【摘要】：前列腺癌是一個多病因且復雜的常見病,是威脅男性健康的常見腫瘤。在我國,隨著人口老齡化的加劇,前列腺癌的發(fā)病率逐年遞增。前列腺癌已成為威脅中國男性健康的主要疾病之一。近年來,隨著miRNA的發(fā)現(xiàn),經(jīng)典的分子生物學理論發(fā)生了重大變革,也使人們對惡性腫瘤的發(fā)生發(fā)展機制有了新的認識。在這個過程中,miRNA以其重要的診斷和治療價值給前列腺癌的研究帶來了新的曙光。本課題組前期通過miRNAs芯片篩選發(fā)現(xiàn),相對于正常前列腺組織和雄激素依賴性前列腺癌組織而言,去勢抵抗性前列腺癌組織中miR-146a的表達水平偏低,并且通過定量PCR手段在臨床前列腺癌切除標本上驗證了這一結(jié)果。后通過分析MSKCC數(shù)據(jù)庫,我們發(fā)現(xiàn)轉(zhuǎn)移性前列腺癌中miR-146a表達水平低于局限性癌,同時Gleason=8分癌組織中miR-146a表達水平低于Gleason8分的癌組織。并且癌組織低表達miR-146a的患者在根治手術(shù)后生化復發(fā)風險更高。我們通過細胞生物學實驗驗證了 miR-146a能夠抑制前列腺癌的增殖、侵襲及遷移等生物學功能,以及誘導癌細胞凋亡的能力,并且抑制癌細胞的體外成瘤能力。糖酵解代謝作為前列腺癌細胞的重要代謝特征之一,其過程中的分子調(diào)控機制暫時不完全明確。且miR-146a分子對前列腺癌糖代謝功能的影響尚未有研究和定論。在第一部分中,我們通過向前列腺癌細胞(PC3和DU145)內(nèi)轉(zhuǎn)染miR-146a mimics后進行細胞內(nèi)ATP檢測、上清液乳酸生成量和葡萄糖濃度檢測、糖酵解速度檢測等手段檢測了 miR-146a對前列腺癌細胞糖代謝能力的影響。結(jié)果發(fā)現(xiàn)miR-146a能夠降低前列腺癌細胞的葡萄糖攝取能力,同時降低細胞的ATP生成量,并且降低糖酵解代謝生成的乳酸量。通過XF96儀器檢測細胞外酸化速度發(fā)現(xiàn)miR-146a抑制了前列腺癌細胞的糖酵解能力。整理和分析GEO數(shù)據(jù)庫中GSE35988數(shù)據(jù)發(fā)現(xiàn),已糖激酶2 (HK2)在前列腺良性增生切除的標本中的表達水平顯著性低于雄激素依賴性前列腺癌和去勢抵抗性前列腺癌,同時去勢抵抗性前列腺癌組織中HK2的表達水平高于雄激素依賴性前列腺癌組織。在前列腺癌細胞中用siRNA下調(diào)HK2蛋白質(zhì)的表達后,發(fā)現(xiàn)細胞的ATP生成量減少,糖酵解產(chǎn)生的乳酸量下降,葡萄糖攝取能力下降,上機檢測細胞外酸化速度發(fā)現(xiàn),細胞的糖酵解能力下降。我們通過熒光素酶報告基因及Western bloting證實HK2是miR-146a的靶基因,通過向細胞中轉(zhuǎn)染miR-146a后恢復性表達HK2蛋白,發(fā)現(xiàn)癌細胞的葡萄糖攝取能力回升,同時糖酵解生成的乳酸量回升,ATP產(chǎn)量回升,機器檢測發(fā)現(xiàn)癌細胞的糖酵解能力得到了恢復。由此我們證明在前列腺癌細胞中,miR-146a通過靶向下調(diào)HK2蛋白的表達,抑制細胞糖酵解能力。聯(lián)合本課題組的所有實驗結(jié)果,miR-146a的抑癌作用已被研究和證明。但miR-146a在前列腺癌中的表達調(diào)控機制仍不十分明確,因此第二部分將研究重心放在了 miR-146a的表達機制的調(diào)控上。第二部分中,我們通過UCSC生物信息數(shù)據(jù)庫,預(yù)測了 miR-146a轉(zhuǎn)錄因子YY1。通過分析ONCOMINE數(shù)據(jù)庫分析發(fā)現(xiàn)前列腺癌組織的YY1表達水平高于正常前列腺組織,轉(zhuǎn)移性前列腺癌組織中YY1表達水平亦高于原位前列腺癌。分析Nakagawa上傳的數(shù)據(jù)發(fā)現(xiàn)且前列腺癌根治術(shù)后生化復發(fā)的患者癌組織中YY1的表達水平顯著高于非生化復發(fā)組,且YY1的表達水平隨著Gleason評分升高。我們選取45例臨床前列腺癌組織,利用免疫組化技術(shù)檢測石蠟切片中YY1蛋白的表達,同時利用原位雜交技術(shù)檢測石蠟切片中miR-146a分子的表達,量化統(tǒng)計結(jié)果后分析發(fā)現(xiàn)miR-146a與YY1的表達呈負相關(guān)(P = 0.007)。通過敲低PC3細胞YY1表達后,提取細胞總RNA,測序獲得全轉(zhuǎn)錄組RNA數(shù)據(jù),并通過基因探針富集分析(GSEA)發(fā)現(xiàn)miR-146a靶基因RNA水平呈現(xiàn)了負向富集(NES=-1.039, FDR=0.267,P=0.15),而EZH2抑制的基因集發(fā)生正向富集(NES=1.34, FDR=0.02,P=0.02)。同時通過熒光定量PCR檢測發(fā)現(xiàn)下調(diào)YY1表達后,miR-146a的表達水平顯著升高(P0.05)。我們構(gòu)建miR-146a初始轉(zhuǎn)錄本上游序列報告基因質(zhì)粒(-1226～36bp),通過熒光素報告基因?qū)嶒灪腿旧|(zhì)免疫共沉淀實驗驗證了 YY1對miR-146a的轉(zhuǎn)錄抑制調(diào)控。熒光定量PCR檢測發(fā)現(xiàn)YY1和EZH2同時下調(diào)后miR-146a的表達進一步升高。最后通過提取細胞核蛋白,實施蛋白質(zhì)共沉淀實驗驗證了 YY1聯(lián)合EZH2參與miR-146a的轉(zhuǎn)錄抑制過程。綜上第二部分結(jié)果,我們證明了在前列腺癌細胞中,YY1能夠招募EZH2抑制miR-146a的轉(zhuǎn)錄過程。
[Abstract]:Prostate cancer is a common disease of multiple causes and complex. It is a common tumor that threatens the health of men. In China, the incidence of prostate cancer is increasing year by year with the aging of the population. Prostate cancer has become one of the major diseases that threaten the health of men in China. In recent years, with the discovery of miRNA, the classic molecular biology theory has been developed. In this process, miRNA has brought new dawn to the research of prostate cancer with its important diagnostic and therapeutic