本文選題：IDH1R132H + 前列腺癌��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：前列腺癌(Prostate cancer,PCa)是歐美男性生殖系統(tǒng)常見的惡性腫瘤,其死亡率位居男性惡性腫瘤的第二位。近年來,隨著血清前列腺特異性抗原(Prostate specific antigen,PSA)篩查的逐步推廣,前列腺癌發(fā)病率快速上升,正日益成為嚴(yán)重威脅我國老年男性健康的疾病。目前臨床治療的重大挑戰(zhàn)之一是如何在前列腺癌中區(qū)分哪些腫瘤生長緩慢,哪些腫瘤可能迅速進(jìn)展,威脅生命。即如何判定腫瘤的"惰性(indolent)"與"侵襲性(aggressive)",并在此基礎(chǔ)上進(jìn)行個體化治療。目前,前列腺癌的過度診斷和過度治療非常嚴(yán)重�，F(xiàn)在普遍應(yīng)用的臨床病理學(xué)參數(shù):Gleason評分,PSA水平,以及臨床病理分期等尚不能很好的判斷預(yù)后。因此,基于分子分型基礎(chǔ)上,對前列腺癌病人進(jìn)行危險分級,來區(qū)分惰性以及侵襲性腫瘤,從而進(jìn)行精準(zhǔn)治療,對前列腺癌的診治至關(guān)重要。異檸檬酸脫氫酶(Isocitrate dehydrogenase,IDH)是體內(nèi)物質(zhì)代謝的關(guān)鍵酶,可以催化異檸檬酸轉(zhuǎn)化成α酮戊二酸(α-ketoglutarate,α-KG),同時增加NADPH的產(chǎn)生。很多腫瘤中都存在異檸檬酸脫氫酶,特別是IDH1/IDH2的突變,這種可能的原癌性質(zhì)的突變可以改變細(xì)胞內(nèi)代謝,產(chǎn)生異常代謝產(chǎn)物以及廣泛的表觀遺傳學(xué)改變。有研究表明,IDH1突變陽性前列腺癌患者代表一種獨(dú)特的分子亞型(以IDH1R132H最常見),但其在前列腺癌發(fā)生發(fā)展中的作用尚不清楚。通過本研究發(fā)現(xiàn),IDH1R132H在前列腺癌中的發(fā)生率在0.6%(2/336)。體外實驗證實具有IDH1R132H的良性前列腺上皮細(xì)胞遷移能力更強(qiáng),在低細(xì)胞因子情況下,有明顯的增殖優(yōu)勢(前列腺癌細(xì)胞中的作用相反)。進(jìn)一步的研究表明IDH1R132H可以通過表觀遺傳學(xué)修飾的改變抑制miR-141-3p,miR-7-5p,miR-223-3p的表達(dá),從而使胰島素樣生因子受體1(Insulin-like Growth Factor 1 Receptor,IGF1R)表達(dá)水平升高,進(jìn)而激活A(yù)KT/STAT3信號通路促使良性前列腺上皮細(xì)胞發(fā)生惡性轉(zhuǎn)化。本研究首次較為系統(tǒng)的闡述了 IDH1R132H在良性前列腺上皮細(xì)胞中所發(fā)揮的作用以及作用機(jī)制,為IDH1R132H在前列腺癌中的診治提供了一定的依據(jù)。研究目的:1、檢測IDH1R132H在前列腺癌患者中的發(fā)生頻度2、研究IDH1R132H在良性前列腺上皮細(xì)胞及前列腺癌細(xì)胞中所發(fā)揮的功能3、探討IDH1R132H促進(jìn)良性前列腺上皮細(xì)胞惡性轉(zhuǎn)化的作用機(jī)制研究方法:1、檢測IDH1R132H在前列腺癌患者中的發(fā)生頻度及突變陽性前列腺癌患者的臨床分子病理特征1.1免疫組織化學(xué)法初步篩查IDH1R132H在前列腺癌組織中的陽性病例。1.2將初步篩查得到的IDH1R132H陽性病例,通過石蠟組織提取DNA,PCR后進(jìn)行測序分析,進(jìn)一步確定IDH1R132H突變。分析IDH1R132H突變陽性前列腺癌患者的臨床分子病理特征。1.3 一代測序檢測前列腺癌及正常細(xì)胞系中IDH1突變情況。2、研究IDH1R132H在良性前列腺上皮細(xì)胞、前列腺癌細(xì)胞中的生物學(xué)功能2.1 慢病毒轉(zhuǎn)染 RWPE-1、LNCAP 細(xì)胞,形成 IDH1R132H,IDH1WT,VECTOR穩(wěn)定表達(dá)的細(xì)胞株,嘌呤霉素進(jìn)行抗性篩選,Western Blot、RT-qPCR檢測其表達(dá)效率。2.2MTS檢測穩(wěn)定表達(dá)IDH1R132H,IDH1WT,VECTOR后,細(xì)胞增殖能力的變化。2.3 Transwell及劃痕實驗檢測穩(wěn)定表達(dá)IDH1R132H,IDH1WT,VECTOR后,細(xì)胞遷移能力的變化。2.4 RT-qPCR檢測前列腺癌干細(xì)胞以及分化相關(guān)指標(biāo)CD133,CD44,α 2 γ 1,AR,PSA等的表達(dá)變化。2.5 AGI5198處理細(xì)胞后,MTS以及Transwell實驗檢測細(xì)胞的增殖及遷移能力的變化。3、探討IDH1R132H促進(jìn)良性前列腺上皮惡性轉(zhuǎn)化的作用機(jī)制3.1 檢測 IDH1R132H 所調(diào)控的 microRNA。1)MicroRNA芯片初步篩選IDH1R132H所調(diào)控的microRNA。2)RT-qPCR 驗證 microRNA 的變化。3)染色質(zhì)免疫共沉淀(ChIP)實驗檢測在microRNAs啟動子區(qū)H3K4me3和H3K27me3富集情況。3.2IDH1R132H 通過下調(diào) miR-141-3p,miR-7-5p,miR-223-3p 促進(jìn) IGF1R 的表達(dá)。1)Targetscan 軟件預(yù)測 miR-141-3p,miR-7-5p,miR-223-3p 的共同靶基因。2)Western Blot及免疫熒光實驗檢測穩(wěn)定表達(dá)IDH1R132H,IDH1WT,VECTOR后,IGF1R的蛋白表達(dá)水平。3)在穩(wěn)定表達(dá)IDH1R132H的RWPE-1細(xì)胞中加入microRNA的mimic和陰性對照,Western Blot檢測其靶基因IGF1R的變化。4)構(gòu)建IGF1R-3'UTR熒光素酶報告基因表達(dá)載體,將microRNA的mimic以及報告基因表達(dá)載體共同轉(zhuǎn)染入HEK293T細(xì)胞中,檢測熒光素酶活性。5)MRNA表達(dá)譜芯片檢測IDH1R132H及VECTOR的差異性基因,通過生物信息學(xué)分析IGF1R相關(guān)基因的變化。3.3驗證IDH1R132H通過IGF1R促進(jìn)良性前列腺上皮細(xì)胞RWPE-1惡性轉(zhuǎn)化。