本文選題：核干因子 + 核糖體生物合成��； 參考：《天津醫(yī)科大學(xué)》2015年博士論文

【摘要】：目的探討NS調(diào)節(jié)核糖體蛋白合成對(duì)前列腺癌細(xì)胞增殖的影響,并明確其分子機(jī)制。方法首先,利用Oncomine數(shù)據(jù)庫逐個(gè)檢索RP(ribosomal protein,RP)在良性前列腺增生(Benign prostatic hyperplasia,BPH)組織與PCa(Prostate cancer,PCa)組織中表達(dá)的差異;然后,收集國人的PCa及增生組織標(biāo)本,提取RNA反轉(zhuǎn)錄行RT-qPCR檢測(cè)以上核糖體蛋白mRNA的表達(dá)水平;進(jìn)而,挑選了核糖體大小亞基蛋白中RP差異表達(dá)最為明顯的兩個(gè),通過western blot檢測(cè)二者在PCa組織中的蛋白表達(dá)情況,并分析其與PCa患者臨床病理特征以及PSA生化復(fù)發(fā)的關(guān)系。構(gòu)建NS低表達(dá)PC3細(xì)胞系,通過蔗糖梯度離心檢測(cè)NS低表達(dá)后對(duì)核糖體大小亞基生物合成的影響;通過RT-qPCR檢測(cè)NS低表達(dá)后核糖體蛋白mRNA水平表達(dá)情況;篩選變化最明顯的核糖體蛋白,通過體內(nèi)、外實(shí)驗(yàn)探討該核糖體蛋白對(duì)PCa發(fā)生發(fā)展中的作用。構(gòu)建c-Myc高表達(dá)PC3細(xì)胞系,再使用c-Myc小分子抑制劑10058-F4處理PC3細(xì)胞,通過RT-qPCR及Western blot檢測(cè)細(xì)胞中NS的表達(dá)水平,使用DCFH-DA探針檢測(cè)細(xì)胞內(nèi)ROS的水平;用H2O2上調(diào)PC3細(xì)胞內(nèi)的ROS,過氧化氫酶Catalase降低PC3細(xì)胞內(nèi)的ROS,進(jìn)而行western blot及qPCR檢測(cè)細(xì)胞內(nèi)的ROS對(duì)NS表達(dá)的影響;通過細(xì)胞計(jì)數(shù)法及MTT分別檢測(cè)c-Myc、ROS及NS對(duì)PC3細(xì)胞增殖能力的影響。結(jié)果通過Oncomine網(wǎng)絡(luò)數(shù)據(jù)庫分析,我們發(fā)現(xiàn)在PCa組織內(nèi)存在核糖體大、小亞基蛋白的高表達(dá),其中RPS蛋白有15種、RPL蛋白有14種。我們通過RT-PCR檢測(cè)這些核糖體蛋白在國人PCa及前列腺增生組織內(nèi)的表達(dá)時(shí),發(fā)現(xiàn)在國人PCa組織中各有11種RPS和RPL存在差異性表達(dá),其中RPS2和RPL36的表達(dá)差異性最為顯著。我們分別分析了RPS2和RPL36與PCa患者臨床病理學(xué)資料的關(guān)系,發(fā)現(xiàn)此二種蛋白與患者的病理及預(yù)后密切相關(guān)。降低PC3細(xì)胞中NS的表達(dá)水平后核糖體大亞基合成受抑制;RT-qPCR結(jié)果顯示NS低表達(dá)后大亞基核糖體蛋白mRNA水平均下調(diào);其中RPL19下調(diào)最為明顯,其在蛋白水平的變化和NS具有一致性;靶向沉默PC3細(xì)胞中的RPL19后,發(fā)現(xiàn)PC3細(xì)胞增殖減慢、凋亡增加且侵襲能力減弱;體內(nèi)實(shí)驗(yàn)顯示,沉默RPL19后,PC3在裸鼠體內(nèi)的成瘤能力下降。上調(diào)PC3細(xì)胞內(nèi)的c-Myc可同時(shí)上調(diào)細(xì)胞內(nèi)ROS和NS的mRNA及蛋白質(zhì)表達(dá)水平;細(xì)胞內(nèi)的ROS水平可增加NS的蛋白表達(dá)水平。細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示:c-Myc高表達(dá)促進(jìn)PC3細(xì)胞的增殖;提高細(xì)胞內(nèi)的ROS水平可促進(jìn)PC3細(xì)胞的增殖;降低PC3細(xì)胞中的NS的水平,可以抑制PC3細(xì)胞的增殖。結(jié)論在前列腺癌中存在c-Myc-ROS-NS信號(hào)通路調(diào)節(jié)NS的異常表達(dá),NS的異常表達(dá)進(jìn)而通過調(diào)節(jié)核糖體大亞基蛋白(尤其是RPL19)促進(jìn)前列腺癌細(xì)胞的增殖。
[Abstract]:Objective to investigate the effects of NS on the proliferation of prostate cancer cells and its molecular mechanism. Methods first, the difference of the expression of RP(ribosomal protein Oncomine in benign prostatic hyperplasia (BPH) and PCa(Prostate cancer-PCA (BPH) was searched by Oncomine database, and then the specimens of PCa and proliferative tissue were collected from Chinese people. RNA reverse transcription RT-qPCR was extracted to detect the mRNA expression level of ribosomal protein, and then two of the ribosome size subunit proteins were selected to detect the expression of RP in PCa tissues by western blot. The clinicopathological features and biochemical recurrence of PSA in patients with PCa were analyzed. PC3 cell line with low expression of NS was constructed and the effect of low expression of NS on biosynthesis of ribosomal subunit was detected by sucrose gradient