本文選題：Elf5　切入點：轉(zhuǎn)化生長因子-β　出處：《天津醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目的探討Elf5在TGF-β誘導(dǎo)的前列腺癌上皮間質(zhì)轉(zhuǎn)化的相關(guān)機制。方法收集并納入124例前列腺癌病理樣本,通過免疫組化染色(IHC)和ImageJ軟件分析Elf5與E-鈣粘蛋白和N-鈣粘蛋白表達的關(guān)系。在幾種常見前列腺癌細(xì)胞系中Western Blot檢測Elf5的表達,在LNCaP細(xì)胞中用siRNA瞬時轉(zhuǎn)染將Elf5沉默使其低表達,在22Rv1細(xì)胞中通過HA-Elf5轉(zhuǎn)染將Elf5穩(wěn)定地高表達;在有/無TGF-β處理的情況下,分組觀察LNCaP和22Rv1細(xì)胞的形態(tài)學(xué)變化,同時Western Blot檢測這些分組之間E-鈣粘蛋白以及反映EMT的生物標(biāo)志物如N-鈣粘蛋白、Snail1、Snail2和ZEB1表達量的變化。對LNCa P和22Rv1細(xì)胞進行球形成實驗和遷移實驗觀察其錨定-非依賴生長和遷移侵襲能力是否增加。用熒光素酶報告基因?qū)嶒?Luciferase)將CAGA12或PAI-1報告基因質(zhì)粒分別在HEK293和22Rv1細(xì)胞中和HA-Elf5共轉(zhuǎn)染,在LNCaP細(xì)胞中和siElf5共轉(zhuǎn)染,然后用TGF-β處理并測定熒光素酶活性來判斷Elf5是否調(diào)節(jié)TGF-β信號轉(zhuǎn)導(dǎo)。在LNCaP細(xì)胞中檢測內(nèi)源性p-SMAD2、SMAD2、p-SMAD3、SMAD3和Elf5的表達并在22Rv1細(xì)胞中驗證。在無內(nèi)源性表達Elf5和SMADs的HEK293細(xì)胞中分組共轉(zhuǎn)染HA-Elf5質(zhì)粒及Flag標(biāo)記的vector、SMAD2、SMAD3或SMAD4質(zhì)粒,免疫共沉淀判斷Elf5是否與SMADs結(jié)合,并在內(nèi)源性表達Elf5和SMADs的LNCaP細(xì)胞中進一步驗證。最后,對原發(fā)前列腺癌病理樣本進行p-SMAD3和TGF-β染色,分析p-SMAD3陽性率,并對Elf5和p-SMAD3與疾病進展結(jié)果之間進行生存分析。結(jié)果對124例人前列腺癌成組匹配免疫組化染色發(fā)現(xiàn),Elf5可使E-鈣粘蛋白表達上調(diào),而使N-鈣粘蛋白表達下調(diào),初步提示前列腺癌組織中Elf5可能抑制EMT的發(fā)生。Elf5在LNCaP細(xì)胞高表達,在22Rv1細(xì)胞低表達;在LNCa P和22Rv1細(xì)胞中,Elf5與TGF-β對細(xì)胞形態(tài)影響呈負(fù)相關(guān),沉默Elf5和/或用TGF-β處理可使細(xì)胞喪失極性,EMT標(biāo)志物表達增加,從而使其具有更強的轉(zhuǎn)移侵襲能力;且可以促進細(xì)胞的球形成能力和遷移能力,使其多向分化能力和惡性遷移能力增加。Elf5和TGF-β對EMT的作用呈負(fù)相關(guān),Elf5可以抑制TGF-β驅(qū)動的前列腺癌上皮間質(zhì)轉(zhuǎn)化。在HEK293、LNCaP和22Rv1中,Elf5均可以抑制CAGA12和PAI-1報告基因熒光素酶活性,提示Elf5可以抑制TGF-β信號通路;在LNCaP細(xì)胞中Western Blot結(jié)果顯示沉默Elf5可以促進SMAD3的磷酸化,而22Rv1細(xì)胞中過表達Elf5則可以抑制SMAD3磷酸化;在HEK293和LNCaP細(xì)胞中,免疫共沉淀結(jié)果均顯示Elf5可以與SMAD3結(jié)合。p-SMAD3在Elf5-/TGF-βhigh前列腺癌樣本中表達顯著增高,Elf5缺失與TGF-β陽性的前列腺癌病人的無轉(zhuǎn)移生存(MFS)有統(tǒng)計學(xué)意義,p-SMAD3表達水平與無轉(zhuǎn)移生存(MFS)有顯著統(tǒng)計學(xué)意義,表明了Elf5缺失可致TGF-β陽性前列腺癌病人轉(zhuǎn)移增加,Elf5聯(lián)合TGF-β的狀態(tài)可能作為前列腺癌轉(zhuǎn)移的標(biāo)志物。結(jié)論Elf5與EMT呈負(fù)相關(guān),其通過與SMAD3結(jié)合抑制其磷酸化進而抑制TGF-β驅(qū)動的前列腺癌EMT,Elf5可能作為TGF-β陽性前列腺癌患者無轉(zhuǎn)移生存的預(yù)后標(biāo)志物。
[Abstract]:Objective to investigate the mechanism of Elf5 in TGF- 尾 -induced epithelial interstitial transformation of prostate cancer. Methods 124 cases of prostate cancer were collected and included in this study. The relationship between Elf5 and expression of E-cadherin and N-cadherin was analyzed by immunohistochemical staining and ImageJ software. The expression of Elf5 was detected by Western Blot in several common prostate cancer cell lines, and Elf5 was silenced by transient transfection of siRNA in LNCaP cells. The expression of Elf5 was stably overexpressed in 22Rv1 cells by HA-Elf5 transfection, and the morphological changes of LNCaP and 22Rv1 cells were observed in groups with and without TGF- 尾 treatment. At the same time, Western Blot was used to detect the expression of E-cadherin and its biomarkers such as N- cadherin 1, Snail1, Snail2 and ZEB1 between these groups. The expression of Ecadherin 2 and ZEB1 in LNCa P and 22Rv1 cells were observed by the experiment of ball formation and migration of LNCa P and 22Rv1 cells. CAGA12 or PAI-1 reporter gene plasmids were co-transfected into HEK293 and 22Rv1 cells by luciferase assay. Cotransfection with siElf5 in LNCaP cells. Then the luciferase