【作者】 程文俊；

【导师】 王心如； 王守林； 夏彦恺；

【作者基本信息】 南京医科大学 ， 生殖毒理， 2010， 博士

【摘要】 卵巢上皮性癌是最常见的女性生殖系统肿瘤之一,死亡率居妇科恶性肿瘤之首,亦是导致女性死亡的第五位病因。卵巢癌的病理类型复杂,且具有易于转移和广泛播散的生物学行为,预后极差。卵巢上皮性癌包括卵巢浆液性癌、子宫内膜样癌及粘液性癌三种主要亚型,其发生机制至今尚不清楚,已越来越受到研究人员的重视。同源盒基因(homeobox gene, HOX)是指一类含有一段180 bp高度保守同源盒序列的基因。哺乳类动物的HOX基因分两大类:一类成簇地排列在不同染色体上,按照胚胎发育期的前-后轴方式(A-P)表达,称Ⅰ类HOX基因;另一类散在分布于不同染色体上,称为非Ⅰ类HOX基因。近年来许多研究表明,同源盒基因的异常表达与白血病、乳腺癌、结肠癌、前列腺癌、肾癌(包括Wilms瘤)等癌症的发生密切相关。我的既往研究结果表明(Cheng W, et al. Nature Medicine, 2005): HOXA9、HOXA10、HOXA11的异常表达与卵巢癌的三种主要亚型(卵巢浆液性癌、子宫内膜样癌及粘液性癌)的发生相关。其中HOXA10特异性地与卵巢子宫内膜样癌的发生相关。DNA甲基化改变在各种肿瘤包括卵巢癌的发生过程中起重要作用。DNA甲基化所致基因失表达及去甲基化所致的基因表达增强是表观遗传学上研究最深入的机制,现已确切证明基因启动子异常甲基化与肿瘤的发生关系密切,基因启动子区CpG岛甲基化可影响转录因子与其识别序列结合,直接抑制基因表达。Yoshida等指出HOXA10在子宫内膜癌中表达下调与肿瘤恶性程度紧密相关,并与HOXA10启动子区甲基化有关。因此我们设想: HOXA10在卵巢癌中的高度表达,可能与HOXA10基因的异常甲基化有关。本研究观察了HOXA10基因启动子区低甲基化改变与卵巢上皮性癌发生的相关性,为临床预防和诊断卵巢癌提供一个新思路。同时,近年来有文献报道,孕激素可抑制卵巢癌细胞增殖,促进细胞凋亡。孕激素类药物之一醋酸甲羟孕酮(Medroxyprogesterone Acetate, MPA),可作为绝经期妇女激素替代疗法中孕激素的组分,也可用作避孕药物,或用来治疗子宫内膜异位症、痛经和闭经。PI3K/Akt通路作为细胞生存信号通路重要的调控位点,在细胞生存和凋亡调控过程中发挥重要作用。孕激素很可能通过磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路发挥其诱导凋亡的作用。卵巢癌发病机制可能与PI3K/Akt表达或功能异常存在关联,大部分卵巢癌中都出现Akt过表达现象。PI3K/Akt信号转导通路是Bcl-2介导的细胞凋亡通路的上游调控程序之一, PI3K/Akt通路活化状态直接影响细胞的增殖和凋亡。大样本的流行病学研究和长期的临床实践均表明,孕激素可以拮抗雌激素的作用,保护卵巢,预防卵巢癌的发生。但是否可将孕激素作为辅助药物运用于卵巢癌的临床治疗目前还没有达成共识。本项研究尝试探索醋酸甲羟孕酮体外诱导卵巢癌细胞凋亡可能存在的机制,为孕激素治疗卵巢癌提供新的理论依据。第一部分HOXA10基因启动子区低甲基化改变与卵巢上皮性癌发生的相关性及其作用机制目的:1.通过观察HOXA10基因在卵巢上皮性癌中的高表达,探讨其在卵巢上皮性癌发生中的作用。2.进一步探索HOXA10基因启动子区低甲基化改变导致的HOXA10基因高表达在卵巢上皮性癌发生中的作用机制,并分析HOXA10基因低甲基化与卵巢上皮性癌临床病理因素的关系。3.研究甲基化转移酶抑制剂5-杂氮-2’脱氧胞苷(5-Aza-dC)对卵巢上皮性癌细胞株SKOV-3和HEY的作用,为5-Aza-dC治疗卵巢上皮性癌提供实验依据。方法:1.抽提卵巢上皮性癌和正常卵巢组织中的RNA及蛋白。用RT-PCR法检测HOXA10基因mRNA表达量变化;用免疫印迹法(Western Blot)检测各组织中HOXA10蛋白表达水平。2.抽提卵巢上皮性癌和正常卵巢组织中的DNA。采用甲基化特异性聚合酶链反应(Methylation specific polyerase chain reaction, MSP)分析HOXA10启动子区低甲基化状态。3.卵巢上皮性癌细胞株:SKOV-3和HEY分别接种于含10%胎牛血清的RPMI-1640培养液中常规培养24h后,换无血清培养基继续培养24h,分别加入0.5、5、50μmol/L 5-杂氮-2’脱氧胞苷(5-Aza-dC),药物作用24h、48h、72h后,四甲基偶氮唑盐(MTT)比色法检测细胞生长增殖。同时分别提取细胞株RNA,用Real-Time PCR法检测HOXA10基因mRNA表达量变化。结果:1.RT-PCR实验结果显示: HOXA10基因mRNA在几乎所有的卵巢上皮性癌中阳性表达,但在正常卵巢组织中表达减少甚至缺失; WB实验结果显示: HOXA10蛋白在卵巢上皮性癌中高表达,而在正常卵巢组织中表达降低。2.用MSP法检测29例卵巢上皮性癌组织样本中,有17例组织中HOXA10启动子区发生低甲基化;而在16例正常卵巢组织中,仅4例组织中HOXA10启动子区发生低甲基化,差异有统计学意义(p<0.05)。HOXA10基因甲基化与卵巢上皮性癌的病理类型、临床分级无关,但与临床期别有关。结合第一部分实验,我们分析得出以下结果: (1)在正常卵巢组织中HOXA10基因启动子区处于正常的甲基化状态,而在大部分卵巢上皮性癌组织中HOXA10基因启动子区发生低甲基化。(2)HOXA10基因在卵巢上皮性癌中阳性表达,而在正常卵巢组织中表达降低甚至缺失。(3)对比HOXA10启动子区发生甲基化和低甲基化的卵巢组织(包括正常卵巢组织和卵巢上皮性癌组织)时发现: HOXA10基因mRNA及蛋白在发生甲基化的组织中表达降低,而在发生低甲基化的组织中表达升高。3.在卵巢上皮性癌细胞株SKOV-3中, HOXA10基因启动子区表现为甲基化状态,运用去甲基化试剂5-杂氮-2’脱氧胞苷(5-Aza-dC)处理细胞株后,卵巢癌细胞株SKOV-3中HOXA10基因的表达升高; MTT法检测发现经5-Aza-dC处理后SKOV-3细胞增殖速度增加。结论:1.特异性甲基转移酶DNMTs抑制剂—5-Aza-dC通过改变卵巢上皮性癌细胞中HOXA10启动子区甲基化状态,从而影响细胞的增殖状态。2.在卵巢上皮性癌中, HOXA10基因启动子区低甲基化改变导致了HOXA10基因在转录和翻译水平的表达升高,从而参与了卵巢上皮性癌的发生。