TSP50在EMT发生中的作用及相关机制研究

发布时间：2018-06-30 06:24

本文选题：TSP50 + EMT　；参考：《东北师范大学》2017年硕士论文

【摘要】：睾丸特异性蛋白酶50(Testes-specific protease 50,TSP50)是一种苏氨酸蛋白酶,在肿瘤的发生和发展中扮演重要角色。以往人们对TSP50的研究主要集中于其对细胞增殖和转移等细胞生物学特性的影响,而关于TSP50在上皮间质转化(Epithelial-mesenchymal transition,EMT)发生发展中的作用研究尚未见报道。本研究主要探讨了TSP50在EMT中的作用,并对其机制进行了初步研究。1.不同细胞系中TSP50和EMT标志物的表达分析为了确定TSP50对EMT的影响,我们首先利用RT-PCR和Western blot方法分析了不同细胞系中TSP50和EMT相关标志物的表达情况。结果发现,在高表达TSP50的细胞中EMT间质标志物Vimentin的表达量亦较高,而上皮标志物E-cadherin的表达则较低;但是在低表达TSP50的细胞中EMT相关标志物的表达量则正好相反。此结果提示,TSP50和EMT可能呈正相关。2.TSP50和EMT的相关性研究(1)过表达TSP50对EMT标志物表达的影响为了验证我们的假设,我们通过过表达或敲低TSP50表达的方法探讨了TSP50的表达量与EMT之间的相关性。我们首先在低表达TSP50的MCF-10A细胞中过表达TSP50,利用Western blot实验分析了过表达TSP50对细胞EMT相关标志物表达的影响。结果显示,过表达TSP50可以使细胞中EMT间质表型标志物N-cadherin和Vimentin的表达上调,EMT上皮表型标志物E-cadherin的表达下调。为了探讨TSP50对EMT的影响是否具有剂量依赖效应,我们在内源高表达TSP50的MDA-MB-231细胞中的进一步过表达TSP50,之后检测EMT标志物的表达情况。结果显示,随着TSP50的表达增高,EMT间质表型标志物N-cadherin和Vimentin的表达进一步上调,EMT上皮表型标志物E-cadherin的表达则进一步下调。以上结果提示,TSP50可促进EMT,且此促进作用具有TSP50表达量依赖性。(2)敲低TSP50的表达对EMT标志物表达的影响为了进一步证实以上结果,我们在高表达TSP50的MDA-MB-231细胞中通过RNAi方法敲低TSP50的表达,之后利用Western blot方法分析了EMT相关标志物的表达情况。结果显示,敲低TSP50的表达可以使细胞中EMT间质标志物N-cadherin和Vimentin的表达下调,EMT上皮标志物E-cadherin的表达上调。此结果进一步证明了TSP50对EMT的促进作用。(3)TSP50对细胞侵袭性的影响细胞发生EMT后会具有一定的侵袭性,因此我们探讨了TSP50对细胞侵袭性的影响。Transwell实验结果显示,过表达TSP50可以增强细胞的侵袭能力,反之敲低TSP50则会抑制细胞的侵袭能力。综上所述,我们发现TSP50的表达量与EMT的发生呈正相关。3.TSP50促进EMT的机制研究为了探究TSP50影响EMT的分子机制。我们通过过表达或敲低TSP50表达的方法探讨了TSP50对细胞内信号通路的影响。Western blot结果显示,过表达TSP50后磷酸化的IKK和IκB的水平上升,p65的入核增加;同时磷酸化的AKT和ERK1/ERK2水平亦明显增加;但是Smad2/3的入核则明显降低。敲低细胞中TSP50的表达则Smad2/3的入核明显增加,而磷酸化的AKT和ERK1/2水平则明显降低。以上结果说明,TSP50可以激活NF-kB、AKT及ERK/MAPK信号,同时抑制TGF-β信号。为了进一步探讨这些信号与TSP50所诱导的EMT的相关性,我们在过表达TSP50的细胞中利用抑制剂分别阻断了NF-kB、ERK/MAPK及AKT信号,之后检测了EMT相关标志物的表达变化。结果显示,抑制NF-kB、AKT及ERK/MAPK信号均可抑制由过表达TSP50所诱导的EMT。我们又在过表达TSP50的细胞中过表Smad2/3,之后检测了EMT相关标志物的表达变化。结果显示,过表达Smad可以抑制由过表达TSP50所诱导的EMT。以上结果提示,NF-kB、AKT、ERK/MAPK和TGF-β信号均参与了TSP50所诱导的EMT。4.TGF-β与TSP50在诱导EMT过程中的相互作用研究因为在之前的报道中,TGF-β可以刺激乳腺上皮细胞发生EMT,而我们的研究则显示,TSP50可以抑制TGF-β信号,但可诱导EMT,这些矛盾的结果促使我们进一步探讨了TGF信号与TSP50所诱导的EMT之间的相关性。我们用TGF-β刺激过表达TSP50的细胞,发现长时间的刺激会引起由TSP50诱导的EMT细胞发生间质上皮转化(Mesenchymal-epithelial transition,MET)。同时,我们还发现,TGF-β在抑制TSP50所诱导的EMT的同时,也抑制了AKT和ERK的激活。此结果提示,TSP50和TGF-β在调控细胞发生EMT的过程中存在着复杂的信号调控网络。本研究证实了原癌基因TSP50可以诱导EMT的发生,并从细胞内信号角度初步探讨了TSP50促进EMT的分子机制以及TGF-β与TSP50在EMT发生过程中的复杂作用关系,从而为全面阐明原癌基因TSP50在肿瘤发生及发展中的作用及机制奠定实验基础。
[Abstract]:Testicular specific protease 50 (Testes-specific protease 50, TSP50) is a kind of threonine protease, which plays an important role in the development and development of tumor. Previous research on TSP50 was mainly focused on its effects on cell biological characteristics such as cell proliferation and metastasis, and TSP50 on epithelial transformation (Epithelial-mesenchym). The role of Al transition, EMT) in the development of TSP50 has not yet been reported. This study mainly discussed the role of TSP50 in EMT, and a preliminary study of the mechanism of the expression of TSP50 and EMT markers in different cell lines in order to determine the effect of TSP50 on EMT. We first analyzed the difference between RT-PCR and Western methods. The expression of TSP50 and EMT related markers in the cell lines showed that the expression of the EMT stromal marker Vimentin in the cells with high expression of TSP50 was also higher, while the expression of the epithelial marker E-cadherin was lower, but the expression of EMT related markers in the cells with low expression of TSP50 was just the opposite. This result suggests TSP50 and EMT. A possible correlation study of positive correlation between.2.TSP50 and EMT (1) overexpressing the effect of TSP50 on the expression of EMT markers to verify our hypothesis, we explored the correlation between the expression of TSP50 and the EMT by expressing or knocking low TSP50 expression. We first overexpressed TSP50 in the MCF-10A cells of lower TSP50, using West. The ERN blot experiment analyzed the effect of overexpression of TSP50 on the expression of EMT related markers. The results showed that overexpression of TSP50 could up-regulated the expression of EMT interstitial markers N-cadherin and Vimentin, and the expression of EMT epithelial phenotype marker E-cadherin was down regulated. We further