本文選題：前列腺癌　切入點(diǎn)：長鏈非編碼RNA　出處：《第二軍醫(yī)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景:中國前列腺癌(Prostate cancer,PCa)的發(fā)病率和死亡率急劇上升,其增長率位居腫瘤之首。同時(shí),PCa的防治面臨著兩大臨床難題,即早期診斷指標(biāo)特異性差和晚期治療方法效果不佳。因此,臨床上亟需找到新型的敏感性和特異較高的PCa診斷標(biāo)志物或診斷策略來代替血清PSA或彌補(bǔ)血清PSA在早期診斷上的不足;基礎(chǔ)研究上亟需闡明PCa進(jìn)展的關(guān)鍵分子機(jī)制,從而研發(fā)出抑制去勢(shì)抵抗性前列腺癌(Castration-resistant prostate cancer,CRPC)的新型藥物,實(shí)現(xiàn)CRPC治療上的突破。研究目的:研究尿液中長鏈非編碼RNA MALAT1和PCA3評(píng)分在前列腺初次穿刺患者中的診斷作用,建立基于尿液MALAT1或PCA3評(píng)分的綜合診斷模型;探索新型mi R-n5在PCa進(jìn)展中的作用機(jī)制,尋找CRPC干預(yù)的新靶點(diǎn)。研究方法:全轉(zhuǎn)錄組擴(kuò)增試劑盒擴(kuò)增尿沉渣RNA,q RT-PCR檢測(cè)MALAT1和PCA3的表達(dá),單因素logistic回歸預(yù)測(cè)PCa的危險(xiǎn)因素,多因素logistic回歸建立PCa診斷模型,Spearman rank檢測(cè)分析尿液MALAT1或PCA3評(píng)分與其它臨床變量之間的關(guān)系,運(yùn)用受試者工作曲線(Receiver operating characteristic curve,ROC)確定診斷指標(biāo)的曲線下面積(Area under the ROC,AUC)、最佳截?cái)嘀怠⑻禺惗群兔舾卸?決策曲線分析(Decision curve analysis,DCA)來估算尿液MALAT1和PCA3評(píng)分的臨床價(jià)值。Northern blot檢測(cè)新型mi RNA的存在與表達(dá),細(xì)胞增殖、遷移、侵襲和克隆形成實(shí)驗(yàn)來評(píng)價(jià)mi R-n5在進(jìn)展性PCa細(xì)胞系中的生物學(xué)功能,生物信息學(xué)軟件預(yù)測(cè)mi R-n5的靶基因,雙熒光素酶報(bào)告基因法驗(yàn)證靶基因,Graph Pad Prism5進(jìn)行數(shù)據(jù)展示并計(jì)算藥物的IC50,皮下種植和脛骨內(nèi)注射建立CRPC小鼠移植瘤模型。用SPSS v.17.0(SPSS Inc.,Chicago,IL,USA),Med Calc v.10.4.7.0(Med Calc Software bvba,Mariakerke,Belgium)和R軟件包v.3.1.1(The R Foundation for Statistical Computing)對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析,運(yùn)用Hem I軟件進(jìn)行數(shù)據(jù)的Heatmap分析;以P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:尿液MALAT1和PCA3評(píng)分可以明確區(qū)分腫瘤組和穿刺陰性組患者并與PCa的檢出率高度相關(guān),但進(jìn)一步的相關(guān)性分析發(fā)現(xiàn),尿液MALAT1和PCA3評(píng)分與臨床危險(xiǎn)因素以及Gleason評(píng)分沒有相關(guān)性。年齡、血清總PSA(Total PSA,t PSA)、前列腺體積(Prostate volume,PV)、游離PSA比值(Percentage of free PSA,%f PSA)和直腸指診結(jié)果(Digital rectal examination,DRE)是總體人群的獨(dú)立預(yù)測(cè)因子,而在PSA=4-10ng/ml(PSA“診斷灰區(qū)”)人群中,年齡、PV、%f PSA和DRE是其獨(dú)立預(yù)測(cè)因子。尿液MALAT1評(píng)分在總體人群中的PCa診斷效能與t PSA相比雖然稍高但沒有統(tǒng)計(jì)學(xué)意義(p=0.510),但在PSA“診斷灰區(qū)”人群中則明顯優(yōu)于t PSA(p=0.034)。PCA3評(píng)分在PSA“診斷灰區(qū)”人群中的PCa診斷效能則明顯優(yōu)于t PSA(p=0.0407)和%f PSA(p=0.046)。進(jìn)一步的logistic回歸分析得出,在發(fā)現(xiàn)組的PSA“診斷灰區(qū)”人群中,基于尿液MALAT1評(píng)分綜合模型的預(yù)測(cè)準(zhǔn)確性為79.79%和AUC為0.853,預(yù)測(cè)準(zhǔn)確性比基礎(chǔ)模型提高了5.32%,AUC提高了0.0318。將這一模型應(yīng)用到驗(yàn)證組也得到了相同的結(jié)果。同樣,在PSA“診斷灰區(qū)”人群中,基于尿液PCA3評(píng)分綜合模型的AUC也高達(dá)0.819,高于基礎(chǔ)模型的0.788。應(yīng)用DCA,在PSA“診斷灰區(qū)”人群中,不管是在發(fā)現(xiàn)組還是在驗(yàn)證組,基于尿液MALAT1評(píng)分綜合模型的臨床預(yù)測(cè)能力都優(yōu)于基礎(chǔ)模型。將臨床穿刺風(fēng)險(xiǎn)定為25%,發(fā)現(xiàn)組人群中基于尿液MALAT1評(píng)分綜合模型的總獲益率高于基礎(chǔ)模型(13.97%vs.11.47%),且可以避免更多不必要的前列腺穿刺(47.32%vs.39.78%);驗(yàn)證組人群基于尿液MALAT1評(píng)分綜合模型的總獲益率同樣高于基礎(chǔ)模型(15.73%vs.13.11%),且可以避免更多不必要的前列腺穿刺(30.33%vs.22.47%),更為重要的是,綜合模型沒有漏診1例高級(jí)別PCa而基礎(chǔ)模型則漏診1例高級(jí)別PCa。同樣,在PSA“診斷灰區(qū)”人群中,基于PCA3評(píng)分綜合模型幾乎在所有臨床穿刺閾值上優(yōu)于基礎(chǔ)模型,特別是在25%-40%這一區(qū)間顯示出了更好的預(yù)測(cè)作用。應(yīng)用30%這一臨床穿刺閾值,基于PCA3綜合模型可以避免60.3%不必要的穿刺活檢,但以漏診4例PCa為代價(jià),其中2例為高級(jí)別PCa。此外,應(yīng)用65對(duì)PCa及其癌旁正常組織進(jìn)行RNA-seq發(fā)現(xiàn)300多個(gè)新型mi RNA,并使用Northern blot驗(yàn)證了其中表達(dá)量最高的8個(gè)新型mi RNA。