本文關(guān)鍵詞：TGF-β1在惡性黑素瘤中對SKP2的調(diào)控機(jī)制及生物學(xué)功能研究　出處：《西安交通大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Akt1 c-Myc 上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)換 SKP2 惡性黑素瘤 TGF-β1

【摘要】：惡性黑素瘤嚴(yán)重威脅著人類健康,由于其高侵襲、高轉(zhuǎn)移、高致死率等特點(diǎn),患者的平均生存期只有8-18個(gè)月。研究顯示,遺傳因素和環(huán)境因素是惡性黑素瘤發(fā)病的主要原因,其中伴隨著多種癌基因信號通路的活化和抑癌基因的失活,如RAS/RAF/MAPK通路、PI3K/Akt通路等。盡管黑素瘤發(fā)生的機(jī)制已經(jīng)逐漸為人們所認(rèn)識,但是現(xiàn)有的臨床治療策略仍不能取得滿意的效果。因此,尋找新的分子靶點(diǎn)對于惡性黑素瘤的早期診斷和靶向治療均具有重要意義。E3連接酶SKP2和TGF-β1信號通路在腫瘤的轉(zhuǎn)移過程中都發(fā)揮重要作用,但是機(jī)制卻不盡相同。TGF-β1通過誘導(dǎo)腫瘤細(xì)胞發(fā)生上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)換的過程(EMT),從而促進(jìn)腫瘤的轉(zhuǎn)移;而SKP2則通過降解細(xì)胞周期抑制劑p27 kip1促進(jìn)腫瘤的生長。最近的研究發(fā)現(xiàn),c-Myc和Akt1都在腫瘤轉(zhuǎn)移過程中發(fā)揮重要作用。我們在本篇研究中證明這些信號通路的相互作用以及相互影響。本研究的主要目的:1.探討TGF-β1對黑素瘤EMT過程的調(diào)節(jié)以及對細(xì)胞侵襲和轉(zhuǎn)移的影響;2.明確TGF-β1調(diào)控SKP2的機(jī)制;3.研究Akt通路和c-Myc是否參與TGF-β1對SKP2的調(diào)控;4.分析SKP2和c-Myc表達(dá)對黑素瘤發(fā)生發(fā)展的臨床意義。本研究所取得的實(shí)驗(yàn)結(jié)果:1.TGF-β1刺激黑素瘤細(xì)胞促進(jìn)SKP2的表達(dá)并誘導(dǎo)EMT的發(fā)生我們首先創(chuàng)建TGF-β1(10 ng/ml)刺激A375和SK-MEL-28兩株黑素瘤細(xì)胞發(fā)生EMT的細(xì)胞模型。在EMT發(fā)生的過程中,其上皮標(biāo)志分子E-cadherin的表達(dá)水平降低,間質(zhì)標(biāo)志分子N-cadherin/snail/Fironectin的表達(dá)水平升高。與此同時(shí),SKP2的蛋白質(zhì)水平、m RNA水平和啟動(dòng)子相對活性都表達(dá)升高。2.TGF-β1通過激活A(yù)kt信號通路誘導(dǎo)SKP2的表達(dá)TGF-β1誘導(dǎo)細(xì)胞發(fā)生EMT過程中,SKP2的表達(dá)上調(diào),Akt1的磷酸化水平也相應(yīng)增加。我們首先通過si RNA干涉Akt1,發(fā)現(xiàn)SKP2的啟動(dòng)子活性,m RNA水平和蛋白水平均顯著下調(diào)。進(jìn)一步,通過過表達(dá)Akt1和豆蔻酰化的Akt1,發(fā)現(xiàn)SKP2的表達(dá)顯著上調(diào)。我們證明Akt1在TGF-β1誘導(dǎo)SKP2表達(dá)增加的過程中是必要條件。3.TGF-β1通過c-Myc誘導(dǎo)SKP2轉(zhuǎn)錄和表達(dá)TGF-β1誘導(dǎo)黑素瘤細(xì)胞發(fā)生EMT過程中,c-Myc的表達(dá)量相應(yīng)增加。我們通過過表達(dá)或干涉轉(zhuǎn)錄因子c-Myc,發(fā)現(xiàn)SKP2的表達(dá)也相應(yīng)的升高或降低。同時(shí),我們使用了兩種c-Myc抑制劑,發(fā)現(xiàn)SKP2的表達(dá)降低。最后,我們又分析了SKP2的啟動(dòng)子,發(fā)現(xiàn)有四個(gè)c-Myc的潛在結(jié)合位點(diǎn)。通過多種實(shí)驗(yàn)方法證明,c-Myc直接結(jié)合到SKP2的啟動(dòng)子,從而促進(jìn)SKP2的轉(zhuǎn)錄。4.c-Myc和SKP2在黑素瘤組織中的相關(guān)性分析為了進(jìn)一步證明以上的結(jié)果,我們在25例人體病例標(biāo)本中對c-Myc、SKP2表達(dá)與腫瘤的惡性程度做了相關(guān)性分析,其中包括正常皮膚、黑色素痣以及惡性黑素瘤。結(jié)果顯示,c-Myc和SKP2的表達(dá)存在正相關(guān)。此外,SKP2和c-Myc在轉(zhuǎn)移的惡性黑素瘤標(biāo)本中表達(dá)最高,在原位的惡性黑素瘤標(biāo)本中表達(dá)次之,在正常的皮膚和黑色素痣中表達(dá)最低。我們的結(jié)果證明,SKP2在TGF-β1誘導(dǎo)黑素瘤細(xì)胞發(fā)生EMT的過程中發(fā)揮重要作用。TGF-β1通過激活A(yù)kt信號通路以及轉(zhuǎn)錄因子c-Myc的轉(zhuǎn)錄調(diào)節(jié)能力,上調(diào)了SKP2的表達(dá),進(jìn)而誘導(dǎo)黑素瘤細(xì)胞發(fā)生EMT。本研究不僅豐富了TGF-β1的信號調(diào)節(jié)網(wǎng)絡(luò),而且發(fā)現(xiàn)了SKP2除了通過調(diào)節(jié)p21和p27的表達(dá)影響細(xì)胞周期外,還可以參與對細(xì)胞EMT過程的調(diào)控,揭示了SKP2的新功能并初步探討了相關(guān)機(jī)制。
[Abstract]:Malignant melanoma is a serious threat to human health. Due to its high invasion, high metastasis and high mortality, the average survival time of the patients is only 8-18 months. Studies have shown that genetic factors and environmental factors are the main causes of malignant melanoma, which is accompanied by activation of many oncogene signaling pathways and inactivation of tumor suppressor genes, such as RAS/RAF/MAPK pathway and PI3K/Akt pathway. Although the mechanism of the occurrence of melanoma has gradually been recognized, the existing clinical treatment strategies can not achieve satisfactory results. Therefore, it is of great significance to find new molecular targets for the early diagnosis and target treatment of malignant melanoma. E3 ligase SKP2 and TGF- beta 1 signaling pathways play an important role in tumor metastasis, but the mechanisms are not the same. TGF- beta 1 promotes tumor metastasis by inducing the transformation process of epithelial cells to stromal cells (EMT), while SKP2 promotes tumor growth by degrading cell cycle inhibitor p27 kip1. Recent studies have found that both c-Myc and Akt1 play an important role in tumor metastasis. In this study, we demonstrate the interaction and interaction of these signaling pathways. The