1)Western Blot檢測IGF 1R下游信號通路AKT,STAT3及磷酸化活性形式的變化。2)siRNA干擾IGF1R后檢測AKT,STAT3以及其磷酸化活性形式的變化。3)siRNA干擾IGF1R,MTS及Transwell實驗檢測細(xì)胞增殖遷移能力的變化。研究結(jié)果:1、IDH1R132H在前列腺癌中的發(fā)生率為0.6%(2/336),IDH1R132H前列腺癌患者缺乏常見的基因突變表型1.1免疫組織化學(xué)(IHC)檢測前列腺癌病例標(biāo)本共計336例,結(jié)果顯示IDH1R132H表達(dá)強(qiáng)陽性(2+,3+)的病例有4個,IDH1R132H表達(dá)弱陽性(1+)的病例有11個。IDH1R13H主要表達(dá)于前列腺癌組織的細(xì)胞漿和細(xì)胞核中。1.2通過PCR基礎(chǔ)上的測序證實IDH1R132H的發(fā)生率為0.6%(2/336)。免疫組化結(jié)果顯示其他重要的前列腺癌相關(guān)的分子特征性改變?nèi)鏓RG重排,SPINK1過表達(dá)等均為陰性。1.3通過PCR基礎(chǔ)上的測序結(jié)果顯示,前列腺癌細(xì)胞系LNCAP,VCAP,22RV1以及良性前列腺上皮細(xì)胞RWPE-1中均未發(fā)現(xiàn)IDH1R132H突變。2、IDH1R132H促進(jìn)良性前列腺上皮細(xì)胞RWPE-1惡性轉(zhuǎn)化2.1 IDH1R132H促進(jìn)RWPE-1細(xì)胞株細(xì)胞因子的非依賴性生長,促進(jìn)RWPE-1細(xì)胞遷移。2.2 IDH1R132H抑制LNCAP細(xì)胞的增殖和遷移能力。2.3 IDH1R132H促進(jìn)干細(xì)胞指標(biāo)CD133的表達(dá)。2.4 AGI5198抑制IDH1R132H突變的RWPE-1細(xì)胞增殖及遷移能力。3、IDH1R132H 通過改變組蛋白修飾抑制 miR-141-3p,miR-223-3p,miR-7-5p 的表達(dá),使IGF1R的表達(dá)水平增高,激活A(yù)KT/STAT3信號通路,從而促進(jìn)RWPE-1細(xì)胞惡性轉(zhuǎn)化3.1 IDH1R132H 調(diào)控 microRNA 的表達(dá)。1)MicroRNA芯片顯示,將IDH1R132H/VECTOR差異兩倍以上的microRNA和IDH1R132H/IDH1WT差異兩倍以上的microRNA取交集。IDH1R132H中共同上調(diào)的microRNA有4個,共同下調(diào)的microRNA有68個。2)通過查找文獻(xiàn),在IDH1R132H下調(diào)的microRNA中篩選前列腺癌中具有抑癌功能的 microRNA,進(jìn)行 PCR 驗證其表達(dá),其中 miR-141-3p,miR-223-3p,miR-7-5p下調(diào)倍數(shù)最明顯。3)ChIP實驗證明,在miR-141啟動子區(qū)上游P3、P4,miR-7-1的P2、P4,以及miR-223的P2、P3、P4引物擴(kuò)增位置有H3K4me3富集的降低,此外在miR-7-1的P2以及miR-223的P2位置,有H3K27me3的富集升高。3.2 IDH1R132H 通過抑制 miR-141-3p,miR-7-5p,miR-223-3p 的表達(dá)從而使IGF1R的表達(dá)水平升高。1)Targetscan 預(yù)測發(fā)現(xiàn) IGF1R 為 miR-141-3p,miR-7-5p,miR-223-3p 的共同靶基因。2)IDH1R132H促進(jìn)RWPE-1細(xì)胞中IGF1R的蛋白表達(dá)。3)穩(wěn)定表達(dá)IDH1R132H的RWPE-1細(xì)胞加入microRNA的mimics后IGF1R的蛋白水平表達(dá)降低。4)雙熒光素酶實驗證實IGF1R為miR-141-3p,miR-7-5p,miR-223-3p的靶基因。5)GSEA分析顯示IGF1R所上調(diào)的基因富集在IDH1R132H中。3.3 IDH1R132H通過上調(diào)IGF1R使RWPE-1細(xì)胞惡性轉(zhuǎn)化。1)IDH1R132H促進(jìn)IGF1R下游AKT,STAT3信號通路激活。2)表達(dá) IDH1R132H 的 RWPE-1 細(xì)胞中,干擾 IGF1R 后 P-AKT,P-STAT3 的表達(dá)水平降低。3)干擾IGF1R后,表達(dá)IDH1R132H的RWPE-1細(xì)胞增殖能力以及遷移能力均被抑制。研究結(jié)論:1、IDH1R132H在前列腺癌病人中的發(fā)生率為0.6%(2/336),IDH1突變陽性的前列腺癌病例缺乏其他前列腺癌重要的分子病理特征改變。2、IDH1R132H可以促進(jìn)良性前列腺上皮細(xì)胞發(fā)生惡性轉(zhuǎn)化—細(xì)胞因子的非依賴性生長,遷移能力增強(qiáng)。3、IDH1R132H 通過抑制 miR-141-3p,miR-7-5p,miR-223-3p 的表達(dá),促使 IGF1R—AKT/STAT3信號通路的激活從而促進(jìn)良性前列腺上皮細(xì)胞惡性轉(zhuǎn)化。
[Abstract]:Prostate cancer (PCa) is a common malignant tumor in the male reproductive system in Europe and America. Its mortality rate is the second of the male malignant tumor. In recent years, with the gradual promotion of the serum prostate specific antigen (Prostate specific antigen, PSA) screening, the incidence of prostatic adenocarcinoma is rising rapidly and is becoming a serious threat to our country. One of the major challenges for clinical treatment is how to distinguish which tumors grow slowly in prostate cancer and which may develop rapidly and threaten life. That is, how to determine the tumor 's "indolent" and "aggressive", and