overexpressed TSP50 in the MDA-MB-231 cells expressing TSP50, and then detected the expression of EMT markers. The results showed that the expression of N-cadherin and Vimentin in the interstitial markers of EMT was further up-regulated with the increase of TSP50 expression, and the expression of EMT epithelial phenotype marker E-cadherin was further downregulated. The results suggest that TSP50 promotes EMT and this promotion is dependent on TSP50 expression. (2) the effect of the expression of low TSP50 on the expression of EMT markers to further confirm the above results, we knock low TSP50 expression by RNAi method in the MDA-MB-231 cells with high expression of TSP50, and then analyze EMT related markers by Western blot method. The results showed that the expression of the EMT stromal markers, N-cadherin and Vimentin, was down regulated by the expression of TSP50, and the expression of E-cadherin was up regulated by the EMT epithelial marker. The results further demonstrated the promotion of TSP50 to EMT. (3) the effect of TSP50 on cell invasiveness may have a certain invasion of the cell after EMT. So we explored the effect of TSP50 on cell invasiveness..Transwell experiments showed that overexpression of TSP50 could enhance cell invasiveness, and conversely, knocking down TSP50 would inhibit cell invasiveness. To sum up, we found that the expression of TSP50 is positively related to the mechanism of EMT in the mechanism of.3.TSP50 to promote EMT. TSP50 affects the molecular mechanism of EMT. We explored the effect of TSP50 on intracellular signaling pathway by expressing or knocking low TSP50 expression..Western blot results showed that IKK and I kappa B increased after overexpression of TSP50, p65 nucleation increased; meanwhile, phosphorylation AKT and levels were also significantly increased. The nucleation of TSP50 decreased significantly. The expression of Smad2/3 in the knockout cells increased significantly, while the levels of phosphorylated AKT and ERK1/2 decreased significantly. The above results indicated that TSP50 could activate NF-kB, AKT and ERK/MAPK signals and inhibit TGF- beta signals. To further explore the correlation between these signals and EMT, TSP50 induced. NF-kB, ERK/MAPK and AKT signals were blocked by inhibitors in the cells overexpressing TSP50, and then the expression changes of EMT related markers were detected. The results showed that the inhibition of NF-kB, AKT and ERK/MAPK signals could inhibit EMT. which was induced by overexpressed TSP50. We also overexpressed the Smad2/3 in the cells that overexpressed the TSP50. The results showed that overexpression of Smad could inhibit the results suggested by over expression of TSP50, and that NF-kB, AKT, ERK/MAPK and TGF- beta signals were involved in the interaction of EMT.4.TGF- beta and TSP50 induced by TSP50 in the induction of EMT process because in previous reports, the TGF- beta could stimulate the mammary gland. EMT, and our study shows that TSP50 can inhibit TGF- beta signal, but can induce EMT. These contradictory results have prompted us to further explore the correlation between the TGF signal and the EMT induced by TSP50. We use TGF- beta to stimulate the expression of TSP50 cells, and a long time stimulation will lead to EMT cells induced by TSP50. Interstitial epithelial transformation (Mesenchymal-epithelial transition, MET). Meanwhile, we also found that TGF- beta inhibited the EMT induced by TSP50 and inhibited the activation of AKT and ERK. This results suggest that there is a complex signal regulation network in the process of regulating cell occurrence by TSP50 and TGF- beta. This study confirmed the proto oncogene TSP. 50 can induce the occurrence of EMT, and preliminarily discuss the molecular mechanism of TSP50 promoting EMT and the complex relationship between TGF- beta and TSP50 during the occurrence of EMT from the angle of intracellular signal, thus laying an experimental foundation for the comprehensive elucidation of the role and mechanism of the proto oncogene TSP50 in the occurrence and development of the tumor.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.23

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