IDH1R132H促进良性前列腺上皮细胞恶性转化及其作用机制的研究

发布时间：2018-04-28 07:49

本文选题：IDH1R132H + 前列腺癌　；参考：《山东大学》2017年硕士论文

【摘要】：前列腺癌(Prostate cancer,PCa)是欧美男性生殖系统常见的恶性肿瘤,其死亡率位居男性恶性肿瘤的第二位。近年来,随着血清前列腺特异性抗原(Prostate specific antigen,PSA)筛查的逐步推广,前列腺癌发病率快速上升,正日益成为严重威胁我国老年男性健康的疾病。目前临床治疗的重大挑战之一是如何在前列腺癌中区分哪些肿瘤生长缓慢,哪些肿瘤可能迅速进展,威胁生命。即如何判定肿瘤的"惰性(indolent)"与"侵袭性(aggressive)",并在此基础上进行个体化治疗。目前,前列腺癌的过度诊断和过度治疗非常严重。现在普遍应用的临床病理学参数:Gleason评分,PSA水平,以及临床病理分期等尚不能很好的判断预后。因此,基于分子分型基础上,对前列腺癌病人进行危险分级,来区分惰性以及侵袭性肿瘤,从而进行精准治疗,对前列腺癌的诊治至关重要。异柠檬酸脱氢酶(Isocitrate dehydrogenase,IDH)是体内物质代谢的关键酶,可以催化异柠檬酸转化成α酮戊二酸(α-ketoglutarate,α-KG),同时增加NADPH的产生。很多肿瘤中都存在异柠檬酸脱氢酶,特别是IDH1/IDH2的突变,这种可能的原癌性质的突变可以改变细胞内代谢,产生异常代谢产物以及广泛的表观遗传学改变。有研究表明,IDH1突变阳性前列腺癌患者代表一种独特的分子亚型(以IDH1R132H最常见),但其在前列腺癌发生发展中的作用尚不清楚。通过本研究发现,IDH1R132H在前列腺癌中的发生率在0.6%(2/336)。体外实验证实具有IDH1R132H的良性前列腺上皮细胞迁移能力更强,在低细胞因子情况下,有明显的增殖优势(前列腺癌细胞中的作用相反)。进一步的研究表明IDH1R132H可以通过表观遗传学修饰的改变抑制miR-141-3p,miR-7-5p,miR-223-3p的表达,从而使胰岛素样生因子受体1(Insulin-like Growth Factor 1 Receptor,IGF1R)表达水平升高,进而激活AKT/STAT3信号通路促使良性前列腺上皮细胞发生恶性转化。本研究首次较为系统的阐述了 IDH1R132H在良性前列腺上皮细胞中所发挥的作用以及作用机制,为IDH1R132H在前列腺癌中的诊治提供了一定的依据。研究目的:1、检测IDH1R132H在前列腺癌患者中的发生频度2、研究IDH1R132H在良性前列腺上皮细胞及前列腺癌细胞中所发挥的功能3、探讨IDH1R132H促进良性前列腺上皮细胞恶性转化的作用机制研究方法:1、检测IDH1R132H在前列腺癌患者中的发生频度及突变阳性前列腺癌患者的临床分子病理特征1.1免疫组织化学法初步筛查IDH1R132H在前列腺癌组织中的阳性病例。1.2将初步筛查得到的IDH1R132H阳性病例,通过石蜡组织提取DNA,PCR后进行测序分析,进一步确定IDH1R132H突变。分析IDH1R132H突变阳性前列腺癌患者的临床分子病理特征。1.3 一代测序检测前列腺癌及正常细胞系中IDH1突变情况。2、研究IDH1R132H在良性前列腺上皮细胞、前列腺癌细胞中的生物学功能2.1 慢病毒转染 RWPE-1、LNCAP 细胞,形成 IDH1R132H,IDH1WT,VECTOR稳定表达的细胞株,嘌呤霉素进行抗性筛选,Western Blot、RT-qPCR检测其表达效率。2.2MTS检测稳定表达IDH1R132H,IDH1WT,VECTOR后,细胞增殖能力的变化。2.3 Transwell及划痕实验检测稳定表达IDH1R132H,IDH1WT,VECTOR后,细胞迁移能力的变化。2.4 RT-qPCR检测前列腺癌干细胞以及分化相关指标CD133,CD44,α 2 γ 1,AR,PSA等的表达变化。2.5 AGI5198处理细胞后,MTS以及Transwell实验检测细胞的增殖及迁移能力的变化。3、探讨IDH1R132H促进良性前列腺上皮恶性转化的作用机制3.1 检测 IDH1R132H 所调控的 microRNA。1)MicroRNA芯片初步筛选IDH1R132H所调控的microRNA。2)RT-qPCR 验证 microRNA 的变化。3)染色质免疫共沉淀(ChIP)实验检测在microRNAs启动子区H3K4me3和H3K27me3富集情况。3.2IDH1R132H 通过下调 miR-141-3p,miR-7-5p,miR-223-3p 促进 IGF1R 的表达。1)Targetscan 软件预测 miR-141-3p,miR-7-5p,miR-223-3p 的共同靶基因。2)Western Blot及免疫荧光实验检测稳定表达IDH1R132H,IDH1WT,VECTOR后,IGF1R的蛋白表达水平。3)在稳定表达IDH1R132H的RWPE-1细胞中加入microRNA的mimic和阴性对照,Western Blot检测其靶基因IGF1R的变化。