on the basis of the individualized treatment. Excessive diagnosis and overtreatment are very serious. The commonly used clinicopathological parameters, such as Gleason score, PSA level, and clinicopathological staging, are not well judged. Therefore, based on molecular typing, the risk classification of prostate cancer patients to distinguish between inert and invasive tumors is accurate. Treatment is very important for the diagnosis and treatment of prostate cancer. Isocitrate dehydrogenase (IDH) is a key enzyme in substance metabolism in the body. It can catalyze ISO citrate into alpha ketopamyl diacid (alpha -ketoglutarate, alpha -KG) and increase the production of NADPH. There are many ISO citrate dehydrogenases in many swelling tumors, especially the process of IDH1/IDH2. Change, this possible primary cancer mutation can change intracellular metabolism, produce abnormal metabolites and extensive epigenetic changes. Studies have shown that IDH1 positive prostate cancer patients represent a unique molecular subtype (most common in IDH1R132H), but its role in the development of prostate cancer is not yet clear. The previous study found that the incidence of IDH1R132H in prostate cancer was 0.6% (2/336). In vitro experiments confirmed that the benign prostatic epithelial cells with IDH1R132H were more capable of migrating. In the case of low cytokine, there was an obvious proliferation advantage (the reaction in the prostate cancer cells). Further research showed that IDH1R132H could be apparent through the apparent view. The changes in genetic modification inhibit the expression of miR-141-3p, miR-7-5p and miR-223-3p, thus increasing the expression level of insulin like factor receptor 1 (Insulin-like Growth Factor 1 Receptor, IGF1R), and then activating the AKT/STAT3 signaling pathway to induce the malignant transformation of benign prostatic epithelial cells. This study was the first to systematically elaborate IDH. The role of 1R132H in benign prostatic epithelial cells and the mechanism of action provide some basis for the diagnosis and treatment of IDH1R132H in prostate cancer. 1, the frequency of the occurrence of IDH1R132H in the prostate cancer patients was 2, and the function of IDH1R132H in benign prostatic skin cells and prostate cancer cells was studied 3. The study of the mechanism of IDH1R132H to promote the malignant transformation of benign prostatic epithelial cells: 1. Detection of the frequency of IDH1R132H in the patients with prostate cancer and the clinical molecular pathological features of the patients with positive prostate cancer; the preliminary screening of the positive cases of IDH1R132H in the prostate cancer tissue by immunohistochemical method, 1.1 IDH1R132H positive cases were screened by step screening, DNA was extracted from paraffin tissue, and PCR was sequenced to further determine the IDH1R132H mutation. The clinical molecular