miR-139-5p在前列腺癌中的作用及分子机制的研究

发布时间：2018-04-25 07:01

本文选题：miR-139-5p + 前列腺癌　；参考：《北京协和医学院》2017年博士论文

【摘要】：第一部分临床相关性研究:miR-139-5p在前列腺癌与良性前列腺增生和健康人群中表达的差异性目的:微小核糖核酸(microRNAs,miRNAs)是一类长度约为22个核苷酸的单链非编码RNA分子,参与基因转录后调控,在各种物种中广泛分布并起重要的生物学作用。miRNAs的异常表达参与肿瘤的增殖、凋亡、转移和侵袭,并与抗肿瘤药物抵抗密切相关。研究表明,miRNAs可能参与前列腺癌的发生、发展,因此探讨miRNAs在前列腺癌中的表达情况及相关机制对前列腺癌的防治具有重要意义。miR-139-5p基因位于11q13.4,是一种最常见的肿瘤相关miRNAs,在多种恶性肿瘤的发生发展中起重要作用,然而miR-139-5p在前列腺癌中的作用及相关机制尚未阐明。本部分研究旨在明确miR-139-5p在前列腺癌、前列腺增生及健康男性外周血中的表达情况;探讨人体外周血miR-139-5p的表达与前列腺癌的关系;进一步结合前列腺癌患者血清前列腺特异性抗原(Prostate specific antigen,PSA)、临床分期及Gleason评分,分析miR-139-5p与前列腺癌患者疾病侵袭及进展的关系;评价miR-139-5p对于前列腺癌的诊断价值。方法:1.收集北京医院2014年3月-2016年7月收治的45例不同分期的前列腺癌患者、45例前列腺增生患者及50例门诊体检的健康男性的临床资料,包括患者血清PSA、年龄、体质指数(body mass index,BMI)、影像学资料及病理资料,接受治疗情况等。所有入组前列腺癌患者均未接受雄激素剥夺治疗或放化疗。提取入组人群外周血标本中RNA并进行逆转录PCR,用real time PCR检测miR-139-5p在不同分组中的表达情况;2.进一步结合前列腺癌患者临床分期及Gleason评分,用real time PCR检测不同临床分期及Gleason评分前列腺癌患者中miR-139-5p的表达水平,分析miR-139-5p与前列腺癌患者疾病侵袭及进展的关系;3.应用受试者工作曲线(receiver operating characteristic,ROC)评价 miR-139-5p作为分子标志物诊断前列腺癌的效力。结果:1.三组患者年龄及BMI无统计学差异(P0.05)。前列腺癌组患者血清PSA水平明显高于前列腺增生组和健康人群组(P0.001)。2.用real time PCR检测发现,与前列腺增生组及健康人群组相比,前列腺癌组患者外周血miR-139-5p表达水平明显升高(P0.001)。前列腺癌组、前列腺增生组及健康人群组患者外周血miR-139-5p表达水平分别为5.3±3.8、1.4±0.8及1.0±0.8。3.分别对前列腺癌患者的PSA、TNM分期及Gleason评分进行分层,发现随着PSA水平、TNM分期及Gleason评分的升高,miR-139-5p表达水平也随之升高,差异有统计学意义(P0.05)。4.以病理结果为参考进行ROC曲线分析,外周血miR-139-5p能够很好的区分前列腺癌患者和健康人群及前列腺增生患者,曲线下面积(Area Under the Curve,AUC)分别为0.915,0.936,差异有统计学意义(P0.001)。结论:1.外周血miR-139-5p在前列腺癌患者中表达水平明显升高,提示外周血miR-139-5p与前列腺癌具有相关性。2.外周血miR-139-5p表达水平随着血清PSA、临床分期及Gleason评分的升高而升高,提示外周血miR-139-5p与前列腺癌的恶性程度相关。3.外周血miR-139-5p能够较好的区分前列腺癌患者与健康人群及前列腺增生患者,有望成为诊断前列腺癌的新型分子标志物。第二部分分子机制研究:miR-139-5p通过作用于PTEN基因抑制前列腺癌细胞凋亡目的:研究表明miRNAs在前列腺癌的发生发展过程中起重要的调控作用,多种miRNAs的异常表达可能导致前列腺癌进展为去势抵抗性前列腺癌,这是导致患者死亡的重要原因。前期研究已经证明miR-139-5p与前列腺癌密切相关,但是miR-139-5p在前列腺癌中的具体作用机制尚无相关功能实验验证报道。本部分研究通过体外细胞功能实验,探讨miR-139-5p在前列腺癌发生发展中的作用,分析前列腺癌细胞中miR-139-5p表达的改变及其对凋亡信号通路的影响;明确miR-139-5p的下游靶基因并揭示miR-139-5p参与前列腺癌细胞凋亡的分子机制,为理解前列腺癌发生发展的内在机理以及前列腺癌的诊断与治疗提供新的思路。方法:1.合成 miR-139-5p 模拟类似物(miR-139-5p mimic)和 miR-139-5p 抑制物(miR-139-5pinhibitor),转染PC3细胞,建立miR-139-5p高/低表达的细胞模型;2.实时定量PCR测定miR-139-5p高/低表达的细胞模型中miR-139-5p的表达水平,CCK-8测定细胞活性,western blot检测凋亡相关蛋白Bc12及Bax表达情况;3.用生物信息学方法预测miR-139-5p的下游靶基因,进一步以双荧光素酶报告分析、western blot和免疫荧光确定miR-139-5p的下游靶基因PTEN;4.以miR-139-5p inhibitor和si-PTEN过表达质粒共转染PC3细胞验证miR-139-5p通过调控PTEN进而调控Bc12和Bax表达变化,最终促进前列腺癌的发生发展。结果:1.成功建立miR-139-5p高/低表达的细胞模型;2.用miR-139-5pinhibitor转染PC3细胞,可降低前列腺癌细胞活性,使抑凋亡因子Bc12的表达降低,促凋亡因子Bax的表达升高;3.双荧光素酶报告分析证实miR-139-5p直接与PTEN 3'-UTR结合,western blot和免疫荧光显示miR-139-5p下调PTEN蛋白表达;4.在PC3细胞中敲低PTEN的表达,可逆转miR-139-5p inhibitor对前列腺癌细胞的促凋亡作用。结论:1.miR-139-5p能够促进前列腺癌PC3的增殖,抑制细胞凋亡,发挥促癌基因的作用;2.沉默miR-139-5p的表达可以促进前列腺癌细胞的凋亡;3.PTEN是miR-139-5p的靶基因,miR-139-5p通过调控靶基因PTEN进而影响前列腺癌细胞的凋亡。
[Abstract]:The first part of the clinical study: the differential expression of miR-139-5p in prostate cancer and benign prostatic hyperplasia and healthy people: microRNAs (miRNAs) is a single strand non coded RNA molecule with a length of about 22 nucleotides, involved in post transcriptional regulation, widely distributed and important in a variety of species. The abnormal expression of biological action of.MiRNAs participates in tumor proliferation, apoptosis, metastasis and invasion, which is closely related to anti tumor drug resistance. The study shows that miRNAs may be involved in the occurrence and development of prostate cancer. Therefore, it is of great significance to explore the expression of miRNAs in prostate