4)构建IGF1R-3'UTR荧光素酶报告基因表达载体,将microRNA的mimic以及报告基因表达载体共同转染入HEK293T细胞中,检测荧光素酶活性。5)MRNA表达谱芯片检测IDH1R132H及VECTOR的差异性基因,通过生物信息学分析IGF1R相关基因的变化。3.3验证IDH1R132H通过IGF1R促进良性前列腺上皮细胞RWPE-1恶性转化。1)Western Blot检测IGF 1R下游信号通路AKT,STAT3及磷酸化活性形式的变化。2)siRNA干扰IGF1R后检测AKT,STAT3以及其磷酸化活性形式的变化。3)siRNA干扰IGF1R,MTS及Transwell实验检测细胞增殖迁移能力的变化。研究结果:1、IDH1R132H在前列腺癌中的发生率为0.6%(2/336),IDH1R132H前列腺癌患者缺乏常见的基因突变表型1.1免疫组织化学(IHC)检测前列腺癌病例标本共计336例,结果显示IDH1R132H表达强阳性(2+,3+)的病例有4个,IDH1R132H表达弱阳性(1+)的病例有11个。IDH1R13H主要表达于前列腺癌组织的细胞浆和细胞核中。1.2通过PCR基础上的测序证实IDH1R132H的发生率为0.6%(2/336)。免疫组化结果显示其他重要的前列腺癌相关的分子特征性改变如ERG重排,SPINK1过表达等均为阴性。1.3通过PCR基础上的测序结果显示,前列腺癌细胞系LNCAP,VCAP,22RV1以及良性前列腺上皮细胞RWPE-1中均未发现IDH1R132H突变。2、IDH1R132H促进良性前列腺上皮细胞RWPE-1恶性转化2.1 IDH1R132H促进RWPE-1细胞株细胞因子的非依赖性生长,促进RWPE-1细胞迁移。2.2 IDH1R132H抑制LNCAP细胞的增殖和迁移能力。2.3 IDH1R132H促进干细胞指标CD133的表达。2.4 AGI5198抑制IDH1R132H突变的RWPE-1细胞增殖及迁移能力。3、IDH1R132H 通过改变组蛋白修饰抑制 miR-141-3p,miR-223-3p,miR-7-5p 的表达,使IGF1R的表达水平增高,激活AKT/STAT3信号通路,从而促进RWPE-1细胞恶性转化3.1 IDH1R132H 调控 microRNA 的表达。1)MicroRNA芯片显示,将IDH1R132H/VECTOR差异两倍以上的microRNA和IDH1R132H/IDH1WT差异两倍以上的microRNA取交集。IDH1R132H中共同上调的microRNA有4个,共同下调的microRNA有68个。2)通过查找文献,在IDH1R132H下调的microRNA中筛选前列腺癌中具有抑癌功能的 microRNA,进行 PCR 验证其表达,其中 miR-141-3p,miR-223-3p,miR-7-5p下调倍数最明显。3)ChIP实验证明,在miR-141启动子区上游P3、P4,miR-7-1的P2、P4,以及miR-223的P2、P3、P4引物扩增位置有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 通过抑制 miR-141-3p,miR-7-5p,miR-223-3p 的表达从而使IGF1R的表达水平升高。1)Targetscan 预测发现 IGF1R 为 miR-141-3p,miR-7-5p,miR-223-3p 的共同靶基因。2)IDH1R132H促进RWPE-1细胞中IGF1R的蛋白表达。3)稳定表达IDH1R132H的RWPE-1细胞加入microRNA的mimics后IGF1R的蛋白水平表达降低。4)双荧光素酶实验证实IGF1R为miR-141-3p,miR-7-5p,miR-223-3p的靶基因。5)GSEA分析显示IGF1R所上调的基因富集在IDH1R132H中。3.3 IDH1R132H通过上调IGF1R使RWPE-1细胞恶性转化。1)IDH1R132H促进IGF1R下游AKT,STAT3信号通路激活。2)表达 IDH1R132H 的 RWPE-1 细胞中,干扰 IGF1R 后 P-AKT,P-STAT3 的表达水平降低。3)干扰IGF1R后,表达IDH1R132H的RWPE-1细胞增殖能力以及迁移能力均被抑制。研究结论:1、IDH1R132H在前列腺癌病人中的发生率为0.6%(2/336),IDH1突变阳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变。2、IDH1R132H可以促进良性前列腺上皮细胞发生恶性转化—细胞因子的非依赖性生长,迁移能力增强。3、IDH1R132H 通过抑制 miR-141-3p,miR-7-5p,miR-223-3p 的表达,促使 IGF1R—AKT/STAT3信号通路的激活从而促进良性前列腺上皮细胞恶性转化。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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