pathological features of IDH1R132H positive prostate cancer patients were analyzed by.1.3 1 sequencing to detect the IDH1 mutation in prostate cancer and normal cell lines, and the IDH1R132H was before benign. Adenosine epithelial cells, biological function of prostate cancer cells 2.1 lentivirus transfected RWPE-1, LNCAP cells, forming IDH1R132H, IDH1WT, VECTOR stable cell lines, purinamycin resistance screening, Western Blot, RT-qPCR detection of the expression efficiency.2.2MTS detection for IDH1R132H, IDH1WT, VECTOR, cell proliferation ability Change.2.3 Transwell and scratch test to detect the cell migration ability after IDH1R132H, IDH1WT, VECTOR,.2.4 RT-qPCR detection of prostate cancer stem cells and differentiation related indicators CD133, CD44, alpha 2, 1, AR, PSA, etc. Changes in capacity.3, mechanism of action of IDH1R132H to promote malignant transformation of benign prostatic epithelium 3.1 detection of microRNA.1 regulated by IDH1R132H) MicroRNA chip screening IDH1R132H regulated microRNA.2) RT-qPCR verifying microRNA's change.3) chromatin immunoprecipitation (ChIP) test in microRNAs promoter region And H3K27me3 enrichment.3.2IDH1R132H through down-regulation of miR-141-3p, miR-7-5p, miR-223-3p to promote IGF1R expression.1) Targetscan software predicts miR-141-3p, miR-7-5p, miR-223-3p common target gene.2) and the stable expression of immunofluorescence test. The RWPE-1 cells expressing IDH1R132H were added to microRNA mimic and negative control, Western Blot was used to detect the change.4 of the target gene IGF1R, and the IGF1R-3'UTR luciferase reporter gene expression vector was constructed. The mimic of microRNA and the reporter gene expression vector were transfected into the HEK293T cells, and the luciferase expression profile chip was detected. Detection of the differentially genes of IDH1R132H and VECTOR, through bioinformatics analysis of the changes in IGF1R related genes.3.3 verification of IDH1R132H through IGF1R to promote the RWPE-1 malignant transformation of.1 in benign prostatic epithelial cells, Western Blot detection of IGF 1R downstream signal pathway T3 and changes in its phosphorylation activity.3) siRNA interference with IGF1R, MTS and Transwell tests for cell proliferation and migration. 1, the incidence of IDH1R132H in prostate cancer is 0.6% (2/336), and IDH1R132H prostate cancer patients lack common gene mutation phenotype 1.1 immuno histochemistry (IHC) for the detection of prostate cancer The total of 336 cases showed that there were 4 cases of strong positive IDH1R132H expression (2+, 3+), and 11.IDH1R13H in the cases of IDH1R132H expression (1+) were expressed mainly in the cytoplasm and nucleus of the prostate cancer tissue and the.1.2 through PCR based on PCR confirmed that the occurrence rate of IDH1R132H was 0.6% (2/336). The immunohistochemical results showed the others. Important prostate cancer related molecular characteristics