第二部分MPA体外诱导卵巢上皮性癌SKOV-3细胞株凋亡的研究目的:1.观察MPA在体外对卵巢上皮性癌细胞株SKOV-3增殖的抑制作用。2.探索MPA诱导SKOV-3细胞凋亡的机制。3.进一步探究孕激素细胞膜受体在诱导卵巢上皮性癌细胞凋亡中的作用。方法:1.卵巢上皮性癌SKOV-3细胞株接种于盛有含10%胎牛血清的RPMI-1640培养液的96孔板中常规培养至贴壁后,换无血清培养基继续培养24h,分别加入0.1、1、10、100μmol/L的MPA,药物作用12h、24h、48h后,四甲基偶氮唑盐(MTT)比色法检测细胞生长增殖。2.卵巢上皮性癌SKOV-3细胞株接种于盛有含10%胎牛血清的RPMI-1640培养液的96孔板中常规培养至贴壁后,换无血清培养基继续培养24h,分别加入1、5、12.5、25μmol/L的LY294002(PI3K抑制剂)孵育1h后,再分别加入0.1、1、10、100μmol/L的MPA,药物作用12h、24h、48h后,四甲基偶氮唑盐(MTT)色法检测细胞生长增殖。3.收集不同浓度MPA作用24h后的SKOV-3细胞,裂解细胞提取总蛋白, Western Blot法检测各组织中Akt、p-Akt及Bcl-2蛋白表达水平。4. SKOV-3细胞经不同浓度MPA分别作用24、48h, Annexin V/PI双染法流式细胞仪检测细胞凋亡。结果:1.MPA以时间-剂量依赖方式抑制SKOV-3细胞的体外增殖,药物作用时间越长、药物浓度越大,对细胞的毒性作用越明显(P<0.05)。2.MPA抑制SKOV-3细胞的体外增殖作用可被LY294002所阻断。3.随着MPA使用剂量的增大, SKOV-3细胞内p-Akt和Bcl-2蛋白表达水平逐步下降(P<0.05)。与对照组相比,醋酸甲羟孕酮浓度为0.1μmol/L时p-Akt表达量下降约28%, Bcl-2表达量下降约10%;当醋酸甲羟孕酮浓度上升至100μmol/L时, p-Akt表达量下降约35%, Bcl-2表达量下降约27%。不同药物浓度组间Akt表达水平无明显变化(P>0.05)。4.MPA以时间-剂量依赖方式诱导SKOV-3细胞凋亡,对比不同时间的统计数据可发现, MPA作用时间为24h时早期凋亡率较晚期凋亡率上升明显(P< 0.05);当药物作用延长至48h后,晚期凋亡在凋亡总体中所占比重上升,早期凋亡上升趋势不明显。结论:1. MPA以时间-剂量依赖方式抑制SKOV-3细胞的体外增殖。2. MPA可抑制PI3K/Akt信号转导通路的激活,降低细胞内游离的Bcl-2的水平,体外诱导SKOV-3细胞发生凋亡。3. MPA浓度为1μmol/L~10μmol/L,药物作用时间为24h时,细胞对药物最为敏感,抑制细胞增殖促进细胞凋亡的作用最强;再加大药物浓度或延长药物作用时间不能明显提高细胞的凋亡率。更多还原

【Abstract】 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. Ovarian cancer has a high incidence of metastasis that generally remains localized within the peritoneal cavity. EOC is not a single disease entity, but instead comprises a heterogeneous group of tumors. These tumors are classified according to their patterns of histological differentiation. The most common histological subtype are-serous、mucinous、and endometrioid tumors. The mechanism of regulation of subtype is unknown, which attract much attention from researchers. During the past hundred years, changes in body structure and development, such as the replacement of antennae with legs, were observed in Drosophila melanogaster mutants. Hox genes would be confirmed and highly conserved homologues identified in most animals. In vertebrates, the Hox genes are located contiguously in clusters, with the number of clusters varying according to anatomic complexity. HOX compromises a series of advanced conserved genes. Hox genes are integral to normal temporospatial limb and organ development along the anterior-posterior (A-P) axis. First, the position of a Hox gene 3’to 5’within a Hox cluster corresponds to its expression in the animal along the A-P axis. One group of Hox gene expression according to the arrangement of A-P named I-group Hox; and another group is scattered on different chromosomes named II- group. Numerous examples of aberrant HOX gene expression have been found in different cancers. The oncogenesis is related to