表观遗传药物逆转OCT2表达抑制和增敏肾细胞癌治疗的机理研究

发布时间：2018-03-21 06:00

本文选题：肾细胞癌　切入点：OCT2转运体　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：肾细胞癌(Renal cell carcinoma,RCC)是泌尿系统中最常见的恶性肿瘤,占成人恶性肿瘤的2-3%。RCC也是最容易产生多药耐药(multidrug resistance,MDR)的肿瘤之一,其临床化疗有效率仅有7-10%,是死亡率最高的泌尿系统肿瘤。肾脏中含有丰富的药物转运体,是介导RCC多药耐药的原因之一。有机阳离子转运体OCT2(organic cation transporter, member 2, SLC22A2)主要表达在肾小管上皮细胞基底侧或顶侧,参与分泌和重吸收的功能,是肾脏中含量最多的有机阳离子转运体。我们实验室前期研究表明,OCT2转运体在RCC患者肿瘤组织中蛋白表达丢失,表观遗传修饰中的DNA甲基化和组蛋白修饰均在OCT2转录抑制中发挥重要作用,且两者之间存在紧密联系,初步推测的信号通路为：MLL1负责催化OCT2基因启动子区域组蛋白甲基化激活信号H3K4me3,转录因子MYC能够招募MLL1,从而促使OCT2基因转录激活。在RCC细胞系及患者中OCT2基因转录抑制,诱因之一是启动子区CpG岛(CpG island,CGI)处在高甲基化水平,致使MYC与OCT2基因启动子区E-Box位点结合受阻,MYC招募能力减弱引起MLL1募集降低,最终导致转录激活信号H3K4me3组蛋白修饰在OCT2基因启动子区显著下降,抑制起始转录,下调蛋白表达水平。本课题在前期研究基础上,验证OCT2基因DNA甲基化与组蛋白甲基化之间的相互作用,完善OCT2基因表观遗传调控机理,丰富OCT2基因调节信号通路。我们通过免疫组化(Immunohistochemistry,IHC)检测RCC组织芯片(tissue microarray,TMA)中OCT2转运体蛋白表达水平,进一步验证OCT2转运体在RCC中完全沉默。以RCC细胞系786-O细胞为宿主细胞,采用慢病毒包装感染技术成功构建了沉默靶基因MLL1、MYC的细胞模型,用DNA甲基化抑制剂地西他滨(Decitabine, DAC)分别处理两种细胞模型,体外报告基因测定和体内CHIP实验证实了前期推测的信号调控通路。表观遗传调控另一个重要成员是组蛋白乙酰化修饰,组蛋白乙酰化转移酶(histone acetylase, HATs)和组蛋白去乙酰化酶(histone deacetylase, HDACs)参与决定组蛋白乙酰化状态。已有研究表明,组蛋白乙酰化水平的紊乱与癌症的发生发展有关,HDAC1与RCC患者组织学分级及临床分期存在相关性。为探究组蛋白乙酰化在OCT2基因转录调控中的机制,以Real Time荧光定量PCR (Real time quantitative polymerase chain reaction, RT-qPCR)表征RCC患者的肿瘤组织及配对的癌旁肾组织中HDACs的mRNA水平,筛选出在RCC肿瘤组织中mRNA水平显著提高的HDAC7/9作为研究对象。我们以OCT2基因沉默表达的RCC细胞系786-0和769-P细胞为模型,发现组蛋白去乙酰化酶抑制剂Vorinostat (SAHA)不仅能显著增加786-0中OCT2基因的表达,同时也能提高MYC的表达,但对769-P却无影响。而SAHA合用DAC能使两株细胞系中OCT2的mRNA水平上升为单用DAC时的两倍左右,提示两种细胞系存在不同调控机理。我们由此推测RCC细胞系中DNA甲基化和HDACs同时参与OCT2基因的转录调控,且存在某种相互作用,MYC在两者之间可能起到桥梁的作用。采用SAHA处理稳定干扰MYC的786-0细胞,发现SAHA不能改变该细胞模型中的OCT2基因的表达,SAHA抑制HDACs需要MYC参与,证实了组蛋白乙酰化在逆转RCC细胞系中OCT2表达的重要性。另外,RNA干扰(RNAinterference, RNAi)实验表明靶向HDAC7/9的siRNA均能增加RCC细胞系786-0和769-P中OCT2的表达,表明HDAC7/9为SAHA抑制的靶基因,MYC可能招募HDAC7/9,介导DNA甲基化与组蛋白乙酰化之间的相互作用。我们实验室前期研究表明,在细胞水平上表观遗传药物DAC与化疗药物奥沙利铂(Oxaliplatin, Oxa)联合用药能增加RCC细胞系对Oxa的敏感性。为得到更可靠的结论,构建了RCC细胞系(786-O, Caki-1)裸鼠移植瘤模型,采用联合用药治疗,发现通过表观遗传药物DAC逆转RCC中OCT2蛋白的表达,能够增加Oxa在肿瘤组织的蓄积从而杀死肿瘤细胞。通过上述研究表明,两种表观遗传药物DAC与SAHA均能逆转RCC中OCT2基因的低表达,转录因子MYC则作为DNA甲基化与组蛋白修饰(组蛋白甲基化与组蛋白乙酰化)之间的纽带：1.组蛋白甲基化DAC能够降低DNA甲基化水平,提高MYC在OCT2启动子区的富集,增加MLL1的招募,催化H3K4me3修饰信号,促进OCT2基因的转录。2.组蛋白乙酰化RCC患者中HDAC7/9存在异常高表达,靶向干扰HDAC7/9的siRNA能上调OCT2的]mRNA表达,而SAHA依赖于MYC影响OCT2启动子区HDAC7/9的富集,提高OCT2的转录水平。该机制适用于786-0细胞,在769-P细胞中诱导机制还有待研究。DAC与SAHA合用时OCT2的转录水平较单用时显著升高,可见DNA甲基化水平的降低,有助于MYC对HDAC7/9的调节,起到协同作用。基于以上调控机理,我们采用联合用药的方案治疗RCC细胞系移植瘤裸鼠,能显著抑制肿瘤生长。上述结果,可为临床设计增加RCC对化疗药物的敏感性的新治疗方案提供参考。
[Abstract]:Renal cell carcinoma (Renal cell, carcinoma, RCC) is the most common malignant tumor of urinary system, accounting for 2-3%.RCC of adult malignant tumors is also the most prone to multidrug resistance (multidrug resistance, MDR) of the tumor, the clinical effective rate of chemotherapy is only 7-10%, the highest mortality rate of urinary system tumor. The kidney contains drug transporters the rich, is one of the reasons of RCC Mediated Multidrug resistance. Organic cation transporters (organic OCT2 cation transporter, member 2, SLC22A2) is