ZNF545在多发性骨髓瘤、宫颈癌和食管癌中的表观调控及其抑制肿瘤细胞生长的机制研究

发布时间：2018-04-30 23:31

本文选题：ZNF545 + DNA甲基化　；参考：《重庆医科大学》2016年博士论文

【摘要】：恶性肿瘤已经成为我国最主要的死亡原因和非常重要的公共健康问题,2015年的癌症预计发病人数和死亡人数分别为429.2万和281.4万,即每天约有1.2万人新患癌症和7500人死于癌症。多发性骨髓瘤(MM)是一种常见的血液系统恶性肿瘤,其以肿瘤性浆细胞在骨髓中克隆性增殖为主要特点,目前自体干细胞移植和化疗等传统方法未见良好的治疗获益;宫颈癌作为女性最为常见的恶性肿瘤之一,其死亡率和发病率近年来有明显升高趋势;而食管癌一直是我国发病率和死亡率最高的四大癌症之一。总的来说,对于肿瘤进展期的治疗和如何早期发现尚待进一步探索和研究。然而,表观遗传学为癌症的早期发现、治疗和预后判断等提供了可能的发展方向。其中,DNA甲基化这一重要的表观调控机制,可能对于多种肿瘤的早期诊断及预后判断等均具有临床意义。KRAB锌指蛋白(Krüppel-associated box domain-zinc finger proteins,KRAB-ZFPs)是机体最常见的一类转录因子。锌指蛋白545(ZNF545)是KRAB-ZFP蛋白家族的成员之一。已有文献报道其在胃癌、鼻咽癌、食管癌、结肠癌、宫颈癌和乳腺癌等肿瘤中出现不同程度的甲基化和表达水平的明显降低,并可能具有显著性抑制肿瘤细胞生长的生物学功能,但其在多发性骨髓瘤中尚未见报道,且在宫颈癌和食管癌等肿瘤中的作用分子机制未明。由此我们推测znf545可能作为一个候选抑癌基因在多发性骨髓瘤、宫颈癌和食管癌中影响肿瘤的发生。本研究通过逆转录聚合酶链式反应(reversetranscriptionpolymerasechainreaction,rt-pcr)、实时荧光定量聚合酶链式反应(real-timefluorescencequantitativepolymerasechainreaction,real-timepcr)、甲基化特异性聚合酶链式反应(methylationspecificpcr,msp)、蛋白免疫印迹(westernblot,wb)和在线数据库的方法分析了人多发性骨髓瘤、宫颈癌和食管癌细胞株、肿瘤组织及其正常组织中znf545基因的表达差异及甲基化状态;采用5-氮杂-2-脱氧胞苷(5-aza-dc)联合组蛋白去乙酰化酶抑制剂曲古菌素a(trichostatina,tsa)的方法处理znf545表达沉默或下调的肿瘤细胞株。通过软琼脂或平板克隆形成实验、mts细胞增殖实验和annexinv-fitc/pi流式细胞术等体外实验检测了znf545对多发性骨髓瘤、宫颈癌和食管癌细胞增殖和凋亡的影响,并进一步采用定量pcr和wb的方法检测了znf545对p53分子信号通路的影响。结果发现,相对于正常组织或癌旁组织,多发性骨髓瘤、宫颈癌和食管癌组织或细胞株的znf545的表达水平均有不同程度的下调或沉默。对有znf545甲基化的肿瘤细胞株去甲基化处理后出现了其mrna表达水平的恢复。同时,多发性骨髓瘤细胞株(km3和rpmi8226)、宫颈癌细胞株(hela)和食管癌细胞株(kyse150),以及28.3%(13/46)的多发性骨髓瘤组织和76.6%(49/64)的食管癌组织存在znf545基因启动子的甲基化。体外细胞功能实验结果示,ZNF545能够有效抑制肿瘤细胞的增殖并促进凋亡,从而发挥其抑制肿瘤细胞生长的生物学功能,并且进一步发现其可能是通过靶向转录激活p53通路的活化进而发挥其抑制肿瘤细胞生长的作用。综上所述,ZNF545可能作为一个抑癌基因,参与了多发性骨髓瘤、宫颈癌和食管癌发生等过程,也为进一步在人类肿瘤中找到早期诊断和判断预后的分子指标,或为肿瘤的靶向治疗提供了一定的理论依据。
[Abstract]:Malignant tumors have become the leading cause of death and a very important public health problem in China. In 2015, the number of cancer patients and the number of deaths were 4 million 292 thousand and 2 million 814 thousand respectively, that is, about 12 thousand people with cancer and 7500 people died of cancer each day. Multiple myeloma (MM) is a common malignant tumor of the blood system, which is a common malignant tumor. The clonal proliferation of tumor plasma cells in the bone marrow is the main characteristic, and the traditional methods such as autologous stem cell transplantation and chemotherapy have not benefited well. Cervical cancer is one of the most common malignant tumors in women. The mortality and incidence of cancer have been obviously increased in recent years, and the incidence and mortality of esophageal cancer have always been the incidence and mortality in our country. One of the highest four major cancers. In general, the treatment and early detection of the progression of the tumor remains to be further explored and studied. However, epigenetics provides a possible direction for early detection of cancer, treatment and prognosis. Among them, the important apparent regulatory mechanism of DNA methylation may be for a variety of swelling. The early diagnosis and prognosis of the tumor are all of clinical significance.KRAB zinc finger protein (Kr u ppel-associated box domain-zinc finger proteins, KRAB-ZFPs) is the most common