MiR-129在前列腺癌中的表达及其功能机制研究

发布时间：2018-02-12 11:06

  本文关键词： 前列腺癌 miR-129 Real-time PCR ROC曲线分析 前列腺癌 miIl-129 转染 细胞生物学行为 miR-129 靶基因 MAPK1 STAT3 BCL-XL　出处：《武汉大学》2015年博士论文　论文类型：学位论文

【摘要】：前列腺癌是目前临床发病率上升最快的泌尿生殖系统恶性肿瘤。其病因和发病机制复杂,病变的发生与进展涉及大量基因的表达与调控异常。目前缺乏理想的前列腺癌早期诊断及临床监测指标,现行的治疗方案难以进一步提升前列腺癌治愈率和改善其预后,临床亟待改进和探索。微小RNA(microRNA, miRNA)是一类长度为20-25nt的非编码的小分子RNA,广泛地参与肿瘤细胞的代谢与生长,调节其增殖、凋亡、分化及侵袭等生物学行为。miRNAs主要通过在转录后水平调控其靶基因的表达来发挥癌基因或抑癌基因的功能。基于miRNAs的肿瘤学研究为临床探寻前列腺癌的诊治方案提供了理想的靶点和新的思路。新近的研究显示,miR-129在诸多肿瘤中起着抑癌基因的作用,其表达水平与肿瘤的恶性表型密切相关,预示其可用作肿瘤干预治疗的靶点以及肿瘤临床诊断和预后评估的分子指标。为明确miR-129与前列腺癌病变演进的关系及其在前列腺癌中可能的功能作用与调控机制,我们实验检测了miR-129在前列腺癌组织及其癌旁正常组织中的表达,并分析了miR-129表达与前列腺癌临床病理特征的相关性。我们进一步检测了过表达miR-129对前列腺癌细胞株PC-3细胞增殖、凋亡、侵袭等生物学行为的影响。最后我们对miR-129的可能靶基因及其下游相关信号通路的调控基因进行了检测,初步探讨了miR-129在前列腺中可能的分子作用机制。我们的实验为miR-129用于前列腺癌临床诊治的深入研究奠定了理论基础。三部分实验内容摘要如下：目的：检测miR-129在前列腺癌组织中的表达情况,明确其表达与前列腺癌临床病理特征的关系,探讨其可能的临床意义。方法：采用荧光实时定量PCR (real-time PCR)技术检测37例前列腺癌及其癌旁组织中的miR-129表达水平。设定临床病理参数分组(年龄、BMI指数、血清PSA、Gleason评分、临床分期),统计分析miR-129在各参数不同分组间的表达差异。ROC曲线分析检测其诊断价值。结果：实时定量PCR分析显示,37例前列腺癌组织中内源性miR-129的平均相对表达量为1.0881±0.5785,低于miR-129在相同来源的癌旁对照组织中的平均表达量1.4986±0.7828,两组间均数比较差异有统计学意义(P0.05)。数据经转换后统计分析显示,各临床病理参数分组中miR-129的相对表达量分别为：年龄60岁(-0.6908±1.5132),年龄≥6(-0.3404±1.2669)；BMI24(-0.3380±1.2652),BMI≥24(-0.6007±1.4556)；PSA≤20μgL (-0.3069 ±1.2026),PSA20μg/L(-0.4906±1.4006)；Gleason评分≤6(-0.5160±1.2934),≥7(-0.3791±1.3380)；病理分期Ⅰ～Ⅱ期(-0.5135±1.3324),Ⅲ-Ⅳ期(-0.2133±1.2934)。miR-129在各参数不同分组间的表达差异均没有统计学意义(P0.05)。ROC曲线分析显示,miR-129对应的AUC(ROC曲线下面积)为0.6614±0.0649,低于PSA的ROC曲线下面积0.8240±0.0480。结论：1.miR-129在前列腺癌组织中的表达明显低于癌旁对照组织,提示miR-129的表达下调与前列腺癌病变相关,可能是导致前列腺癌发生的原因之一。2.miR-129的表达与前列腺癌的临床病理特征无明显相关性,提示miR-129可能不是理想的前列腺癌临床监测指标。3.miR-129用于前列腺癌的诊断效能不高,可能不是理想的前列腺癌诊断指标。24(-0.3380±1.2652),BMI≥24(-0.6007±1.4556)；PSA≤20μgL (-0.3069 ±1.2026),PSA20μg/L(-0.4906±1.4006)；Gleason评分≤6(-0.5160±1.2934),≥7(-0.3791±1.3380)；病理分期Ⅰ～Ⅱ期(-0.5135±1.3324),Ⅲ-Ⅳ期(-0.2133±1.2934)。miR-129在各参数不同分组间的表达差异均没有统计学意义(P0.05)。ROC曲线分析显示,miR-129对应的AUC(ROC曲线下面积)为0.6614±0.0649,低于PSA的ROC曲线下面积0.8240±0.0480。目的：检测过表达miR-129后前列腺癌PC-3细胞增殖、细胞周期、凋亡、细胞迁移与侵袭等生物学行为的变化,明确过表达miR-129对前列腺癌细胞的功能调控作用及效应。方法：体外培养前列腺癌PC-3细胞,采用DharmaFECT2转染试剂将has-miR-129 mimic转染入PC-3细胞,Real-time PCR检测转染后miR-129的表达水平以确定转染效能：CCK-8法检测转染后PC-3细胞增殖活性的改变；流式细胞术(FCM)检测细胞周期的变化;AnnexinV/7-AAD双染法检测细胞凋亡的改变；Transwell迁移及侵袭实验分别检测转染后PC-3细胞迁移及侵袭能力的变化。结果：转染has-miR-129 mimic后的PC-3细胞中lmiR-129的相对表达量(435±52)约为对照组(1.0±0.05)的435倍,两组间均数比较差异有统计学意义(P0.01)。CCK-8法检测显示,转染24 h内,转染组细胞增殖能力(0.5565±0.0295)与对照组(0.5279±0.0284)比较无明显差异(P0.05),48h后,转染组细胞的增殖能力(0.7147±0.0721)明显下降,与对照组(0.8784±0.0573)比较差异有统计学意义(P0.05)。FCM检测显示,转染组PC-3细胞G1期细胞比例(67.25±4.50)与对照组(55.54±2.10)比较显著增加(P0.05),其S期细胞比例(28.05±2.90)较对照组(39.61±4.20)则明显减少(P0.05)。流式细胞仪分析显示转染组PC-3细胞的早期凋亡率为(13.52±2.50)%,与对照组(5.81±2.04)%比较明显增加,差异有统计学意义(P0.05)。transwcll迁移实验显示转染组平均穿膜细胞数为(93.17±21.79),较对照组(205.38±35.25)显著减少,差异具有统计学意义(P0.01)。侵袭实验显示转染组侵袭细胞数为(35.16±12.09),显著少于对照组(102.65±22.10),组间比较差异有统计学意义(P0.01)。结论：1.成熟的miR-129模拟物片段可有效地转染入PC-3细胞并获得理想的miR-129过表达状态。2.过表达miR-129可负向调控PC-3细胞的生长：可抑制PC-3细胞增殖；阻滞PC-3细胞于G1期；可促进PC-3细胞凋亡并抑制其迁移及侵袭能力。3.miR-129在前列腺癌中可能发挥着抑癌基因的作用,恢复其表达可发挥抑癌效应,提示miR-129有可能用作前列腺癌干预治疗的靶点。目的：检测PC-3细胞中miR-129的靶基因并探讨其可能的分子作用机制。方法：运用生物信息学分析软件(Targetscan、MIRanda和PicTar)预测,并通过双荧光报告基因实验验证miR-129的靶基因。Real-time PCR及Western blot法分别检测过表达nfiR-129后PC-3细胞中靶基因的mRNA及蛋白表达变化,Western blot检测靶基因下游调控基因的蛋白表达变化。