activity of TGF- 尾 was used to determine whether Elf5 regulated TGF- 尾 signal transduction. The expression of endogenous p-SMAD2P- SMAD3SMAD3 and Elf5 was detected in LNCaP cells and verified in 22Rv1 cells. In HEK293 cells with no endogenous expression of Elf5 and SMADs, the expression of SMAD3 and Elf5 were detected. Group A co-transfected HA-Elf5 plasmid and vectorus SMAD2SMAD3 or SMAD4 plasmid labeled with Flag. Immunoprecipitation was used to determine whether Elf5 binds to SMADs and was further verified in LNCaP cells that express Elf5 and SMADs. Finally, p-SMAD3 and TGF- 尾 staining were used to analyze the positive rate of p-SMAD3 in primary prostate cancer samples. The survival analysis between Elf5 and p-SMAD3 and the results of disease progression was carried out. Results the expression of Ecadherin was up-regulated and the expression of N- cadherin was down-regulated in 124 cases of human prostate cancer by immunohistochemical staining. It was suggested that Elf5 might inhibit the expression of EMT in LNCaP cells, but low expression in 22Rv1 cells, whereas in LNCaP and 22Rv1 cells, there was a negative correlation between Ef5 and TGF- 尾. Silencing Elf5 and / or treatment with TGF- 尾 can increase the expression of EMT markers in cells without polarity, thus making them have stronger ability of metastasis and invasion, and can promote the ability of cell ball formation and migration. There was a negative correlation between the effect of Elf5 and TGF- 尾 on EMT. Elf5 inhibited the epithelial mesenchymal transformation of prostate cancer induced by TGF- 尾. In HEK293 LNCaP and 22Rv1, Elf5 inhibited the luciferase activity of CAGA12 and PAI-1 reporter genes. These results suggest that Elf5 can inhibit the TGF- 尾 signaling pathway, Western Blot in LNCaP cells shows that silencing Elf5 can promote the phosphorylation of SMAD3, while overexpression of Elf5 in 22Rv1 cells can inhibit SMAD3 phosphorylation, while in HEK293 and LNCaP cells, SMAD3 phosphorylation can be inhibited by silencing Elf5. Results of immunoprecipitation showed that Elf5 could bind with SMAD3. P-SMAD3 expression was significantly increased in prostate cancer samples with Elf5 deletion and TGF- 尾 positive prostate cancer. The expression level of p-SMAD3 was significantly higher than that of non-metastasized prostatic cancer patients with positive TGF- 尾 and Elf5 deletion. There was a significant difference between the expression level of p-SMAD3 and non-metastatic prostatic cancer patients. MFS) has significant statistical significance. The results suggest that the loss of Elf5 may increase the metastasis of TGF- 尾 positive prostate cancer patients. The combination of TGF- 尾 and Elf5 may be a marker of prostate cancer metastasis. Conclusion there is a negative correlation between Elf5 and EMT. It may be a prognostic marker of metastasis-free survival in prostate cancer patients with TGF- 尾 positive prostate cancer by inhibiting its phosphorylation by binding with SMAD3 and then inhibiting TGF- 尾 -driven prostate cancer EMT-Elf5.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.25

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相關(guān)期刊論文 前1條

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本文編號：1568764