cancer and the related mechanisms for the prevention and treatment of prostate cancer.MiR-139- The 5P gene, located in 11q13.4, is one of the most common tumor related miRNAs, which plays an important role in the development of a variety of malignant tumors. However, the role and mechanisms of miR-139-5p in prostate cancer have not been elucidated. The purpose of this study is to clarify the expression of miR-139-5p in prostate cancer, prostatic hyperplasia and healthy male peripheral blood. To explore the relationship between the expression of miR-139-5p in human peripheral blood and prostate cancer; to further combine the serum prostatic specific antigen (Prostate specific antigen, PSA), clinical stage and Gleason score in the patients with prostate cancer, to analyze the relationship between the disease invasion and progression of prostate cancer patients, and to evaluate the diagnosis of prostate cancer by miR-139-5p. Methods: 1. the clinical data of 45 patients with different stages of prostate cancer, 45 cases of benign prostatic hyperplasia and 50 healthy men were collected in Beijing Hospital in July -2016 March 2014, including the patients' serum PSA, age, body mass index (body mass index, BMI), imaging data and pathological data, receiving treatment All the patients with prostate cancer were not treated with androgen deprivation therapy or radiotherapy and chemotherapy. RNA and reverse transcriptase PCR were performed in the peripheral blood samples of the group. Real time PCR was used to detect the expression of miR-139-5p in different groups. 2. the clinical stages of the prostate cancer patients and the Gleason score were further combined with the real time PCR to detect the difference. The expression level of miR-139-5p in patients with prostate cancer by clinical staging and Gleason score, analysis of the relationship between miR-139-5p and the disease invasion and progression of prostate cancer patients; 3. the efficacy of miR-139-5p as a molecular marker in the diagnosis of prostate cancer was evaluated by the receiver operating characteristic (ROC). Results: 1. and three groups of patients. There was no statistical difference between age and BMI (P0.05). The serum PSA level of the prostate cancer group was significantly higher than that of the prostatic hyperplasia group and the healthy group (P0.001).2. using real time PCR detection. Compared with the prostatic hyperplasia group and the healthy group, the peripheral blood miR-139-5p expression level of the prostate cancer group was significantly increased (P0.001). The prostate cancer group, The levels of miR-139-5p expression in the peripheral blood of the prostatic hyperplasia group and the healthy group were 5.3 + 3.8,1.4 + 0.8 and 1 + 0.8.3. respectively. The levels of PSA, TNM staging and Gleason score were stratified respectively. The level of TNM staging and Gleason score increased with the PSA level, and the expression level of miR-139-5p increased, the difference was statistically significant. Significance (P0.05).4. was used for ROC curve analysis with pathological results. Peripheral blood miR-139-5p could distinguish between prostate cancer patients and healthy people and patients with benign prostatic hyperplasia. The area under the curve (Area Under the Curve, AUC) was 0.915,0.936, the difference was statistically significant (P0.001). Conclusion: 1. peripheral blood miR-139-5p is in prostate cancer. The level of expression in the peripheral blood miR-139-5p was