本文關(guān)鍵詞： 前列腺癌 miR-129 Real-time PCR ROC曲線分析 前列腺癌 miIl-129 轉(zhuǎn)染 細胞生物學行為 miR-129 靶基因 MAPK1 STAT3 BCL-XL　出處：《武漢大學》2015年博士論文　論文類型：學位論文

【摘要】：前列腺癌是目前臨床發(fā)病率上升最快的泌尿生殖系統(tǒng)惡性腫瘤。其病因和發(fā)病機制復雜,病變的發(fā)生與進展涉及大量基因的表達與調(diào)控異常。目前缺乏理想的前列腺癌早期診斷及臨床監(jiān)測指標,現(xiàn)行的治療方案難以進一步提升前列腺癌治愈率和改善其預后,臨床亟待改進和探索。微小RNA(microRNA, miRNA)是一類長度為20-25nt的非編碼的小分子RNA,廣泛地參與腫瘤細胞的代謝與生長,調(diào)節(jié)其增殖、凋亡、分化及侵襲等生物學行為。miRNAs主要通過在轉(zhuǎn)錄后水平調(diào)控其靶基因的表達來發(fā)揮癌基因或抑癌基因的功能�；趍iRNAs的腫瘤學研究為臨床探尋前列腺癌的診治方案提供了理想的靶點和新的思路。新近的研究顯示,miR-129在諸多腫瘤中起著抑癌基因的作用,其表達水平與腫瘤的惡性表型密切相關(guān),預示其可用作腫瘤干預治療的靶點以及腫瘤臨床診斷和預后評估的分子指標。為明確miR-129與前列腺癌病變演進的關(guān)系及其在前列腺癌中可能的功能作用與調(diào)控機制,我們實驗檢測了miR-129在前列腺癌組織及其癌旁正常組織中的表達,并分析了miR-129表達與前列腺癌臨床病理特征的相關(guān)性。我們進一步檢測了過表達miR-129對前列腺癌細胞株P(guān)C-3細胞增殖、凋亡、侵襲等生物學行為的影響。最后我們對miR-129的可能靶基因及其下游相關(guān)信號通路的調(diào)控基因進行了檢測,初步探討了miR-129在前列腺中可能的分子作用機制。我們的實驗為miR-129用于前列腺癌臨床診治的深入研究奠定了理論基礎。三部分實驗內(nèi)容摘要如下：目的：檢測miR-129在前列腺癌組織中的表達情況,明確其表達與前列腺癌臨床病理特征的關(guān)系,探討其可能的臨床意義。方法：采用熒光實時定量PCR (real-time PCR)技術(shù)檢測37例前列腺癌及其癌旁組織中的miR-129表達水平。設定臨床病理參數(shù)分組(年齡、BMI指數(shù)、血清PSA、Gleason評分、臨床分期),統(tǒng)計分析miR-129在各參數(shù)不同分組間的表達差異。ROC曲線分析檢測其診斷價值。結(jié)果：實時定量PCR分析顯示,37例前列腺癌組織中內(nèi)源性miR-129的平均相對表達量為1.0881±0.5785,低于miR-129在相同來源的癌旁對照組織中的平均表達量1.4986±0.7828,兩組間均數(shù)比較差異有統(tǒng)計學意義(P0.05)。數(shù)據(jù)經(jīng)轉(zhuǎn)換后統(tǒng)計分析顯示,各臨床病理參數(shù)分組中miR-129的相對表達量分別為：年齡60歲(-0.6908±1.5132),年齡≥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評分≤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在各參數(shù)不同分組間的表達差異均沒有統(tǒng)計學意義(P0.05)。ROC曲線分析顯示,miR-129對應的AUC(ROC曲線下面積)為0.6614±0.0649,低于PSA的ROC曲線下面積0.8240±0.0480。結(jié)論：1.miR-129在前列腺癌組織中的表達明顯低于癌旁對照組織,提示miR-129的表達下調(diào)與前列腺癌病變相關(guān),可能是導致前列腺癌發(fā)生的原因之一。2.miR-129的表達與前列腺癌的臨床病理特征無明顯相關(guān)性,提示miR-129可能不是理想的前列腺癌臨床監(jiān)測指標。3.miR-129用于前列腺癌的診斷效能不高,可能不是理想的前列腺癌診斷指標。24(-0.3380±1.2652),BMI≥24(-0.6007±1.4556)；PSA≤20μgL (-0.3069 ±1.2026),PSA20μg/L(-0.4906±1.4006)；Gleason評分≤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在各參數(shù)不同分組間的表達差異均沒有統(tǒng)計學意義(P0.05)。ROC曲線分析顯示,miR-129對應的AUC(ROC曲線下面積)為0.6614±0.0649,低于PSA的ROC曲線下面積0.8240±0.0480。目的：檢測過表達miR-129后前列腺癌PC-3細胞增殖、細胞周期、凋亡、細胞遷移與侵襲等生物學行為的變化,明確過表達miR-129對前列腺癌細胞的功能調(diào)控作用及效應。