hMOF的表达以及其对组蛋白H4K16乙酰基化酶活性在胃癌发病中的作用研究

发布时间：2018-08-11 18:50

【摘要】：组蛋白翻译后修饰是重要的表观遗传学调控机制，包括甲基化、乙酰基化、泛素化、磷酸化和糖基化等。其中，组蛋白乙酰基化是研究较早、最多的表观遗传调控机制之一，它由组蛋白乙酰基转移酶（Histone acetyltransferases, HATs）和组蛋白去乙酰基转移酶（Histone deacetyltransferases, HDACs）共同调控来维持细胞内乙酰基化水平的动态平衡。因此，任何原因引起乙酰基化/去乙酰基化之间的平衡失调，，都将导致细胞内生物过程的异常，甚至产生癌症。最近，越来越多的研究数据表明，全细胞内组蛋白H4第16位赖氨酸K16（H4K16）的乙酰基化可能与某些肿瘤的发生密切相关。细胞内H4K16的乙酰基化水平受多种组蛋白修饰酶的调控，而人源乙酰基转移酶MOF（hMOF）在细胞内主要通过乙酰基化H4K16来参与细胞内的各种生物学功能。我们在之前的研究中已经发现hMOF的基因表达水平可能与一些癌症的发生相关，如肾癌、卵巢癌、结肠癌等。同时，我们在胃癌组织中也发现hMOF的低表达倾向。为了进一步研究和证实hMOF表达以及其所修饰的组蛋白H4K16的乙酰基化水平与胃癌之间的相关性，以及在胃癌中组蛋白H4K16的乙酰基化水平是否还受其他修饰酶的调控，本论文中以胃癌肿瘤组织和胃癌细胞株作为研究对象，通过RT-qPCR、免疫印迹（Western Blot）、细胞染色、过表达和siRNA敲低等技术，系统研究了hMOF的表达以及全细胞组蛋白H4K16的乙酰基化水平与胃癌的发生、临床分期、病理分期、以及病人预后等之间的相关性。（1）我们从52例胃癌患者中同时收集了肿瘤、癌旁和正常组织，利用RT-qPCR检测了hMOF基因的表达水平。统计学分析结果证实81%（42/52）病人的肿瘤组织中hMOF的表达量比正常组织下降了2倍以上。另外，低表达hMOF基因不仅与胃癌的肿瘤分期pT2-T4、淋巴结转移和远处转移相关，而且与患者的生存期也密切相关。但是，hMOF基因的表达量在不同年龄组、不同性别组和不同细胞分化程度组之间没有统计学上的显著性差异。（2）利用RT-qPCR方法对32例胃癌患者的肿瘤、癌旁和正常组织进行了HDAC4基因表达水平的检测。统计学分析发现，在胃癌肿瘤中HDAC4基因表达量虽然有上升趋势，但是没有统计学上的显著性差异。HDAC4的高表达与胃癌的临床分期、病理分期、淋巴结转移、胃癌的远处转移，生存期等均无统计学差异。（3）结合RT-qPCR、WB和细胞染色方法，在两种胃癌细胞系SGC-7901和MGC-803中证实了hMOF的低表达和HDAC4的高表达。同时，与正常人胃粘膜细胞GES-1比较，组蛋白H4K16的乙酰基化水平在SGC-7901和MGC-803胃癌细胞系中明显降低。为了证实H4K16乙酰基化水平的低下与hMOF和HDAC4之间的调控关系，在MGC-803胃癌细胞中进行了过表达和siRNA敲低实验。结果显示，过表达hMOF提高了全细胞中H4K16的乙酰基化水平，但是并没有影响HDAC4的mRNA水平和蛋白水平。相反，siRNA敲低HDAC4，即没有影响hMOF的蛋白表达，也没有因去乙酰基化作用的减少而导致H4K16乙酰化水平的升高。（4）本论文的研究结果证实组蛋白乙酰基转移酶hMOF参与了胃癌的发生机制，胃癌细胞内H4K16乙酰基化水平主要受hMOF乙酰基转移酶，而不是去乙酰基化酶HDAC4的调控。
[Abstract]:Histone acetyltransferases (HATs) and histone deathylation are important epigenetic regulatory mechanisms, including methylation, acetylation, ubiquitination, phosphorylation and glycosylation. Histone deacetyltransferases (HDACs) are co-regulated to maintain the dynamic balance of acetylation levels in cells. Therefore, any imbalance between acetylation and deacetylation caused by any cause will lead to abnormal cellular processes and even cancer. Recently, more and more research data show that all of them are complete. Acetylation of histone H4 at position 16 lysine K16 (H4K16) may be closely related to the development of some tumors. Acetylation of histone H4K16 in cells is regulated by a variety of histone modifying enzymes, while human acetyltransferase MOF (hMOF) participates in various cellular biological functions mainly through acetylation of H4K16. In order to further study and confirm the expression of hMOF and the acetylation level of histone H4K16 in stomach, we have found that the expression of hMOF may be associated with the development of some cancers, such as renal, ovarian and colon cancers. Correlation between cancers and whether the acetylation level of histone H4K16 in gastric cancer is regulated by other modified enzymes. In this study, we systematically investigated the expression of hMOF in gastric cancer tissues and cell lines by RT-qPCR, Western Blot, cell staining, overexpression and siRNA knockdown techniques. The acetylation level of whole cell histone H4K16 was correlated with the occurrence, clinical stage, pathological stage and prognosis of gastric cancer.
(1) The expression of hMOF gene was detected by RT-qPCR in 52 patients with gastric cancer. Statistical analysis showed that the expression of hMOF gene in 81% (42/52) of the cancer tissues was more than two times lower than that in normal tissues. Lymph node metastasis is associated with distant metastasis and is also associated with survival. However, there is no significant difference in the expression of hMOF gene between different age groups, sex groups and different cell differentiation groups.
(2) The expression level of HDAC4 gene was detected by RT-qPCR in 32 cases of gastric cancer, adjacent to cancer and normal tissues. Statistical analysis showed that the expression level of HDAC4 gene in gastric cancer increased, but there was no significant difference between them. There was no significant difference in metastasis, survival rate and distant metastasis of gastric cancer.
(3) The low expression of hMOF and high expression of HDAC4 were confirmed in two gastric cancer cell lines SGC-7901 and MGC-803 by RT-qPCR, WB and cell staining. The acetylation level of histone H4K16 was significantly lower in SGC-7901 and MGC-803 than in normal gastric mucosal cells GES-1. The results showed that the over-expression of hMOF increased the acetylation level of H4K16, but did not affect the mRNA and protein levels of HDAC4. On the contrary, siRNA knocked down HDAC4, which did not affect the protein surface of hMOF. Da did not increase the level of acetylation of H4K16 due to the decrease of deacetylation.
(4) Histone acetyltransferase hMOF is involved in the pathogenesis of gastric cancer. The acetylation level of H4K16 in gastric cancer cells is mainly regulated by hMOF acetyltransferase, not deacetylase HDAC 4.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

【共引文献】