结果：生物信息学软件预测MAPK1为miR-129潜在的靶基因。双荧光报告基因试验显示,过表达miR-129可明显抑制野生型pMIR_ MAPK1WT的荧光素酶活性(0.495±0.070),与Scramble对照组(1.0±0.128)相比差异具有显著的统计学意义(P0.01),但对突变型pMIR_MAPK1_MUT1、pMIR_MAPK1_MUT2和pMIR_MAPK1_MUT12基本没有作用(P0.05)。过表达miR-129后PC-3细胞中MAPK1的mRNA目对表达量为0.72±0.041,与阴性对照组(1.0±0.056)比较差异有统计学意义(P0.01)；其蛋白的相对表达量为0.37±0.09,对照组为1.0±0.19,组间比较差异有统计学意义(P0.01)；过表达miR-129后MAPK1下游调控基因STAT3的蛋白表达变化不明显,但其磷酸化蛋白p-STAT3的表达水平(0.33±0.07)与对照组(1.0±0.109)比较明显下降(P0.01)：下游调控基因BCL-XL的蛋白相对表达量(0.26±0.11)显著低于对照组(1.0±0.12)(P0.01)。结论：1.MAPK1为miR-129潜在的靶基因。2.miR-129可能通过降解mRNA和抑制翻译两种方式下调了MAPK1的表达。3p-STAT3及BCL-XL的表达下调与MAPK1同步,提示miR-129可能通过作用于MAPK1进而影响了STAT3的转录活性,前列腺癌中可能存在MAPK1-STAT3-BCL-XL信号调控途径。
[Abstract]:Prostate cancer is the malignant tumor of the genitourinary system in clinic at present the fastest rising incidence. Its etiology and pathogenesis is complex, the occurrence and progress of disease involving the expression and regulation of a large number of gene abnormalities. Early diagnosis of prostate cancer and clinical monitoring indicators are not ideal, the treatment is difficult to further improve the cure rate and prostate cancer improve the prognosis, clinical improvement and exploration. The small RNA (microRNA, miRNA) is a class of small molecule RNA encoding length is non 20-25nt, widely participate in metabolism and growth of tumor cells, regulating the proliferation, apoptosis, differentiation and biological behavior of invasion of.MiRNAs mainly through the expression in the post transcriptional regulation of its target a gene to play the oncogenes or tumor suppressor genes. Oncology research based on miRNAs for exploring the clinical treatment of prostate cancer provides the ideal target and new Ideas. Recent studies have shown that miR-129 plays a role of tumor suppressor genes in many tumors, malignant phenotype expression and tumor related molecular index indicates that it can be used as the target of tumor therapy, diagnosis and prognosis of tumors in clinical evaluation. In order to elucidate the relationship between miR-129 and prostate cancer lesions and its evolution may be in prostate cancer function and regulation mechanism, we examined the expression of miR-129 in prostate cancer tissues and adjacent normal tissues, and analyze the correlation between miR-129 expression and clinicopathological features of prostate cancer. We further examined the expression of miR-129 on proliferation and apoptosis of prostate cancer cell line, PC-3 cells, biological effects the behavior of invasion genes of miR-129. Finally we may target genes and their downstream signaling pathways were detected, discussed miR- 129 possible molecular mechanism in the prostate. Lay a theoretical foundation for further study of the clinical diagnosis and treatment of prostate cancer. Our experiments for miR-129 for the three part of the experiment are as follows: Abstract Objective: to detect the expression of miR-129 in prostate cancer, a clear relationship between the expression and clinicopathological features of prostate cancer and explore its clinical significance possible. Methods: using real-time fluorescence quantitative PCR (real-time PCR) expression was detected in 37 cases of prostate cancer and adjacent tissues miR-129. The clinical pathological parameters set group (age, BMI