.001). Promoter hypermethylation of tumor suppressor gene CDH13, DK.K3 and FOXL2 in ovarian granulosa cell tumor may be a frequent event and plays an important role in the GCTS development. It could be used as a diagnosis of GCT. (2) The expression rate of ZEH2 is less than CDH13, DKK3 and FOXL2 methylation frequency in GCTS, but significantly higher than that in follicular cyst tissues, suggesting that histone methylation may play a role in the GCTS development and could be used as a reference indicator in the diagnosis of granulosa cell tumor. (3) In this subject, no associations are found between the frequence of CDH13, DKK3, FOXL2 methylation, the rate of EZH2 expression and the clinicopatho-logical parameters of the patients with GCT with respect to age, tumor size, FIGO stage and metastasis. There might be three possible explanations:The first is that the tumor suppressor genes methylation and histone methylation might occur as an early event in GCT and play an important role in GCT development. They may not affect the progress and the prognosis of ovarian grain layer cell tumor. The second possibility is that it might be belonged to the characteristics of low potential malignancy and long-term recurrence of GCT. The small sample size might be the third possibilities and large samples study is needed further. (4) In the present study, no association is found between the expression of EZH2 and methylation of the CDH13, DKK3 and FOXL2 promoters. We speculate that EZH2 may affect DNA methylation by direct interaction with DNA methyltransferases, however, the majority of H3K27me3-marked genes lack DNA methylation in embryonic stem cells, indicating that EZH2 recruitment may not be sufficient to promote DNA methylation. Thus, further investigation is required to assess the functions of the EZH2 protein in GCTs. (5) In this study, we do not find concentrated mutation point in the fourteen exons of CDH13, suggesting that DNA methylation may work alone and demethylation might re-activation the expression activity of CDH13. (6) A-->G mutations are found in exons7 of DKK3 in four of the ten cases of GCT and C-->G mutations are found in FOXL2-C134W in all of the ten cases. Meaning that promoter methylation and gene mutation might occur at the same time. If so, to reverse the methylation perhaps could not activate gene expression and demethylation treatment alone might be invalid. This discovery might be a useful tips for apparent treatment.