value in this process. This group was found by miRNAs chip screening earlier than normal prostate tissue and androgen dependence. In adenocarcinoma tissue, the expression of miR-146a in the castrated resistant prostate cancer tissues was low, and the results were verified by quantitative PCR in the clinical prostatic carcinoma excision specimens. After the analysis of the MSKCC database, we found that the level of miR-146a in metastatic prostate cancer was lower than that of the localized cancer, and the Gleason=8 sub cancer group was also found. The expression level of miR-146a in the tissue is lower than that of Gleason8. The patients with low expression of miR-146a in the cancer tissues have a higher risk of biochemical recurrence after radical operation. We verified that miR-146a can inhibit the biological function of the proliferation, invasion and migration of the prostate cancer and the ability to induce the apoptosis of the cancer cells by the cell biology experiment. As one of the important metabolic characteristics of prostate cancer cells, glycolytic metabolism is one of the important metabolic characteristics of prostate cancer cells, and the molecular regulatory mechanism in the process is not completely clear. And the effect of miR-146a on the glucose metabolism of prostate cancer has not been studied and determined. In the first part, we pass to the prostate cancer cell (PC3). After transfection of miR-146a mimics, intracellular ATP detection, lactic acid production and glucose concentration in the supernatant, glycolysis velocity detection and so on were used to detect the effect of miR-146a on the glucose metabolism of prostate cancer cells. The results showed that miR-146a could reduce the glucose uptake ability of prostate cancer cells and reduce the cells at the same time. The amount of ATP generated and reduced the amount of lactic acid produced by glycolytic metabolism. MiR-146a inhibited the glycolysis of prostate cancer cells through the detection of the rate of extracellular acidification through the XF96 instrument. The GSE35988 data in the GEO database found that the expression of glucokinase 2 (HK2) in specimens of benign prostatic hyperplasia was significant. Sex is lower than androgen dependent prostate cancer and castrated resistant prostate cancer, and the expression of HK2 in the castrated resistant prostate cancer tissues is higher than that of androgen dependent prostate cancer. After the expression of HK2 protein is downregulated by siRNA in prostate cancer cells, the decrease of ATP production in cells and the amount of lactic acid produced by glycolysis are found. The decrease of glucose uptake, the detection of extracellular acidification speed on the machine and the decrease in glycolysis ability of cells. We confirmed that HK2 was the target gene of miR-146a through the luciferase reporter gene and Western bloting. After transfection of miR-146a into the cell, the expression of HK2 protein was recovered, and the ability of glucose uptake in cancer cells was recovered. At the same