本文選題：腎細(xì)胞癌　切入點(diǎn)：OCT2轉(zhuǎn)運(yùn)體　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：腎細(xì)胞癌(Renal cell carcinoma,RCC)是泌尿系統(tǒng)中最常見的惡性腫瘤,占成人惡性腫瘤的2-3%。RCC也是最容易產(chǎn)生多藥耐藥(multidrug resistance,MDR)的腫瘤之一,其臨床化療有效率僅有7-10%,是死亡率最高的泌尿系統(tǒng)腫瘤。腎臟中含有豐富的藥物轉(zhuǎn)運(yùn)體,是介導(dǎo)RCC多藥耐藥的原因之一。有機(jī)陽(yáng)離子轉(zhuǎn)運(yùn)體OCT2(organic cation transporter, member 2, SLC22A2)主要表達(dá)在腎小管上皮細(xì)胞基底側(cè)或頂側(cè),參與分泌和重吸收的功能,是腎臟中含量最多的有機(jī)陽(yáng)離子轉(zhuǎn)運(yùn)體。我們實(shí)驗(yàn)室前期研究表明,OCT2轉(zhuǎn)運(yùn)體在RCC患者腫瘤組織中蛋白表達(dá)丟失,表觀遺傳修飾中的DNA甲基化和組蛋白修飾均在OCT2轉(zhuǎn)錄抑制中發(fā)揮重要作用,且兩者之間存在緊密聯(lián)系,初步推測(cè)的信號(hào)通路為：MLL1負(fù)責(zé)催化OCT2基因啟動(dòng)子區(qū)域組蛋白甲基化激活信號(hào)H3K4me3,轉(zhuǎn)錄因子MYC能夠招募MLL1,從而促使OCT2基因轉(zhuǎn)錄激活。在RCC細(xì)胞系及患者中OCT2基因轉(zhuǎn)錄抑制,誘因之一是啟動(dòng)子區(qū)CpG島(CpG island,CGI)處在高甲基化水平,致使MYC與OCT2基因啟動(dòng)子區(qū)E-Box位點(diǎn)結(jié)合受阻,MYC招募能力減弱引起MLL1募集降低,最終導(dǎo)致轉(zhuǎn)錄激活信號(hào)H3K4me3組蛋白修飾在OCT2基因啟動(dòng)子區(qū)顯著下降,抑制起始轉(zhuǎn)錄,下調(diào)蛋白表達(dá)水平。本課題在前期研究基礎(chǔ)上,驗(yàn)證OCT2基因DNA甲基化與組蛋白甲基化之間的相互作用,完善OCT2基因表觀遺傳調(diào)控機(jī)理,豐富OCT2基因調(diào)節(jié)信號(hào)通路。我們通過免疫組化(Immunohistochemistry,IHC)檢測(cè)RCC組織芯片(tissue microarray,TMA)中OCT2轉(zhuǎn)運(yùn)體蛋白表達(dá)水平,進(jìn)一步驗(yàn)證OCT2轉(zhuǎn)運(yùn)體在RCC中完全沉默。以RCC細(xì)胞系786-O細(xì)胞為宿主細(xì)胞,采用慢病毒包裝感染技術(shù)成功構(gòu)建了沉默靶基因MLL1、MYC的細(xì)胞模型,用DNA甲基化抑制劑地西他濱(Decitabine, DAC)分別處理兩種細(xì)胞模型,體外報(bào)告基因測(cè)定和體內(nèi)CHIP實(shí)驗(yàn)證實(shí)了前期推測(cè)的信號(hào)調(diào)控通路。表觀遺傳調(diào)控另一個(gè)重要成員是組蛋白乙酰化修飾,組蛋白乙�；D(zhuǎn)移酶(histone acetylase, HATs)和組蛋白去乙�；�(histone deacetylase, HDACs)參與決定組蛋白乙酰化狀態(tài)。已有研究表明,組蛋白乙�；降奈蓙y與癌癥的發(fā)生發(fā)展有關(guān),HDAC1與RCC患者組織學(xué)分級(jí)及臨床分期存在相關(guān)性。為探究組蛋白乙�；贠CT2基因轉(zhuǎn)錄調(diào)控中的機(jī)制,以Real Time熒光定量PCR (Real time quantitative polymerase chain reaction, RT-qPCR)表征RCC患者的腫瘤組織及配對(duì)的癌旁腎組織中HDACs的mRNA水平,篩選出在RCC腫瘤組織中mRNA水平顯著提高的HDAC7/9作為研究對(duì)象。