leukocythemia、breast cancer、colorectal carcinoma、prostatic carcinoma、and renal carcinoma (including Wilms tumor). According to our previous study, expression of HOXA9、HOXA10 and HOXA11 were not detected in normal human ovarian surface epithelium. However, coliner expression patterns of these HOX genes that normally occur in Mullerian-duct-derived epithelial were found to be recapitulated in serous、endometrioid and mucinous EOCs according to the pattern of Mullerian-like differentiation of these tumors. Importantly, HOXA10 is related to the ovarian endometrioid cancer specifically.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). Yoshida et al have brought forward that HOXA10 is down-expressed in endometrial carcinoma, which is related to low grade tumor, and also with the methylation of HOXA10. In that way, we hypothesized that the high expression of HOXA10 in ovarian cancer is related to the methylation condition of HOXA10 CpG islands. This study is aiming to finding the underling relationship between the HOXA10 CpG islands methylation and the oncogenesis of ovarian cancer, further seek out the new idea for the prevention and curing the ovarian cancers.Prevention and treatment is the severe problems in the iateria of ovarian cancer. Ovaries are located deep in the pelvic, and because of the special anatomical location, the early detection and diagnosis of ovarian cancer become very difficult. Meanwhile laking of high sensitivity diagnostic indicators caused that only when the ovarian cancer is in a advanced stages, the exact diagnosis can be done. And it often leads to poor prognosis of patients. On the current international principles of treatment of advanced ovarian cancer is a comprehensive treatment including a cytoreductive surgery and paclitaxel and a platinum-based chemotherapy. Due to a high rate of recurrence and high resistance rates in recurrent tumors, five-year survival rate of ovarian carcinoma still in a low level.MPA is one kind of progestin drugs, the most common uses of MPA are as the progestin component of menopausal hormone replacement therapy and also as an oral or depot-injected contraceptive. Medroxyprogesterone is also used as a treatment for endometriosis, dysmenorrhea, and amenorrhea. Because MAP has anti-estrogen function and has capability to promote the proliferative endometrium transform to secretary endometrium, it has been widely used as an adjuvant treatment of estrogen and progesterone receptor-positive endometrial cancer. In recent years, some studies suggested that progesterone can inhibit ovarian cancer cell proliferation and induce cancer cell apoptosis. But the specific mechanism remains unknown.Some researchers pointed that MPA might induce apoptosis via PI3K/Akt