乙酰转移酶p300对脂肪酸合成的调控机制及其在前列腺癌中的作用

发布时间：2018-04-26 18:37

本文选题：脂肪酸合成 + 前列腺癌　；参考：《吉林大学》2015年博士论文

【摘要】：研究表明，前列腺癌表现出对脂肪酸从头合成的强烈需求。通常情况下在正常组织中处于抑制状态的脂肪酸从头合成途径，在前列腺癌细胞中高度激活，并合成大量脂肪酸，促进肿瘤细胞的生存和发展。因此，脂肪酸代谢途径已成为前列腺癌治疗研究的一个新热点。脂肪酸合酶（FASN），是脂肪酸合成中的关键酶，在正常细胞（除肝脏和脂肪组织）中表达水平较低，而在许多癌症，特别是前列腺癌中呈高表达，，并且提示预后不良。然而，FASN在前列腺癌的调控机制尚不完全清楚。p300是一种转录共激活因子，并具有乙酰转移酶活性，是前列腺癌的一个十分重要的促癌基因。但在前列腺癌中，p300是否参与FASN和脂肪酸合成调控还不清楚。目的：研究FASN和p300的关系，及p300对FASN的表达和脂肪酸合成的调控机制；进一步研究p300-FASN通路在前列腺癌中的生物学功能。方法：我们分析了p300的染色质免疫共沉淀测序（CHIP-Seq）的结果，并使用CHIP技术验证p300与FASN的结合情况，及组蛋白乙酰化水平的变化。我们应用RNAi技术敲除p300或使用不同浓度的p300酶抑制剂处理细胞，在不同的前列腺癌细胞系中降调p300的表达，再应用RT-PCR或western blot技术检测FASN的表达。我们应用油红染色检测细胞或新鲜冷冻组织中脂质水平，应用MTS技术鉴定细胞的生存力，应用免疫组织化学染色检测在人体组织中p300和FASN蛋白质的表达水平。传代杂交产生p300基因敲除小鼠，并监测此小鼠前列腺组织中的FASN表达水平及脂质水平变化。为了检测p300-FASN通路的生物学功能，我们建立和培养低表达p300或FASN或同时低表达两者的前列腺癌稳定细胞系。以肿瘤细胞皮下注射建立小鼠异种移植模型，记录移植瘤生长曲线，收获的肿瘤移植物用于制备冷冻和石蜡切片等。结果：（1）在前列腺癌细胞中p300结合于FASN启动子，上调组蛋白乙酰化水平。（2）p300调节FASN mRNA和蛋白表达水平。（3）在人类前列腺癌组织中，p300与FASN的表达呈正相关。（4）无论在体外或在体内，p300均会影响前列腺癌细胞中脂肪酸的合成。（5）p300通过FASN这一通路调节前列腺癌细胞增殖和肿瘤生长。结论：我们的研究确定了，p300作为前列腺癌的一个重要的促癌因子，与FASN呈正相关。它在转录水平上调节FASN表达，并影响前列腺癌的脂肪酸合成，进一步通过FASN这一通路影响前列腺癌细胞的增殖生长。它在以脂肪酸代谢为靶向的前列腺癌的治疗中具有重要的意义。
[Abstract]:Studies have shown that prostate cancer has shown a strong demand for fatty acid ab initio synthesis. Normally, fatty acids in normal tissues, which are inhibited by ab initio synthesis, are highly activated in prostate cancer cells, and a large amount of fatty acids are synthesized to promote the survival and development of tumor cells. Therefore, fatty acid metabolism pathway has become a new research hotspot in prostate cancer treatment. Fatty acid synthase (FASN), a key enzyme in fatty acid synthesis, has a low expression level in normal cells (except liver and adipose tissue), but is highly expressed in many cancers, especially prostate cancer, indicating poor prognosis. However, the regulatory mechanism of FASN in prostate cancer is not fully understood. P300 is a transcriptional co-activator with acetyltransferase activity, which is a very important oncopromoting gene in prostate cancer. However, it is not clear whether the p300 is involved in the regulation of FASN and fatty acid synthesis in prostate cancer. Aim: to study the relationship between FASN and p300, the regulation mechanism of p300 on FASN expression and fatty acid synthesis, and to study the biological function of p300-FASN pathway in prostate cancer. Methods: we analyzed the results of chromatin immunoprecipitation sequencing (CHIP-Seq) of p300 and used CHIP technique to verify the binding of p300 to FASN and the change of histone acetylation level. We used RNAi to knockout p300 or treated cells with different concentrations of p300 enzyme inhibitors, and then down-regulated the expression of p300 in different prostate cancer cell lines, and then detected the expression of FASN by RT-PCR or western blot. Oil red staining was used to detect lipid levels in cells or fresh frozen tissues, MTS technique was used to determine the viability of cells, and immunohistochemical staining was used to detect the expression of p300 and FASN proteins in human tissues. The p300 gene knockout mice were produced by subculture hybridization, and the expression level of FASN and the changes of lipid level in the prostate tissues of the mice were monitored. In order to investigate the biological function of p300-FASN pathway, we established and cultured prostate cancer stable cell lines with low expression of p300 or FASN or both. The xenotransplantation model of mice was established by subcutaneous injection of tumor cells, the growth curve of transplanted tumor was recorded, and the harvested tumor grafts were used to prepare frozen and paraffin sections. Results: 1) p300 binds to FASN promoter in prostate cancer cells. Upregulation of histone acetylation level. P300 regulates FASN mRNA and protein expression. P300 is positively correlated with FASN expression in human prostate cancer tissues.) both in vitro and in vivo, p300 affects fatty acid syntheses in prostate cancer cells. The FASN pathway regulates the proliferation and tumor growth of prostate cancer cells. Conclusion: our study confirmed that p300 is an important tumor promoter in prostate cancer, which is positively correlated with FASN. It regulates the expression of FASN at the transcriptional level and affects the synthesis of fatty acids in prostate cancer. It further affects the proliferation and growth of prostate cancer cells through the FASN pathway. It plays an important role in the treatment of prostate cancer targeting fatty acid metabolism.
【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】：R737.25

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