通過在進(jìn)展性PCa與惰性PCa細(xì)胞系中的表達(dá)情況比較,篩選出4個(gè)與PCa進(jìn)展相關(guān)的新型mi RNA。再通過臨床樣本的驗(yàn)證,最終選取mi R-n5作為后續(xù)的研究對(duì)象。體外的功能實(shí)驗(yàn)表明,mi R-n5能夠明顯抑制進(jìn)展性PCa細(xì)胞的增殖、遷移、侵襲和克隆形成能力,說明mi R-n5的缺失或低表達(dá)可能促進(jìn)了PCa的進(jìn)展。進(jìn)一步的體內(nèi)實(shí)驗(yàn)證明了腫瘤內(nèi)注射mi R-n5能夠抑制小鼠移植瘤的生長。此后,生物信息學(xué)分析并找到了mi R-n5的作用靶基因KDM6B,同時(shí),通過雙熒光素酶報(bào)告基因及q RT-PCR進(jìn)行驗(yàn)證。進(jìn)一步的研究表明,KDM6B的特異性抑制劑——小分子化合物GSK-J4能夠抑制KDM6B的活性并明顯抑制PCa細(xì)胞的生長,體內(nèi)實(shí)驗(yàn)也證明了這一小分子化合物能夠明顯抑制小鼠移植瘤的生長。結(jié)論:尿液MALAT1和PCA3是PCa的獨(dú)立預(yù)測(cè)因子,能明確區(qū)分前列腺穿刺陽性和陰性患者,基于尿液MALAT1或PCA3建立的臨床綜合診斷模型可大大提高PCa的臨床診斷效能。尤其在PSA“診斷灰區(qū)”,尿液MALAT1和PCA3的診斷效能明顯高于血清總PSA,基于尿液MALAT1或PCA3建立的臨床綜合診斷模型的預(yù)測(cè)能力高于應(yīng)用臨床危險(xiǎn)因素建立的基礎(chǔ)模型,并能夠避免更多不必要的前列腺穿刺且不會(huì)漏診更多的高級(jí)別PCa。此外,本研究證明了mi R-n5在體內(nèi)和體外都能明顯抑制進(jìn)展性PCa的生長,它通過抑制其靶基因KDM6B的表達(dá)發(fā)揮抗腫瘤作用。同時(shí),GSK-J4能夠抑制mi R-n5的靶基因KDM6B的活性并明顯抑制PCa細(xì)胞的生長。新型mi R-n5有望成為一個(gè)新的CRPC干預(yù)的靶點(diǎn),而GSK-J4也有望成為治療進(jìn)展性PCa的一種新型靶向藥物。
[Abstract]:Background: prostate cancer Chinese (Prostate cancer, PCa) incidence and mortality rates rose sharply, the growth rate ranked first in cancer prevention and treatment of PCa. At the same time, facing two major clinical problems, namely early diagnosis effect index of poor specificity and late treatment is poor. Therefore, the clinical need to find high sensitivity and specificity PCa diagnosis of new markers or diagnostic strategies instead of serum PSA or serum PSA in early diagnosis for lack of the basic research to clarify the PCa; the key advances in molecular mechanism, thus developed a suppression of castration resistant prostate cancer (Castration-resistant prostate, cancer, CRPC) of the new drug, to achieve a breakthrough in the treatment of CRPC. Objective: To study the effect of long chain non diagnostic MALAT1 encoding RNA and PCA3 score in patients with primary prostate puncture in the urine, urine MALAT1 or PCA3 score is established based on Ensemble Synthetic diagnosis model; to explore the mechanism of the new mi R-n5 in the progression of PCa, a new target for CRPC treatment. Methods: whole transcriptome amplification kit amplified urine RNA, expression of Q MALAT1 and PCA3 RT-PCR test, single factor Logistic regression prediction of PCa risk factors, logistic regression to establish PCa diagnosis Spearman model, rank test to analyze the relationship between urine MALAT1 or PCA3 score and other clinical variables, using the receiver operating curve (Receiver operating characteristic curve, ROC) to determine the diagnostic indexes of the area under the curve (Area under the ROC, AUC), the optimal cut-off value, the sensitivity and specificity of decision curve analysis (Decision curve analysis, DCA) to estimate the clinical value of.Northern blot mi RNA to detect new urine MALAT1 and PCA3 scores of the existence and expression, cell proliferation, migration, formation of MI R experiment to evaluate the invasion and clone The biological