class of transcription factors. Zinc finger protein 545 (ZNF545) is one of the members of the KRAB-ZFP protein family. It has been reported in the literature on gastric cancer, nasopharyngeal carcinoma, esophageal and colon cancer. There is a significant reduction in methylation and expression levels in cervical and breast cancer, and it may have a significant biological function to inhibit tumor cell growth, but it has not been reported in multiple myeloma, and the molecular mechanism of the role in cervical and esophageal cancer is not clear. Therefore, we speculate that znf545 A candidate tumor suppressor gene may affect the occurrence of tumors in multiple myeloma, cervical and esophageal cancer. The present study uses reversetranscriptionpolymerasechainreaction (RT-PCR), real-time quantitative polymerase chain reaction (real-timefluorescencequantitativepolymerasechainreaction,) Real-timepcr), methylation specific polymerase chain reaction (methylationspecificpcr, MSP), protein immunoblotting (Westernblot, WB) and online database analysis of human multiple myeloma, cervical and esophageal cancer cell lines, the difference in the expression of znf545 gene and the methylation status in the tumor tissues and their normal groups, and the use of 5- nitrogen heterozygosity. 2- deoxycytidine (5-aza-dC) combined with histone deacetylase inhibitor treacycin a (trichostatina, TSA) to treat znf545 expressing silent or down regulated tumor cell lines. Experiments were made by soft agar or flat clones, MTS cell proliferation and annexinv-fitc/pi flow cytometry were used to detect znf545 to multiple bone in vitro. The effect of the proliferation and apoptosis of myeloma, cervical and esophageal cancer cells, and further using quantitative PCR and WB methods to detect the effect of znf545 on the p53 molecular signaling pathway. Results showed that the expression level of znf545 in multiple myeloma, cervical and esophageal cancer tissues, or cell lines, relative to normal tissue or Para cancerous tissue were varying degrees. Downregulation or silence. After demethylation of tumor cell lines with znf545 methylation, the level of mRNA expression was recovered. Multiple myeloma cell lines (KM3 and RPMI8226), cervical cancer cell lines (Hela) and esophageal cancer cell lines (kyse150), and 28.3% (13/46) multiple myeloma tissue and 76.6% (49/64) esophageal cancer group The results show that ZNF545 can effectively inhibit the proliferation and promote apoptosis of the tumor cells in vitro, and thus play its biological function in inhibiting the growth of the tumor cells, and further discover that it may be activated by the activation of the p53 pathway by target transcription and then to inhibit the tumor. In summary, ZNF545 may be a tumor suppressor gene, involved in multiple myeloma, cervical and esophageal carcinogenesis, and also to find molecular indicators for early diagnosis and prognosis in human tumors, or to provide a theoretical basis for the target treatment of tumor.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.3;R737.33;R735.1

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