我們以O(shè)CT2基因沉默表達(dá)的RCC細(xì)胞系786-0和769-P細(xì)胞為模型,發(fā)現(xiàn)組蛋白去乙酰化酶抑制劑Vorinostat (SAHA)不僅能顯著增加786-0中OCT2基因的表達(dá),同時(shí)也能提高M(jìn)YC的表達(dá),但對(duì)769-P卻無(wú)影響。而SAHA合用DAC能使兩株細(xì)胞系中OCT2的mRNA水平上升為單用DAC時(shí)的兩倍左右,提示兩種細(xì)胞系存在不同調(diào)控機(jī)理。我們由此推測(cè)RCC細(xì)胞系中DNA甲基化和HDACs同時(shí)參與OCT2基因的轉(zhuǎn)錄調(diào)控,且存在某種相互作用,MYC在兩者之間可能起到橋梁的作用。采用SAHA處理穩(wěn)定干擾MYC的786-0細(xì)胞,發(fā)現(xiàn)SAHA不能改變?cè)摷?xì)胞模型中的OCT2基因的表達(dá),SAHA抑制HDACs需要MYC參與,證實(shí)了組蛋白乙�；谀孓D(zhuǎn)RCC細(xì)胞系中OCT2表達(dá)的重要性。另外,RNA干擾(RNAinterference, RNAi)實(shí)驗(yàn)表明靶向HDAC7/9的siRNA均能增加RCC細(xì)胞系786-0和769-P中OCT2的表達(dá),表明HDAC7/9為SAHA抑制的靶基因,MYC可能招募HDAC7/9,介導(dǎo)DNA甲基化與組蛋白乙酰化之間的相互作用。我們實(shí)驗(yàn)室前期研究表明,在細(xì)胞水平上表觀遺傳藥物DAC與化療藥物奧沙利鉑(Oxaliplatin, Oxa)聯(lián)合用藥能增加RCC細(xì)胞系對(duì)Oxa的敏感性。為得到更可靠的結(jié)論,構(gòu)建了RCC細(xì)胞系(786-O, Caki-1)裸鼠移植瘤模型,采用聯(lián)合用藥治療,發(fā)現(xiàn)通過表觀遺傳藥物DAC逆轉(zhuǎn)RCC中OCT2蛋白的表達(dá),能夠增加Oxa在腫瘤組織的蓄積從而殺死腫瘤細(xì)胞。通過上述研究表明,兩種表觀遺傳藥物DAC與SAHA均能逆轉(zhuǎn)RCC中OCT2基因的低表達(dá),轉(zhuǎn)錄因子MYC則作為DNA甲基化與組蛋白修飾(組蛋白甲基化與組蛋白乙�；�)之間的紐帶：1.組蛋白甲基化DAC能夠降低DNA甲基化水平,提高M(jìn)YC在OCT2啟動(dòng)子區(qū)的富集,增加MLL1的招募,催化H3K4me3修飾信號(hào),促進(jìn)OCT2基因的轉(zhuǎn)錄。2.組蛋白乙酰化RCC患者中HDAC7/9存在異常高表達(dá),靶向干擾HDAC7/9的siRNA能上調(diào)OCT2的]mRNA表達(dá),而SAHA依賴于MYC影響OCT2啟動(dòng)子區(qū)HDAC7/9的富集,提高OCT2的轉(zhuǎn)錄水平。該機(jī)制適用于786-0細(xì)胞,在769-P細(xì)胞中誘導(dǎo)機(jī)制還有待研究。DAC與SAHA合用時(shí)OCT2的轉(zhuǎn)錄水平較單用時(shí)顯著升高,可見DNA甲基化水平的降低,有助于MYC對(duì)HDAC7/9的調(diào)節(jié),起到協(xié)同作用。基于以上調(diào)控機(jī)理,我們采用聯(lián)合用藥的方案治療RCC細(xì)胞系移植瘤裸鼠,能顯著抑制腫瘤生長(zhǎng)。上述結(jié)果,可為臨床設(shè)計(jì)增加RCC對(duì)化療藥物的敏感性的新治療方案提供參考。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.11

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