Elf5抑制TGF-β诱导的前列腺癌上皮间质转化的机制研究

发布时间：2018-03-05 04:30

  本文选题：Elf5　切入点：转化生长因子-β　出处：《天津医科大学》2015年博士论文　论文类型：学位论文

【摘要】：目的探讨Elf5在TGF-β诱导的前列腺癌上皮间质转化的相关机制。方法收集并纳入124例前列腺癌病理样本,通过免疫组化染色(IHC)和ImageJ软件分析Elf5与E-钙粘蛋白和N-钙粘蛋白表达的关系。在几种常见前列腺癌细胞系中Western Blot检测Elf5的表达,在LNCaP细胞中用siRNA瞬时转染将Elf5沉默使其低表达,在22Rv1细胞中通过HA-Elf5转染将Elf5稳定地高表达;在有/无TGF-β处理的情况下,分组观察LNCaP和22Rv1细胞的形态学变化,同时Western Blot检测这些分组之间E-钙粘蛋白以及反映EMT的生物标志物如N-钙粘蛋白、Snail1、Snail2和ZEB1表达量的变化。对LNCa P和22Rv1细胞进行球形成实验和迁移实验观察其锚定-非依赖生长和迁移侵袭能力是否增加。用荧光素酶报告基因实验(Luciferase)将CAGA12或PAI-1报告基因质粒分别在HEK293和22Rv1细胞中和HA-Elf5共转染,在LNCaP细胞中和siElf5共转染,然后用TGF-β处理并测定荧光素酶活性来判断Elf5是否调节TGF-β信号转导。在LNCaP细胞中检测内源性p-SMAD2、SMAD2、p-SMAD3、SMAD3和Elf5的表达并在22Rv1细胞中验证。在无内源性表达Elf5和SMADs的HEK293细胞中分组共转染HA-Elf5质粒及Flag标记的vector、SMAD2、SMAD3或SMAD4质粒,免疫共沉淀判断Elf5是否与SMADs结合,并在内源性表达Elf5和SMADs的LNCaP细胞中进一步验证。最后,对原发前列腺癌病理样本进行p-SMAD3和TGF-β染色,分析p-SMAD3阳性率,并对Elf5和p-SMAD3与疾病进展结果之间进行生存分析。结果对124例人前列腺癌成组匹配免疫组化染色发现,Elf5可使E-钙粘蛋白表达上调,而使N-钙粘蛋白表达下调,初步提示前列腺癌组织中Elf5可能抑制EMT的发生。Elf5在LNCaP细胞高表达,在22Rv1细胞低表达;在LNCa P和22Rv1细胞中,Elf5与TGF-β对细胞形态影响呈负相关,沉默Elf5和/或用TGF-β处理可使细胞丧失极性,EMT标志物表达增加,从而使其具有更强的转移侵袭能力;且可以促进细胞的球形成能力和迁移能力,使其多向分化能力和恶性迁移能力增加。Elf5和TGF-β对EMT的作用呈负相关,Elf5可以抑制TGF-β驱动的前列腺癌上皮间质转化。在HEK293、LNCaP和22Rv1中,Elf5均可以抑制CAGA12和PAI-1报告基因荧光素酶活性,提示Elf5可以抑制TGF-β信号通路;在LNCaP细胞中Western Blot结果显示沉默Elf5可以促进SMAD3的磷酸化,而22Rv1细胞中过表达Elf5则可以抑制SMAD3磷酸化;在HEK293和LNCaP细胞中,免疫共沉淀结果均显示Elf5可以与SMAD3结合。p-SMAD3在Elf5-/TGF-βhigh前列腺癌样本中表达显著增高,Elf5缺失与TGF-β阳性的前列腺癌病人的无转移生存(MFS)有统计学意义,p-SMAD3表达水平与无转移生存(MFS)有显著统计学意义,表明了Elf5缺失可致TGF-β阳性前列腺癌病人转移增加,Elf5联合TGF-β的状态可能作为前列腺癌转移的标志物。结论Elf5与EMT呈负相关,其通过与SMAD3结合抑制其磷酸化进而抑制TGF-β驱动的前列腺癌EMT,Elf5可能作为TGF-β阳性前列腺癌患者无转移生存的预后标志物。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25

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