本文選題：TSP50 + EMT　； 參考：《東北師范大學(xué)》2017年碩士論文

【摘要】：睪丸特異性蛋白酶50(Testes-specific protease 50,TSP50)是一種蘇氨酸蛋白酶,在腫瘤的發(fā)生和發(fā)展中扮演重要角色。以往人們對TSP50的研究主要集中于其對細(xì)胞增殖和轉(zhuǎn)移等細(xì)胞生物學(xué)特性的影響,而關(guān)于TSP50在上皮間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)發(fā)生發(fā)展中的作用研究尚未見報(bào)道。本研究主要探討了TSP50在EMT中的作用,并對其機(jī)制進(jìn)行了初步研究。1.不同細(xì)胞系中TSP50和EMT標(biāo)志物的表達(dá)分析為了確定TSP50對EMT的影響,我們首先利用RT-PCR和Western blot方法分析了不同細(xì)胞系中TSP50和EMT相關(guān)標(biāo)志物的表達(dá)情況。結(jié)果發(fā)現(xiàn),在高表達(dá)TSP50的細(xì)胞中EMT間質(zhì)標(biāo)志物Vimentin的表達(dá)量亦較高,而上皮標(biāo)志物E-cadherin的表達(dá)則較低;但是在低表達(dá)TSP50的細(xì)胞中EMT相關(guān)標(biāo)志物的表達(dá)量則正好相反。此結(jié)果提示,TSP50和EMT可能呈正相關(guān)。2.TSP50和EMT的相關(guān)性研究(1)過表達(dá)TSP50對EMT標(biāo)志物表達(dá)的影響為了驗(yàn)證我們的假設(shè),我們通過過表達(dá)或敲低TSP50表達(dá)的方法探討了TSP50的表達(dá)量與EMT之間的相關(guān)性。我們首先在低表達(dá)TSP50的MCF-10A細(xì)胞中過表達(dá)TSP50,利用Western blot實(shí)驗(yàn)分析了過表達(dá)TSP50對細(xì)胞EMT相關(guān)標(biāo)志物表達(dá)的影響。結(jié)果顯示,過表達(dá)TSP50可以使細(xì)胞中EMT間質(zhì)表型標(biāo)志物N-cadherin和Vimentin的表達(dá)上調(diào),EMT上皮表型標(biāo)志物E-cadherin的表達(dá)下調(diào)。為了探討TSP50對EMT的影響是否具有劑量依賴效應(yīng),我們在內(nèi)源高表達(dá)TSP50的MDA-MB-231細(xì)胞中的進(jìn)一步過表達(dá)TSP50,之后檢測EMT標(biāo)志物的表達(dá)情況。結(jié)果顯示,隨著TSP50的表達(dá)增高,EMT間質(zhì)表型標(biāo)志物N-cadherin和Vimentin的表達(dá)進(jìn)一步上調(diào),EMT上皮表型標(biāo)志物E-cadherin的表達(dá)則進(jìn)一步下調(diào)。以上結(jié)果提示,TSP50可促進(jìn)EMT,且此促進(jìn)作用具有TSP50表達(dá)量依賴性。(2)敲低TSP50的表達(dá)對EMT標(biāo)志物表達(dá)的影響為了進(jìn)一步證實(shí)以上結(jié)果,我們在高表達(dá)TSP50的MDA-MB-231細(xì)胞中通過RNAi方法敲低TSP50的表達(dá),之后利用Western blot方法分析了EMT相關(guān)標(biāo)志物的表達(dá)情況。結(jié)果顯示,敲低TSP50的表達(dá)可以使細(xì)胞中EMT間質(zhì)標(biāo)志物N-cadherin和Vimentin的表達(dá)下調(diào),EMT上皮標(biāo)志物E-cadherin的表達(dá)上調(diào)。此結(jié)果進(jìn)一步證明了TSP50對EMT的促進(jìn)作用。