mainly expressed in renal tubular epithelial cells in the basal side or top side, function and secretion and reabsorption, is the most abundant organic cation transporter in the kidney. Our previous study show that OCT2 transporters in tumor tissue of RCC patients in the loss of expression, epigenetic modification of DNA methylation and histone modification in OCT2 play an important role in transcriptional repression There is a close relationship between the two, and between the signaling pathways inferred for MLL1 responsible for the catalytic promoter region of OCT2 gene histone methylation activation signal H3K4me3, transcription factor MYC can recruit MLL1, so as to promote the activation of OCT2 gene transcription. The inhibition of OCT2 gene transcription in RCC cells and patients, one of the incentives is to start CpG island the sub district (CpG Island, CGI) in the high level of methylation, resulting in MYC and OCT2 gene promoter E-Box binding site is blocked, MYC caused MLL1 to raise the ability to recruit weakened decreased, eventually lead to transcriptional activation of signal H3K4me3 histone modifications in promoter region were significantly decreased in OCT2 gene, inhibit transcription initiation, down-regulation of protein expression level. Study on the basis of previous study, the interaction between the validation of OCT2 gene DNA methylation and histone methylation, OCT2 gene epigenetic regulation mechanism of OCT2 gene regulation, rich channel No. pathway. We by immunohistochemistry (Immunohistochemistry, IHC) detection of RCC tissue microarray (tissue microarray, TMA) OCT2 transporter protein expression level, further validation of OCT2 transporter RCC in complete silence. In 786-O cells RCC cells as host cells, packaging infection was successfully constructed silencing target gene MLL1 by lentivirus. MYC cell model, DNA methylation inhibitors decitabine (Decitabine, DAC) were treated two cell model in vitro reporter gene assay and in vivo CHIP experiments confirmed the signal that the pre control pathway. Epigenetic regulation is another important member of histone acetylation, histone acetylation transferase (histone acetylase, HATs) and histone deacetylases (histone, deacetylase, HDACs) involved in the decision of histone acetylation. Studies have shown that the level of histone acetylation. Mess with the occurrence and development of cancer, HDAC1 and tissue of patients with RCC grade and clinical stage correlated. For research into the mechanism of histone acetylation in OCT2 gene transcription regulation, using Real Time fluorescence quantitative PCR (Real time quantitative polymerase chain reaction, RT-qPCR) HDACs tumor tissues and paired noncancerous characterization of RCC patients the level of mRNA in renal tissue, screened in tumor tissues of RCC mRNA significantly increased the level of HDAC7/9 as the research object. We expressed by the OCT2 gene silencing of 786-0 RCC cell lines and 769-P cells as a model, found that histone deacetylase inhibitor