pathway. Being a important regulatory sites in cell survival pathway, PI3K/Akt pathway play a vital role in cell’s proliferation and apoptosis. Numerous reports suggest a role of PI3K signaling in invasion and metastasis in various kinds of carcinomas. Akt has been also shown to be amplified or over-expressed in ovarian cancer, implying that it also plays a role in ovarian oncogenesis. Survival factors induce Bcl-2 pathway activation via several protein kinase signaling pathways including activation of phosphatidylinositol 3-kinase (PI3K)-Akt, which directly determined the cell proliferation or apoptosis.Epidemiological studies and long-term clinical practice show that progesterone may antagonize the effect of estrogen to protect the ovary, preventing the occurrence of ovarian cancer. Currently, there is no consensus about whether progesterone can be applied as adjuvant treatment of ovarian cancer. The present study attempts to reveal the mechanisms of medroxyprogesterone acetate induced apoptosis in ovarian cancer cells, and provide a new theoretical basis for the treatment of ovarian cancer.Part I The effects of hypomethylation of HOXA10 CpG promoter in ovarian cancerObjective The purpose of this study was to find the highly expression of HOXA10 in ovarian carcinoma and to determine the relationship between hypomethylation of HOXA10 gene promoter and highly expression in malignant ovarian tissues, which could induce the ovarian carcinogenesis, and finally to confirm the function of 5-Aza-dC on epithelial ovarian cell lines to provide the evidence of the treatment of 5-Aza-dC in clinic.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 confirmed our present study using SKOV-3 and HEY ovarian cancer cell lines treated with the demethylation 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 RT–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. HOXA10 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) HOXA10 promoter is normally methylated in most normal ovarian samples, whereas hypomethylated in abundant epithelial ovarian cancers. (2) HOXA10 is highly expressed 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-CdR treatment. The results showed that after the treatment of the SKOV-3 cell line with 5-Aza-dC, demethylation of HOXA10 and concomitant increasing HOXA10 expression occurred.Conclusion:1. In epithelial ovarian carcinoma, the HOXA10 gene promoter hypomethylation condition could bring about the highly expression of HOXA10 both in the transcript and translate levels, which could induce the ovarian carcinogenesis.2. By 5-Aza-dC treatment, the HOXA10 gene promoter could be demethylated, then concomitant increasing HOXA10 expression occurred, further stimulate the ovarian cell lines to proliferate and differentiate.Part II Medroxyprogesterone Acetate Induce Human Ovarian Cancer Cell Line SKOV-3 Apoptosis in VitroObjective 1. The purpose of this study was to observation of MPA depressed SKOV-3 cells proliferation in vitro.2. Explore the mechanism of MPA induced SKOV-3 cells apoptosis in vitro. 