such as ERG rearrangement, SPINK1 overexpression and negative.1.3 sequencing on the basis of PCR showed that no IDH1R132H mutation.2 was found in the prostate cancer cell lines LNCAP, VCAP, 22RV1, and benign prostatic epithelial RWPE-1, and IDH1R132H promotes benign prostatic epithelial cell RWPE-1. Sexual transformation of 2.1 IDH1R132H promotes the non dependent growth of cytokines in RWPE-1 cells, promotes the migration of RWPE-1 cells by.2.2 IDH1R132H, inhibits the proliferation and migration of LNCAP cells,.2.3 IDH1R132H promotes the expression of CD133 in stem cells,.2.4 AGI5198 inhibits the proliferation and migration of IDH1R132H mutations. Histone modification inhibits the expression of miR-141-3p, miR-223-3p, and miR-7-5p, increases the expression level of IGF1R, activates the AKT/STAT3 signaling pathway, and thus promotes the malignant transformation of RWPE-1 cells by 3.1 IDH1R132H to regulate the expression of microRNA, and MicroRNA chips display two times the difference of microRNA from IDH1R132H/VECTOR difference over two times. There are 4 microRNA together up.IDH1R132H in the above microRNA, and 68.2 for the common downregulation. Through the search of the literature, the microRNA that has the tumor suppressor function in the prostate cancer is screened in the microRNA of the IDH1R132H downregulation, and PCR is used to verify the expression of the microRNA. MiR-141-3p, miR-223-3p, and miR-7-5p downregulation are the most obvious. The experimental results show that the amplification position of P3, P4, miR-7-1, P2, P3, P4 primers in the upstream of the miR-141 promoter region is lower than that of H3K4me3 enrichment. Targetscan prediction found that IGF1R is the common target gene.2 of miR-141-3p, miR-7-5p, miR-223-3p,.2) IDH1R132H promotes the protein expression of IGF1R in RWPE-1 cells. -5p, miR-223-3p target gene.5) GSEA analysis shows that the gene of IGF1R is enriched in IDH1R132H.3.3 IDH1R132H by up regulation of IGF1R makes RWPE-1 cell malignant transformation.1) IDH1R132H promotes the downstream of the signaling pathway. After interfering with IGF1R, the proliferation and migration ability of RWPE-1 cells expressing IDH1R132H were suppressed. 1, the incidence of IDH1R132H in prostate cancer patients was 0.6% (2/336), and the IDH1 mutation positive prostate cancer cases lacked the other important molecular pathophysiological characteristics of prostate cancer,.2, IDH1R132H could promote the benign prostatic hyperplasia. The malignant transformation of the skin cells - the non dependent growth of cytokine, the mobility of.3, and the inhibition of the expression of miR-141-3p, miR-7-5p, and miR-223-3p by IDH1R132H, promote the activation of IGF1R AKT/STAT3 signaling pathway to promote the malignant transformation of benign prostatic epithelial cells.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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