Vorinostat (SAHA) not only can significantly increase the expression of OCT2 gene in 786-0. At the same time it can improve the expression of MYC, but had no effect on 769-P. SAHA combined with DAC can make OCT2 two cell lines in the level of mRNA increased by two times for single DAC about two tips Cell lines have different regulation mechanism of transcriptional regulation. We hypothesized that the methylation of DNA and HDACs in RCC cells in the OCT2 gene, and there is some interaction, MYC may play the role of a bridge between the two. 786-0 cells treated with SAHA stable knockdown of MYC, found that SAHA can not change the expression of OCT2 gene in the cells in the model, SAHA HDACs need MYC in inhibition of histone acetylation, confirmed the importance of the expression of OCT2 in reverse RCC cells. In addition, RNA interference (RNAinterference, RNAi) experiments showed that HDAC7/9 targeting siRNA can increase RCC cells expression of OCT2 786-0 and 769-P, showed that the target gene HDAC7/9 the inhibition of SAHA, MYC may recruit HDAC7/9 mediated interaction between DNA methylation and histone acetylation. Our previous study showed that epigenetic drugs DAC and chemotherapy form at the cellular level Asha Leigh Per (Oxaliplatin, Oxa) drug combination can increase the sensitivity of RCC cells to Oxa. In order to obtain more reliable conclusions, constructed RCC cell lines (786-O, Caki-1) in nude mice, the combination therapy, found by epigenetic drugs DAC inverse OCT2 protein in RCC, can increase the accumulation of Oxa in tumor tissue and kill tumor cells. These results indicated that two kinds of epigenetic drugs DAC and SAHA could reverse the low expression of OCT2 RCC gene, the transcription factor MYC as DNA methylation and histone modifications (histone methylation and histone acetylation) link between the 1.: histone methylation of DAC can reduce the DNA methylation level, increased MYC in the OCT2 promoter enrichment sub area increased MLL1, recruitment, catalytic modification of H3K4me3 signal, promote HDAC7/9 transcription.2. histone acetylation of OCT2 gene in RCC patients The expression of abnormally high, siRNA interference targeting HDAC7/9 can upregulate the expression of OCT2]mRNA and SAHA MYC dependent effects of OCT2 promoter enrichment promoter HDAC7/9, transcription of OCT2. This mechanism applies to 786-0 cells in 769-P cells induced by the mechanism remains to be studied together with SAHA and.DAC at the transcriptional level of OCT2 compared with significantly increased, visible DNA methylation level decreased, contribute to the regulation of MYC on HDAC7/9, has a synergistic effect. Based on the above regulation mechanism, treatment of transplanted tumor RCC cell line in nude mice by combination therapy, can inhibit tumor growth. The above results can provide a reference for clinical design add a new treatment sensitivity of RCC to chemotherapeutic drugs.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.11

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