function of -n5 in the progression of PCa cell lines, target genes were predicted by bioinformatics software mi R-n5, dual luciferase assay validation of target gene, Graph Pad Prism5 for data display and calculation of the drug IC50, the establishment of CRPC transplantation tumor mouse model of subcutaneous implantation and intratibia injection. Using SPSS (SPSS Inc., v.17.0 Chicago, IL, USA), Med Calc v.10.4.7.0 (Med Calc Software BVBA, Mariakerke, Belgium) and R (The R Foundation software v.3.1.1 for Statistical Computing) for statistical analysis of data using Hem I software for data analysis by P0.05 Heatmap; the difference was statistically significant. Results: the urine MALAT1 and PCA3 the score can clearly distinguish the tumor group and negative group patients with PCa puncture and the detection rate was highly correlated, but further correlation analysis found that the urine MALAT1 and PCA3 score and the clinical risk factors and Gl The score of eason. No correlation between age, serum total PSA (Total PSA, t PSA), prostate volume (Prostate volume, PV), the ratio of PSA (Percentage of free free PSA,%f PSA) and rectal examination results (Digital rectal examination, DRE) were independent predictors of the overall population, and in the PSA=4-10ng/ml (PSA "diagnostic gray zone") population, age, PV,%f PSA and DRE are the independent predictors of MALAT1 score PSA. Urine PCa diagnostic efficacy and T in the general population than although slightly higher but not statistically significant (p=0.510), but in PSA "diagnostic gray zone" in the crowd was significantly better than t PSA (p=0.034) PSA ".PCA3 score in the diagnosis of PCa diagnostic gray zone" in the crowd was significantly better than t PSA (p=0.0407) and%f PSA (p=0.046). Further logistic regression analysis, found in the group of PSA diagnosis of "gray zone" in the population, based on urine MALAT1 score model The prediction accuracy is 79.79% and AUC is 0.853, the prediction accuracy is 5.32% more than the base model, AUC improved 0.0318. applied this model to the validation group also got the same result. Similarly, in the PSA diagnosis of "gray zone" in the crowd, urine PCA3 scores model of AUC is as high as 0.819 based on the above basis the application of 0.788. model in DCA, PSA diagnosis of "gray zone" in the crowd, no matter is found in groups or in the validation group, the clinical prediction ability of urine MALAT1 scores were better than the model based on the basic model. The clinical risk of puncture was 25% and the total benefit based on MALAT1 score found urine synthesis model is higher than the rate of group based model the crowd (13.97%vs.11.47%), and can avoid more unnecessary prostate biopsy (47.32%vs.39.78%); the total benefit groups based on the verification of the integrated model of the rate of urine MALAT1 score higher than that of basic models (15.73% Vs.13.11%), and can avoid more unnecessary prostate biopsy (30.33%vs.22.47%), more importantly, no comprehensive model of misdiagnosis of 1 