main purpose of this study: 1. to investigate the regulation of TGF- beta 1 on melanoma EMT and the process of metastasis and invasion of cells; 2. TGF- beta 1 clear mechanism of regulation of SKP2; 3. of the Akt pathway and whether c-Myc is involved in regulation of TGF- beta 1 on SKP2; 4. analysis of SKP2 and c-Myc on the expression and clinical significance of melanoma. The development of the. The results obtained in this study are as follows: 1.TGF- beta 1 stimulates the expression of SKP2 and induces the occurrence of EMT in melanoma cells. We first create TGF- TGF- 1 (10 ng/ml) to stimulate A375 and SK-MEL-28 to form EMT cell models of melanoma cells. In the process of EMT, the expression level of the epithelial marker molecule E-cadherin is reduced, and the expression level of the interstitial marker molecule N-cadherin/snail/Fironectin is increased. At the same time, the protein level of SKP2, the level of M RNA and the relative activity of promoter increased. 2.TGF- beta 1 induces the expression of SKP2 through activating Akt signaling pathway, and SKP2 expression is upregulated, and the level of Akt1 phosphorylation is increased as well as EMT expression is induced by TGF- beta 1. We first detected the promoter activity of SKP2 by interfering with Akt1 by Si RNA, and the m RNA level and protein level were significantly down regulated. Further, through the expression of Akt1 and myrimeylation of Akt1, the expression of SKP2 was significantly up-regulated. We have demonstrated that Akt1 is a necessary condition in the process of increasing the expression of SKP2 in TGF- beta 1. 3.TGF- beta 1 increases the expression of c-Myc during the induction of SKP2 transcription by c-Myc and the expression of TGF- beta 1 in the induction of EMT in melanoma cells. By expressing or interfering with the transcription factor c-Myc, we found that the expression of SKP2 was also increased or reduced accordingly. At the same time, we used two kinds of c-Myc inhibitors, and found that the expression of SKP2 was reduced. Finally, we analyzed the promoter of SKP2 and found that there were four c-Myc potential binding sites. Through a variety of experimental methods, it is proved that c-Myc is directly combined with the promoter of SKP2, thus promoting the transcription of SKP2. The correlation between 4.c-Myc and SKP2 in melanoma tissues analysis in order to prove the above results, we malignant degree on the expression of c-Myc and SKP2 in 25 cases of human samples and tumor with the correlation analysis, including normal skin, melanocytic nevi and malignant melanoma. The results showed that there was a positive correlation between the expression of c-Myc and SKP2. In addition, SKP2 and c-Myc expressed the highest level in metastatic malignant melanoma specimens. The expression level was the second in the malignant melanoma specimens in situ, and the lowest in normal skin and melanocytic nevus. Our results show that SKP2 plays an important role in the induction of EMT by TGF- beta 1 in melanoma cells. TGF- beta 1 up-regulated the expression of SKP2 by activating the Akt signaling pathway and the transcriptional regulation of the transcription factor c-Myc, thus inducing EMT in the melanoma cells. This study not only enriched the signal regulatory network of TGF- beta 1, but also found that SKP2 could regulate the regulation of EMT process through regulating the expression of p21 and p27, revealing the new function of SKP2 and preliminarily exploring the related mechanisms.
【學(xué)位授予單位】：西安交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R739.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Relation of overexpression of S phase kinase-associated protein 2 with reduced expression of p27 and PTEN in human gastric carcinoma[J];World Journal of Gastroenterology;2005年42期

，

本文編號：1343636