time, the amount of lactic acid produced by glycolysis rose, the production of ATP picked up, and the machine detection found that the glycolysis ability of cancer cells was recovered. Thus, we have shown that in prostate cancer cells, miR-146a can inhibit the expression of HK2 by targeting the expression of HK2 protein and inhibit the ability of glycolysis. All experimental results of this group have shown that the tumor suppressor effect of miR-146a has been found. The expression regulation mechanism of miR-146a in prostate cancer is still not very clear, so the second part will focus on the regulation of the expression mechanism of miR-146a. In the second part, we predict that the miR-146a transcription factor YY1. is found before the analysis of the analysis of the ONCOMINE database through the UCSC bioinformation database. The expression level of YY1 in adenocarcinoma tissue is higher than that of normal prostate tissue, and the expression of YY1 in metastatic prostate cancer is also higher than in situ prostate cancer. Analysis of Nakagawa uploaded data found that the expression level of YY1 in the cancer tissues of patients with biochemical recurrence after radical prostatectomy was significantly higher than that of non biochemical recurrence group, and the expression level of YY1 was higher than that of the non biochemical recurrence group. With the increase of Gleason score, we selected 45 cases of clinical prostate cancer tissue, using immunohistochemical technique to detect the expression of YY1 protein in paraffin section, and detect the expression of miR-146a in paraffin section by in situ hybridization. After quantifying the statistical results, we found that the expression of miR-146a and YY1 was negatively correlated (P = 0.007). After the expression of PC3 cell YY1, the total RNA of the cell was extracted and the RNA data of the whole transcriptional group were sequenced, and the RNA level of the miR-146a target gene was negatively enriched (NES=-1.039, FDR=0.267, P=0.15) by the gene probe enrichment analysis (GSEA), while the EZH2 inhibition gene set was positively enriched (NES=1.34, decreasing). The expression level of miR-146a increased significantly after the downregulation of YY1 (P0.05). We constructed the upstream sequence of miR-146a transcriptional gene plasmids (-1226 to 36bp). The transcription inhibition regulation of YY1 on miR-146a was verified by the fluorescein reporter gene experiment and chromatin immunoprecipitation experiment. YY1 and EZH were detected by the fluorescence quantitative PCR detection. 2 at the same time, the expression of miR-146a was further increased. Finally, the transcriptional inhibition process of YY1 combined with EZH2 was verified by the extraction of nuclear protein and protein co precipitation experiment. The results of the second part showed that YY1 could recruit EZH2 to inhibit the transcription of miR-146a in prostate cancer cells.
【學位授予單位】：東南大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R737.25

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