發(fā)布時間：2018-04-25 07:01

  本文選題：miR-139-5p + 前列腺癌��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：第一部分臨床相關(guān)性研究:miR-139-5p在前列腺癌與良性前列腺增生和健康人群中表達(dá)的差異性目的:微小核糖核酸(microRNAs,miRNAs)是一類長度約為22個核苷酸的單鏈非編碼RNA分子,參與基因轉(zhuǎn)錄后調(diào)控,在各種物種中廣泛分布并起重要的生物學(xué)作用。miRNAs的異常表達(dá)參與腫瘤的增殖、凋亡、轉(zhuǎn)移和侵襲,并與抗腫瘤藥物抵抗密切相關(guān)。研究表明,miRNAs可能參與前列腺癌的發(fā)生、發(fā)展,因此探討miRNAs在前列腺癌中的表達(dá)情況及相關(guān)機制對前列腺癌的防治具有重要意義。miR-139-5p基因位于11q13.4,是一種最常見的腫瘤相關(guān)miRNAs,在多種惡性腫瘤的發(fā)生發(fā)展中起重要作用,然而miR-139-5p在前列腺癌中的作用及相關(guān)機制尚未闡明。本部分研究旨在明確miR-139-5p在前列腺癌、前列腺增生及健康男性外周血中的表達(dá)情況;探討人體外周血miR-139-5p的表達(dá)與前列腺癌的關(guān)系;進(jìn)一步結(jié)合前列腺癌患者血清前列腺特異性抗原(Prostate specific antigen,PSA)、臨床分期及Gleason評分,分析miR-139-5p與前列腺癌患者疾病侵襲及進(jìn)展的關(guān)系;評價miR-139-5p對于前列腺癌的診斷價值。方法:1.收集北京醫(yī)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例門診體檢的健康男性的臨床資料,包括患者血清PSA、年齡、體質(zhì)指數(shù)(body mass index,BMI)、影像學(xué)資料及病理資料,接受治療情況等。所有入組前列腺癌患者均未接受雄激素剝奪治療或放化療。提取入組人群外周血標(biāo)本中RNA并進(jìn)行逆轉(zhuǎn)錄PCR,用real time PCR檢測miR-139-5p在不同分組中的表達(dá)情況;2.進(jìn)一步結(jié)合前列腺癌患者臨床分期及Gleason評分,用real time PCR檢測不同臨床分期及Gleason評分前列腺癌患者中miR-139-5p的表達(dá)水平,分析miR-139-5p與前列腺癌患者疾病侵襲及進(jìn)展的關(guān)系;3.應(yīng)用受試者工作曲線(receiver operating characteristic,ROC)評價 miR-139-5p作為分子標(biāo)志物診斷前列腺癌的效力。結(jié)果:1.三組患者年齡及BMI無統(tǒng)計學(xué)差異(P0.05)。前列腺癌組患者血清PSA水平明顯高于前列腺增生組和健康人群組(P0.001)。2.用real time PCR檢測發(fā)現(xiàn),與前列腺增生組及健康人群組相比,前列腺癌組患者外周血miR-139-5p表達(dá)水平明顯升高(P0.001)。前列腺癌組、前列腺增生組及健康人群組患者外周血miR-139-5p表達(dá)水平分別為5.3±3.8、1.4±0.8及1.0±0.8。3.分別對前列腺癌患者的PSA、TNM分期及Gleason評分進(jìn)行分層,發(fā)現(xiàn)隨著PSA水平、TNM分期及Gleason評分的升高,miR-139-5p表達(dá)水平也隨之升高,差異有統(tǒng)計學(xué)意義(P0.05)。4.以病理結(jié)果為參考進(jìn)行ROC曲線分析,外周血miR-139-5p能夠很好的區(qū)分前列腺癌患者和健康人群及前列腺增生患者,曲線下面積(Area Under the Curve,AUC)分別為0.915,0.936,差異有統(tǒng)計學(xué)意義(P0.001)。結(jié)論:1.