centrifugation, and the expression of ribosomal protein mRNA after low expression of NS was detected by RT-qPCR. The role of ribosomal protein in the development of PCa was studied in vivo and in vitro. C-Myc overexpression PC3 cell line was constructed, then PC3 cells were treated with c-Myc small molecule inhibitor 10058-F4. The expression of NS was detected by RT-qPCR and Western blot, and ROS was detected by DCFH-DA probe. H2O2 up-regulated ros in PC3 cells, catalase Catalase decreased rose in PC3 cells, and then western blot and qPCR were used to detect the effect of ROS on NS expression, and the effects of c-Myc Ros and NS on the proliferation of PC3 cells were detected by cell count and MTT, respectively. Results through the analysis of Oncomine network database, we found that there were high expression of ribosomal protein and small subunit protein in PCa tissue, of which there were 15 kinds of RPS protein and 14 species of RPS protein. When we detected the expression of these ribosomal proteins in Chinese PCa and prostatic hyperplasia by RT-PCR, we found that there were 11 different RPS and RPL expressions in Chinese PCa, and RPS2 and RPL36 were the most significant. We analyzed the relationship between RPS2 and RPL36 and the clinicopathological data of PCa patients, and found that these two proteins were closely related to the pathology and prognosis of PCa patients. The results of RT-qPCR showed that the mRNA level of large subunit ribosomal protein was down-regulated after decreasing the expression level of NS in PC3 cells, in which RPL19 down-regulation was the most obvious, and the changes in protein level were consistent with NS. After targeting RPL19 in PC3 cells, the proliferation of PC3 cells slowed down, the apoptosis increased and the invasiveness decreased. In vivo experiments showed that the tumorigenic ability of pPC3 in nude mice was decreased after RPL19 silencing. Upregulation of c-Myc in PC3 cells could increase the expression of mRNA and protein in both ROS and NS cells, while ROS level in cells could increase the protein expression level of NS. The results of cell proliferation assay showed that the high expression of w _ c-Myc could promote the proliferation of PC3 cells, increase the level of ROS in cells could promote the proliferation of PC3 cells, and decrease the level of NS in PC3 cells, which could inhibit the proliferation of PC3 cells. Conclusion there is an abnormal expression of NS regulated by c-Myc-ROS-NS signaling pathway in prostate cancer and the abnormal expression of NS can promote the proliferation of prostate cancer cells by regulating ribosomal large subunit protein (especially RPL19).
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 岳保紅;蔚利納;王園園;;沉默核干因子基因表達(dá)對(duì)HL-60白血病細(xì)胞凋亡的影響[J];中國實(shí)驗(yàn)血液學(xué)雜志;2009年02期

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本文編號(hào)：1857118