TGF-β1在恶性黑素瘤中对SKP2的调控机制及生物学功能研究

发布时间：2017-12-27 23:24

本文关键词：TGF-β1在恶性黑素瘤中对SKP2的调控机制及生物学功能研究　出处：《西安交通大学》2017年博士论文　论文类型：学位论文

【摘要】：恶性黑素瘤严重威胁着人类健康,由于其高侵袭、高转移、高致死率等特点,患者的平均生存期只有8-18个月。研究显示,遗传因素和环境因素是恶性黑素瘤发病的主要原因,其中伴随着多种癌基因信号通路的活化和抑癌基因的失活,如RAS/RAF/MAPK通路、PI3K/Akt通路等。尽管黑素瘤发生的机制已经逐渐为人们所认识,但是现有的临床治疗策略仍不能取得满意的效果。因此,寻找新的分子靶点对于恶性黑素瘤的早期诊断和靶向治疗均具有重要意义。E3连接酶SKP2和TGF-β1信号通路在肿瘤的转移过程中都发挥重要作用,但是机制却不尽相同。TGF-β1通过诱导肿瘤细胞发生上皮细胞向间质细胞转换的过程(EMT),从而促进肿瘤的转移;而SKP2则通过降解细胞周期抑制剂p27 kip1促进肿瘤的生长。最近的研究发现,c-Myc和Akt1都在肿瘤转移过程中发挥重要作用。我们在本篇研究中证明这些信号通路的相互作用以及相互影响。本研究的主要目的:1.探讨TGF-β1对黑素瘤EMT过程的调节以及对细胞侵袭和转移的影响;2.明确TGF-β1调控SKP2的机制;3.研究Akt通路和c-Myc是否参与TGF-β1对SKP2的调控;4.分析SKP2和c-Myc表达对黑素瘤发生发展的临床意义。本研究所取得的实验结果:1.TGF-β1刺激黑素瘤细胞促进SKP2的表达并诱导EMT的发生我们首先创建TGF-β1(10 ng/ml)刺激A375和SK-MEL-28两株黑素瘤细胞发生EMT的细胞模型。在EMT发生的过程中,其上皮标志分子E-cadherin的表达水平降低,间质标志分子N-cadherin/snail/Fironectin的表达水平升高。与此同时,SKP2的蛋白质水平、m RNA水平和启动子相对活性都表达升高。2.TGF-β1通过激活Akt信号通路诱导SKP2的表达TGF-β1诱导细胞发生EMT过程中,SKP2的表达上调,Akt1的磷酸化水平也相应增加。我们首先通过si RNA干涉Akt1,发现SKP2的启动子活性,m RNA水平和蛋白水平均显著下调。进一步,通过过表达Akt1和豆蔻酰化的Akt1,发现SKP2的表达显著上调。我们证明Akt1在TGF-β1诱导SKP2表达增加的过程中是必要条件。3.TGF-β1通过c-Myc诱导SKP2转录和表达TGF-β1诱导黑素瘤细胞发生EMT过程中,c-Myc的表达量相应增加。我们通过过表达或干涉转录因子c-Myc,发现SKP2的表达也相应的升高或降低。同时,我们使用了两种c-Myc抑制剂,发现SKP2的表达降低。最后,我们又分析了SKP2的启动子,发现有四个c-Myc的潜在结合位点。通过多种实验方法证明,c-Myc直接结合到SKP2的启动子,从而促进SKP2的转录。4.c-Myc和SKP2在黑素瘤组织中的相关性分析为了进一步证明以上的结果,我们在25例人体病例标本中对c-Myc、SKP2表达与肿瘤的恶性程度做了相关性分析,其中包括正常皮肤、黑色素痣以及恶性黑素瘤。结果显示,c-Myc和SKP2的表达存在正相关。此外,SKP2和c-Myc在转移的恶性黑素瘤标本中表达最高,在原位的恶性黑素瘤标本中表达次之,在正常的皮肤和黑色素痣中表达最低。我们的结果证明,SKP2在TGF-β1诱导黑素瘤细胞发生EMT的过程中发挥重要作用。TGF-β1通过激活Akt信号通路以及转录因子c-Myc的转录调节能力,上调了SKP2的表达,进而诱导黑素瘤细胞发生EMT。本研究不仅丰富了TGF-β1的信号调节网络,而且发现了SKP2除了通过调节p21和p27的表达影响细胞周期外,还可以参与对细胞EMT过程的调控,揭示了SKP2的新功能并初步探讨了相关机制。
[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.
【学位授予单位】：西安交通大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.5

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