3. Reveal the functions of progesterone membrane receptors in human ovarian cancer cells apoptosis.Methods 1. Epithelial ovarian cancer SKOV-3 cells at exponential phase were subcultured at 7×103 cells/well into 96-well microplates and cultured overnight. Cells were treated with serial concentrations (0.1, 1,10,100μmol/l) of MAP for 12, 24 or 48h. And the cytotoxicity was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay2. SKOV-3 cells were treated with serial concentrations (1, 5, 12.5, 25μmol/L)LY294002 for 1h before treated with different concentrations of MAP. And the cytotoxicity was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay.3. SKOV-3 cells treat with serial concentrations MAP for 24h, lysed cells and collection of protein, Akt, pAkt, pBad and Bcl-2 protein expression was detected by Western blotting.4. SKOV-3 cells were treated with different concentrations of MPA role of 24, 48 h; Annexin V / PI double staining flow cytometry to detect cell apoptosis.Result 1. Medroxyprogesterone acetate inhibited the growth of SKOV-3cell line effectively, in a time-and-dose dependent way (P<0.01).2. MPA inhibition of SKOV-3 cell proliferation effect could be blocked by LY2940023. The expression of p-Akt and Bcl-2 protein in the SKOV-3 cells were decreased in all medroxyprogestogen groups and significant decreased in high dose medroxyprogestogen group (P<0.05). The expression of p-Akt protein was decreased to 72% in the low dose group and 65% in high dose group. The levels of Bcl-2 protein were descended to 90% in low dose group and 73% in high dose group. The expression of Akt protein was no significant difference in both medroxyprogestogen group and control group.4. MPA induced SKOV-3 cell apoptosis in a time-dose dependent manner. Comparison of different time statistics can be found, early apoptosis rate significantly rised(P< 0.05), when the time was 24h; extension of time to 48h, late apoptosis rate increased, and early apoptosis rate increased slowly.Conclusion 1. MPA induced SKOV-3 cell apoptosis in a time-dose dependent manner.2. MPA can inhibit the PI3K/Akt signal pathway, reducing the level of Bad phosphorylation, induced SKOV-3 cells apoptosis in vitro.3. The cells most sensitive to the drug, when the drug effects time was 24h and MPA concentration from 1μmol L to 10μmol / L, increase the drug concentration or drug effect can not be extended significantly improve the cell apoptosis.更多还原