significantly higher in patients with prostate cancer. The level of miR-139-5p expression in peripheral blood.2. in peripheral blood increased with the increase of serum PSA, clinical stage and Gleason score, suggesting that miR-139-5p in peripheral blood associated with the malignancy of prostate cancer,.3. peripheral blood miR-139-5p can distinguish the prostate better. Cancer patients and healthy people and patients with benign prostatic hyperplasia are expected to be a new molecular marker for the diagnosis of prostate cancer. The second part of the molecular mechanism study: miR-139-5p inhibits the apoptosis of prostate cancer cells by acting on the PTEN gene. The study shows that miRNAs plays an important role in the development of prostate cancer, and a variety of miRN The abnormal expression of As may lead to the progression of prostate cancer to prostatic cancer, which is an important cause of death. Earlier studies have shown that miR-139-5p is closely related to prostate cancer, but the specific mechanism of miR-139-5p in prostate cancer has not yet been verified by the related work. To explore the role of miR-139-5p in the development of prostate cancer and to analyze the changes in the expression of miR-139-5p in prostate cancer cells and its influence on the pathway of apoptosis signal, and to clarify the molecular mechanism of the miR-139-5p involved in the apoptosis of prostate cancer cells to understand the development of prostate cancer. The internal mechanism and the diagnosis and treatment of prostate cancer provide new ideas. Methods: 1. synthesis of miR-139-5p analog analogues (miR-139-5p mimic) and miR-139-5p inhibitor (miR-139-5pinhibitor), transfect PC3 cells, establish a miR-139-5p high / low expression cell model, and 2. real-time quantitative PCR for the determination of miR-139-5p high / low expression in the cell model of M. IR-139-5p expression level, CCK-8 assay cell activity, Western blot to detect the expression of apoptosis related protein Bc12 and Bax; 3. using bioinformatics method to predict the downstream target gene of miR-139-5p, further using double Luciferase Report Analysis, Western blot and immunofluorescence determination of miR-139-5p downstream target gene PTEN; 4. with miR-139-5p R and si-PTEN overexpressed plasmids co transfected PC3 cells to verify that miR-139-5p regulates Bc12 and Bax expression by regulating PTEN and ultimately promotes the development of prostate cancer. Results: 1. the cell model of miR-139-5p high / low expression was successfully established, and 2. PC3 fine cells transfected with miR-139-5pinhibitor could reduce the activity of prostate cancer cells and inhibit apoptosis. The expression of factor Bc12 decreased and the expression of apoptotic factor Bax increased; 3. double Luciferase Report analysis confirmed that miR-139-5p was directly associated with PTEN 3'-UTR, Western blot and immunofluorescent miR-139-5p down regulated the expression of PTEN protein; 4. the low PTEN expression in PC3 cells could reverse the apoptosis inducing apoptosis of prostate cancer cells. Conclusion: 1.miR-139-5p can promote the proliferation of PC3 in prostate cancer, inhibit apoptosis and play the role of proto oncogene. 2. silent miR-139-5p expression can promote the apoptosis of prostate cancer cells; 3.PTEN is the target gene of miR-139-5p, and miR-139-5p affects the apoptosis of prostate cancer cells by regulating the target gene PTEN.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

【相似文献】