外周血miR-139-5p在前列腺癌患者中表達(dá)水平明顯升高,提示外周血miR-139-5p與前列腺癌具有相關(guān)性。2.外周血miR-139-5p表達(dá)水平隨著血清PSA、臨床分期及Gleason評分的升高而升高,提示外周血miR-139-5p與前列腺癌的惡性程度相關(guān)。3.外周血miR-139-5p能夠較好的區(qū)分前列腺癌患者與健康人群及前列腺增生患者,有望成為診斷前列腺癌的新型分子標(biāo)志物。第二部分分子機制研究:miR-139-5p通過作用于PTEN基因抑制前列腺癌細(xì)胞凋亡目的:研究表明miRNAs在前列腺癌的發(fā)生發(fā)展過程中起重要的調(diào)控作用,多種miRNAs的異常表達(dá)可能導(dǎo)致前列腺癌進(jìn)展為去勢抵抗性前列腺癌,這是導(dǎo)致患者死亡的重要原因。前期研究已經(jīng)證明miR-139-5p與前列腺癌密切相關(guān),但是miR-139-5p在前列腺癌中的具體作用機制尚無相關(guān)功能實驗驗證報道。本部分研究通過體外細(xì)胞功能實驗,探討miR-139-5p在前列腺癌發(fā)生發(fā)展中的作用,分析前列腺癌細(xì)胞中miR-139-5p表達(dá)的改變及其對凋亡信號通路的影響;明確miR-139-5p的下游靶基因并揭示miR-139-5p參與前列腺癌細(xì)胞凋亡的分子機制,為理解前列腺癌發(fā)生發(fā)展的內(nèi)在機理以及前列腺癌的診斷與治療提供新的思路。方法:1.合成 miR-139-5p 模擬類似物(miR-139-5p mimic)和 miR-139-5p 抑制物(miR-139-5pinhibitor),轉(zhuǎn)染PC3細(xì)胞,建立miR-139-5p高/低表達(dá)的細(xì)胞模型;2.實時定量PCR測定miR-139-5p高/低表達(dá)的細(xì)胞模型中miR-139-5p的表達(dá)水平,CCK-8測定細(xì)胞活性,western blot檢測凋亡相關(guān)蛋白Bc12及Bax表達(dá)情況;3.用生物信息學(xué)方法預(yù)測miR-139-5p的下游靶基因,進(jìn)一步以雙熒光素酶報告分析、western blot和免疫熒光確定miR-139-5p的下游靶基因PTEN;4.以miR-139-5p inhibitor和si-PTEN過表達(dá)質(zhì)粒共轉(zhuǎn)染PC3細(xì)胞驗證miR-139-5p通過調(diào)控PTEN進(jìn)而調(diào)控Bc12和Bax表達(dá)變化,最終促進(jìn)前列腺癌的發(fā)生發(fā)展。結(jié)果:1.成功建立miR-139-5p高/低表達(dá)的細(xì)胞模型;2.用miR-139-5pinhibitor轉(zhuǎn)染PC3細(xì)胞,可降低前列腺癌細(xì)胞活性,使抑凋亡因子Bc12的表達(dá)降低,促凋亡因子Bax的表達(dá)升高;3.雙熒光素酶報告分析證實miR-139-5p直接與PTEN 3'-UTR結(jié)合,western blot和免疫熒光顯示miR-139-5p下調(diào)PTEN蛋白表達(dá);4.在PC3細(xì)胞中敲低PTEN的表達(dá),可逆轉(zhuǎn)miR-139-5p inhibitor對前列腺癌細(xì)胞的促凋亡作用。結(jié)論:1.miR-139-5p能夠促進(jìn)前列腺癌PC3的增殖,抑制細(xì)胞凋亡,發(fā)揮促癌基因的作用;2.沉默miR-139-5p的表達(dá)可以促進(jìn)前列腺癌細(xì)胞的凋亡;3.PTEN是miR-139-5p的靶基因,miR-139-5p通過調(diào)控靶基因PTEN進(jìn)而影響前列腺癌細(xì)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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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