cases of high grade PCa based model of missed diagnosis in 1 cases of high grade PCa., PSA in diagnosis of "grey zone" in the crowd, PCA3 score of the comprehensive model in almost all clinical on the basis of model based on the threshold of puncture is better than 25%-40%, especially in this interval showed better predictors. The clinical application of 30% puncture threshold, PCA3 model can avoid unnecessary biopsy of 60.3% based on 4 cases of misdiagnosis but with the price of PCa, including 2 cases of high grade PCa. in application 65 pairs of PCa and normal tissues were found in RNA-seq more than 300 new mi RNA, and use Northern blot to validate the expression level of the highest of the 8 new mi RNA. through the situation of comparative expression in the progress of PCa and inert PCa cell lines, screen Select the new mi RNA. 4 and PCa related through the validation of clinical samples, the final selection of MI R-n5 as a follow-up study. Functional experiments in vitro showed that MI and R-n5 could significantly inhibit the progression of PCa cell proliferation, migration, invasion and clone formation ability, lack of MI or low expression of R-n5 may promote the progress of PCa. In vivo experiments proved that intratumoral injection of MI R-n5 can inhibit tumor growth. Since then, the bioinformatics analysis and find the target genes of KDM6B, MI and R-n5, were verified by dual luciferase reporter gene Q and RT-PCR. Further studies show that the activity of a specific inhibitor of KDM6B the small molecule compound GSK-J4 can inhibit KDM6B and inhibit the growth of PCa cells in vivo experiments have proved that the small molecular compounds can inhibit the growth of transplanted tumor in mice The growth of MALAT1 and PCA3. Conclusion: the urine was an independent predictor of PCa, can make a clear distinction between positive and negative prostate biopsy in patients with clinical diagnosis of urinary MALAT1 or PCA3 model established can greatly improve the diagnostic efficiency of PCa based on PSA. Especially in the diagnosis of "grey zone", the diagnostic performance of urine MALAT1 and PCA3 were significantly higher than those of the serum total PSA based application of clinical risk factors for the predictive ability of clinical comprehensive diagnosis model established by the PCA3 or MALAT1 in urine was higher than that based on high level PCa. and can avoid more unnecessary prostate biopsy and not missed more in addition, this study demonstrated that MI R-n5 could inhibit the progression of PCa in vivo and in vitro growth, its antitumor effect by inhibiting the expression of the target gene of KDM6B. At the same time, the target gene KDM6B GSK-J4 can inhibit the activity of MI R-n5 and inhibit PCa cell Growth. The new mi R-n5 is expected to be a new target for CRPC intervention, and GSK-J4 is also expected to be a new target drug for the treatment of progressive PCa.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.25

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