(3)TSP50對細(xì)胞侵襲性的影響細(xì)胞發(fā)生EMT后會(huì)具有一定的侵襲性,因此我們探討了TSP50對細(xì)胞侵襲性的影響。Transwell實(shí)驗(yàn)結(jié)果顯示,過表達(dá)TSP50可以增強(qiáng)細(xì)胞的侵襲能力,反之敲低TSP50則會(huì)抑制細(xì)胞的侵襲能力。綜上所述,我們發(fā)現(xiàn)TSP50的表達(dá)量與EMT的發(fā)生呈正相關(guān)。3.TSP50促進(jìn)EMT的機(jī)制研究為了探究TSP50影響EMT的分子機(jī)制。我們通過過表達(dá)或敲低TSP50表達(dá)的方法探討了TSP50對細(xì)胞內(nèi)信號(hào)通路的影響。Western blot結(jié)果顯示,過表達(dá)TSP50后磷酸化的IKK和IκB的水平上升,p65的入核增加;同時(shí)磷酸化的AKT和ERK1/ERK2水平亦明顯增加;但是Smad2/3的入核則明顯降低。敲低細(xì)胞中TSP50的表達(dá)則Smad2/3的入核明顯增加,而磷酸化的AKT和ERK1/2水平則明顯降低。以上結(jié)果說明,TSP50可以激活NF-kB、AKT及ERK/MAPK信號(hào),同時(shí)抑制TGF-β信號(hào)。為了進(jìn)一步探討這些信號(hào)與TSP50所誘導(dǎo)的EMT的相關(guān)性,我們在過表達(dá)TSP50的細(xì)胞中利用抑制劑分別阻斷了NF-kB、ERK/MAPK及AKT信號(hào),之后檢測了EMT相關(guān)標(biāo)志物的表達(dá)變化。結(jié)果顯示,抑制NF-kB、AKT及ERK/MAPK信號(hào)均可抑制由過表達(dá)TSP50所誘導(dǎo)的EMT。我們又在過表達(dá)TSP50的細(xì)胞中過表Smad2/3,之后檢測了EMT相關(guān)標(biāo)志物的表達(dá)變化。結(jié)果顯示,過表達(dá)Smad可以抑制由過表達(dá)TSP50所誘導(dǎo)的EMT。以上結(jié)果提示,NF-kB、AKT、ERK/MAPK和TGF-β信號(hào)均參與了TSP50所誘導(dǎo)的EMT。4.TGF-β與TSP50在誘導(dǎo)EMT過程中的相互作用研究因?yàn)樵谥暗膱?bào)道中,TGF-β可以刺激乳腺上皮細(xì)胞發(fā)生EMT,而我們的研究則顯示,TSP50可以抑制TGF-β信號(hào),但可誘導(dǎo)EMT,這些矛盾的結(jié)果促使我們進(jìn)一步探討了TGF信號(hào)與TSP50所誘導(dǎo)的EMT之間的相關(guān)性。我們用TGF-β刺激過表達(dá)TSP50的細(xì)胞,發(fā)現(xiàn)長時(shí)間的刺激會(huì)引起由TSP50誘導(dǎo)的EMT細(xì)胞發(fā)生間質(zhì)上皮轉(zhuǎn)化(Mesenchymal-epithelial transition,MET)。同時(shí),我們還發(fā)現(xiàn),TGF-β在抑制TSP50所誘導(dǎo)的EMT的同時(shí),也抑制了AKT和ERK的激活。此結(jié)果提示,TSP50和TGF-β在調(diào)控細(xì)胞發(fā)生EMT的過程中存在著復(fù)雜的信號(hào)調(diào)控網(wǎng)絡(luò)。本研究證實(shí)了原癌基因TSP50可以誘導(dǎo)EMT的發(fā)生,并從細(xì)胞內(nèi)信號(hào)角度初步探討了TSP50促進(jìn)EMT的分子機(jī)制以及TGF-β與TSP50在EMT發(fā)生過程中的復(fù)雜作用關(guān)系,從而為全面闡明原癌基因TSP50在腫瘤發(fā)生及發(fā)展中的作用及機(jī)制奠定實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.23

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本文編號(hào)：2085298