miR-146a对前列腺癌糖代谢功能影响的研究及其表达调控机制

发布时间：2018-08-01 13:00

【摘要】：前列腺癌是一个多病因且复杂的常见病,是威胁男性健康的常见肿瘤。在我国,随着人口老龄化的加剧,前列腺癌的发病率逐年递增。前列腺癌已成为威胁中国男性健康的主要疾病之一。近年来,随着miRNA的发现,经典的分子生物学理论发生了重大变革,也使人们对恶性肿瘤的发生发展机制有了新的认识。在这个过程中,miRNA以其重要的诊断和治疗价值给前列腺癌的研究带来了新的曙光。本课题组前期通过miRNAs芯片筛选发现,相对于正常前列腺组织和雄激素依赖性前列腺癌组织而言,去势抵抗性前列腺癌组织中miR-146a的表达水平偏低,并且通过定量PCR手段在临床前列腺癌切除标本上验证了这一结果。后通过分析MSKCC数据库,我们发现转移性前列腺癌中miR-146a表达水平低于局限性癌,同时Gleason=8分癌组织中miR-146a表达水平低于Gleason8分的癌组织。并且癌组织低表达miR-146a的患者在根治手术后生化复发风险更高。我们通过细胞生物学实验验证了 miR-146a能够抑制前列腺癌的增殖、侵袭及迁移等生物学功能,以及诱导癌细胞凋亡的能力,并且抑制癌细胞的体外成瘤能力。糖酵解代谢作为前列腺癌细胞的重要代谢特征之一,其过程中的分子调控机制暂时不完全明确。且miR-146a分子对前列腺癌糖代谢功能的影响尚未有研究和定论。在第一部分中,我们通过向前列腺癌细胞(PC3和DU145)内转染miR-146a mimics后进行细胞内ATP检测、上清液乳酸生成量和葡萄糖浓度检测、糖酵解速度检测等手段检测了 miR-146a对前列腺癌细胞糖代谢能力的影响。结果发现miR-146a能够降低前列腺癌细胞的葡萄糖摄取能力,同时降低细胞的ATP生成量,并且降低糖酵解代谢生成的乳酸量。通过XF96仪器检测细胞外酸化速度发现miR-146a抑制了前列腺癌细胞的糖酵解能力。整理和分析GEO数据库中GSE35988数据发现,已糖激酶2 (HK2)在前列腺良性增生切除的标本中的表达水平显著性低于雄激素依赖性前列腺癌和去势抵抗性前列腺癌,同时去势抵抗性前列腺癌组织中HK2的表达水平高于雄激素依赖性前列腺癌组织。在前列腺癌细胞中用siRNA下调HK2蛋白质的表达后,发现细胞的ATP生成量减少,糖酵解产生的乳酸量下降,葡萄糖摄取能力下降,上机检测细胞外酸化速度发现,细胞的糖酵解能力下降。我们通过荧光素酶报告基因及Western bloting证实HK2是miR-146a的靶基因,通过向细胞中转染miR-146a后恢复性表达HK2蛋白,发现癌细胞的葡萄糖摄取能力回升,同时糖酵解生成的乳酸量回升,ATP产量回升,机器检测发现癌细胞的糖酵解能力得到了恢复。由此我们证明在前列腺癌细胞中,miR-146a通过靶向下调HK2蛋白的表达,抑制细胞糖酵解能力。联合本课题组的所有实验结果,miR-146a的抑癌作用已被研究和证明。但miR-146a在前列腺癌中的表达调控机制仍不十分明确,因此第二部分将研究重心放在了 miR-146a的表达机制的调控上。第二部分中,我们通过UCSC生物信息数据库,预测了 miR-146a转录因子YY1。通过分析ONCOMINE数据库分析发现前列腺癌组织的YY1表达水平高于正常前列腺组织,转移性前列腺癌组织中YY1表达水平亦高于原位前列腺癌。分析Nakagawa上传的数据发现且前列腺癌根治术后生化复发的患者癌组织中YY1的表达水平显著高于非生化复发组,且YY1的表达水平随着Gleason评分升高。我们选取45例临床前列腺癌组织,利用免疫组化技术检测石蜡切片中YY1蛋白的表达,同时利用原位杂交技术检测石蜡切片中miR-146a分子的表达,量化统计结果后分析发现miR-146a与YY1的表达呈负相关(P = 0.007)。通过敲低PC3细胞YY1表达后,提取细胞总RNA,测序获得全转录组RNA数据,并通过基因探针富集分析(GSEA)发现miR-146a靶基因RNA水平呈现了负向富集(NES=-1.039, FDR=0.267,P=0.15),而EZH2抑制的基因集发生正向富集(NES=1.34, FDR=0.02,P=0.02)。同时通过荧光定量PCR检测发现下调YY1表达后,miR-146a的表达水平显著升高(P0.05)。我们构建miR-146a初始转录本上游序列报告基因质粒(-1226～36bp),通过荧光素报告基因实验和染色质免疫共沉淀实验验证了 YY1对miR-146a的转录抑制调控。荧光定量PCR检测发现YY1和EZH2同时下调后miR-146a的表达进一步升高。最后通过提取细胞核蛋白,实施蛋白质共沉淀实验验证了 YY1联合EZH2参与miR-146a的转录抑制过程。综上第二部分结果,我们证明了在前列腺癌细胞中,YY1能够招募EZH2抑制miR-146a的转录过程。
[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.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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