方法：體外培養(yǎng)前列腺癌PC-3細胞,采用DharmaFECT2轉(zhuǎn)染試劑將has-miR-129 mimic轉(zhuǎn)染入PC-3細胞,Real-time PCR檢測轉(zhuǎn)染后miR-129的表達水平以確定轉(zhuǎn)染效能：CCK-8法檢測轉(zhuǎn)染后PC-3細胞增殖活性的改變；流式細胞術(shù)(FCM)檢測細胞周期的變化;AnnexinV/7-AAD雙染法檢測細胞凋亡的改變；Transwell遷移及侵襲實驗分別檢測轉(zhuǎn)染后PC-3細胞遷移及侵襲能力的變化。結(jié)果：轉(zhuǎn)染has-miR-129 mimic后的PC-3細胞中l(wèi)miR-129的相對表達量(435±52)約為對照組(1.0±0.05)的435倍,兩組間均數(shù)比較差異有統(tǒng)計學意義(P0.01)。CCK-8法檢測顯示,轉(zhuǎn)染24 h內(nèi),轉(zhuǎn)染組細胞增殖能力(0.5565±0.0295)與對照組(0.5279±0.0284)比較無明顯差異(P0.05),48h后,轉(zhuǎn)染組細胞的增殖能力(0.7147±0.0721)明顯下降,與對照組(0.8784±0.0573)比較差異有統(tǒng)計學意義(P0.05)。FCM檢測顯示,轉(zhuǎn)染組PC-3細胞G1期細胞比例(67.25±4.50)與對照組(55.54±2.10)比較顯著增加(P0.05),其S期細胞比例(28.05±2.90)較對照組(39.61±4.20)則明顯減少(P0.05)。流式細胞儀分析顯示轉(zhuǎn)染組PC-3細胞的早期凋亡率為(13.52±2.50)%,與對照組(5.81±2.04)%比較明顯增加,差異有統(tǒng)計學意義(P0.05)。transwcll遷移實驗顯示轉(zhuǎn)染組平均穿膜細胞數(shù)為(93.17±21.79),較對照組(205.38±35.25)顯著減少,差異具有統(tǒng)計學意義(P0.01)。侵襲實驗顯示轉(zhuǎn)染組侵襲細胞數(shù)為(35.16±12.09),顯著少于對照組(102.65±22.10),組間比較差異有統(tǒng)計學意義(P0.01)。結(jié)論：1.成熟的miR-129模擬物片段可有效地轉(zhuǎn)染入PC-3細胞并獲得理想的miR-129過表達狀態(tài)。2.過表達miR-129可負向調(diào)控PC-3細胞的生長：可抑制PC-3細胞增殖；阻滯PC-3細胞于G1期；可促進PC-3細胞凋亡并抑制其遷移及侵襲能力。3.miR-129在前列腺癌中可能發(fā)揮著抑癌基因的作用,恢復其表達可發(fā)揮抑癌效應,提示miR-129有可能用作前列腺癌干預治療的靶點。目的：檢測PC-3細胞中miR-129的靶基因并探討其可能的分子作用機制。方法：運用生物信息學分析軟件(Targetscan、MIRanda和PicTar)預測,并通過雙熒光報告基因?qū)嶒烌炞CmiR-129的靶基因。Real-time PCR及Western blot法分別檢測過表達nfiR-129后PC-3細胞中靶基因的mRNA及蛋白表達變化,Western blot檢測靶基因下游調(diào)控基因的蛋白表達變化。結(jié)果：生物信息學軟件預測MAPK1為miR-129潛在的靶基因。雙熒光報告基因試驗顯示,過表達miR-129可明顯抑制野生型pMIR_ MAPK1WT的熒光素酶活性(0.495±0.070),與Scramble對照組(1.0±0.128)相比差異具有顯著的統(tǒng)計學意義(P0.01),但對突變型pMIR_MAPK1_MUT1、pMIR_MAPK1_MUT2和pMIR_MAPK1_MUT12基本沒有作用(P0.05)。過表達miR-129后PC-3細胞中MAPK1的mRNA目對表達量為0.72±0.041,與陰性對照組(1.0±0.056)比較差異有統(tǒng)計學意義(P0.01)；其蛋白的相對表達量為0.37±0.09,對照組為1.0±0.19,組間比較差異有統(tǒng)計學意義(P0.01)；過表達miR-129后MAPK1下游調(diào)控基因STAT3的蛋白表達變化不明顯,但其磷酸化蛋白p-STAT3的表達水平(0.33±0.07)與對照組(1.0±0.109)比較明顯下降(P0.01)：下游調(diào)控基因BCL-XL的蛋白相對表達量(0.26±0.11)顯著低于對照組(1.0±0.12)(P0.01)。結(jié)論：1.MAPK1為miR-129潛在的靶基因。2.miR-129可能通過降解mRNA和抑制翻譯兩種方式下調(diào)了MAPK1的表達。3p-STAT3及BCL-XL的表達下調(diào)與MAPK1同步,提示miR-129可能通過作用于MAPK1進而影響了STAT3的轉(zhuǎn)錄活性,前列腺癌中可能存在MAPK1-STAT3-BCL-XL信號調(diào)控途徑。
[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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