index, serum PSA, Gleason score, clinical stage), statistical analysis of miR-129 in various parameters between the different groups of expression differences the diagnostic value of detection of.ROC curve analysis. Results: real time quantitative PCR analysis showed that the average endogenous miR-129 in 37 cases of prostate carcinoma relative expression quantity was 1.0881 + 0.5785, less than miR-129 in the same source adjacent average the expression amount was 1.4986 + 0.7828, the two groups were statistically significant difference (P0.05). After data conversion analysis of statistics, the relative expression miR-129 clinicopathological parameters in the groups were: age 60 (-0.6908 + 1.5132), aged 6 (-0.3404 + 1.2669); BMI24 (-0.3380 + 1.2652), BMI = 24 (-0.6007 + 1.4556); PSA = 20 gL (-0.3069 + 1.2026), PSA20 g/L (-0.4906 + 1.4006); Gleason score less than 6 (-0.5160 + 1.2934), aged 7 (-0.3791 + 1.3380) pathological stage; stage I-II (-0.5135 + 1.3324), III - IV (-0.2133 + 1.2934).MiR-129 expression difference in parameters between the different groups were not statistically significant (P0.05).ROC curve analysis showed that the miR-129 corresponding to the AUC (area under the ROC curve was 0.6614 + 0.0649), less than the area of ROC curve PSA the 0.8240 + 0 Conclusion: the.0480. expression of 1.miR-129 in prostate cancer tissues was significantly lower than that of the adjacent normal tissues, suggesting that the down-regulation of miR-129 expression is associated with prostate cancer lesions may lead to clinical pathological features of prostate cancer because of the expression of.2.miR-129 and prostate cancer was not related to prostate cancer, clinical monitoring index of.3.miR-129 suggested that miR-129 may not be ideal for prostate cancer diagnostic efficiency is not high, diagnosis index of.24 prostate cancer may not be ideal (-0.3380 + 1.2652), BMI = 24 (-0.6007 + 1.4556); PSA = 20 gL (-0.3069 + 1.2026), PSA20 g/L (-0.4906 + 1.4006); Gleason score less than 6 (-0.5160 + 1.2934), or 7 (-0.3791 + 1.3380); pathological stage I-II (-0.5135 + 1.3324), III - IV (-0.2133 + 1.2934).MiR-129 expression difference in parameters between the different groups were not statistically significant (P0.05.ROC) MiR-129 curve analysis showed that the corresponding AUC (ROC area under the curve) was 0.6614 + 0.0649, less than the area under the PSA curve of ROC 0.8240 + 0.0480.. Objective: to detect the proliferation of prostate cancer cells PC-3 expression after miR-129 cell cycle, apoptosis, changes of cell biological behaviors such as migration and invasion, clear expression regulation function and the effect of miR-129 on prostate cancer cells. Methods: prostate cancer PC-3 cells cultured in vitro by DharmaFECT2 transfection reagent has-miR-129 mimic were transfected into PC-3 cells, Real-time PCR was used to detect the expression of miR-129 after transfection to determine the level of effectiveness: the change of PC-3 cell proliferation was assessed by CCK-8 assay; flow cytometry (FCM) to detect the changes of cell cycle; AnnexinV/7-AAD method to detect cell apoptosis changes; Transwell migration and invasion assay were detected after transfection of PC-3 cells migration and invasion 鑳藉姏鐨勫彉鍖，

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