SARM在肾癌细胞中的功能及其分子机制的初步研究
发布时间：2018-08-29 14:59
【摘要】：肾细胞癌(renal cell carcinoma,RCC)是起源于肾小管上皮的恶性肿瘤,占肾脏恶性肿瘤的80%-90%,是成人泌尿系统常见的恶性肿瘤之一[1]。肾细胞癌的生物学行为及其特征较为复杂,其发生发展的机制尚未完全明确。目前,外科手术是根治肾细胞癌的主要方法,约有1/3的肾细胞癌患者初次就诊时就已发现有转移,无法接受根治性手术治疗[2],约有1/3的局限性肾癌患者行根治性手术后仍会出现远处转移[3],对于局限性肾癌患者,行根治性肾切除术或保留肾单位手术后,约90%以上患者能获得五年无病生存。同时,转移性肾癌(metastatic renal cell carcinoma,mRCC)对传统放化疗均不敏感,其5年生存率小于10%[4]。因此,探索肾细胞癌发生、发展的分子机制,寻找有效的生物学标记物,已成为肾细胞癌研究的热点,以便为肾细胞癌的生物治疗提供新的靶点和新思路。近年来分子靶向药物治疗成为肿瘤治疗的热点,并已在多种肿瘤的治疗中获得了重大进展,已成为抗肿瘤治疗的重要组成部分[5]。分子靶向治疗是以肿瘤细胞中过度表达的某些标志性大分子物质作为靶点,利用药物阻断这些大分子物质相关的信号转导路径,从而得到控制肿瘤的生长、进展及转移等作用。研究发现缺氧诱导因子(hypoxia-inducible factor, HIF)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、血小板衍生生长因子(platelet-derived growth factor, PDGF)、表皮生长因子(epidermal growth factor, EGF)及其受体(EGFR)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)等均为分子靶向治疗的重要靶向分子。这些分子的异常表达与肾癌特别是肾透明细胞癌的预后有着密切关系,使之成为肾细胞癌分子靶向治疗的基础和作用靶点。RCC分子靶向治疗中最为重要的分子信号通路主要包括VEGF通路和mTOR通路。VEGF是肿瘤诱导产生新生血管的关键细胞因子,研究发现在肾细胞癌组织中VEGF、VEGFR-1、VEGFR-2mRNA异常高度表达,同时肿瘤组织中的微血管密度显著大于正常肾组织[6,7]。VEGF与其受体结合后可激活细胞内信号传导通路,包括PI3K/Akt/mTOR和Ras/Raf/丝裂原激活蛋白激酶的激酶(mitogen activated protein kinase kinase,MAPKK)/细胞外信号调节激酶(extracellular signal regulated kinase, ERK)等信号通路,促进内皮细胞的增生和分化,进而广泛参与肾癌的生长、增殖、分化等过程。SARM(Sterile-alpha and HEAT/Armadillo motif containing protein)是 TLR(Toll-like receptor)信号通路中含有 TIR (Toll/interleukin-l receptor)结构域的五个接头蛋白之一,其在进化上非常保守,从线虫、果蝇、文昌鱼到哺乳动物都有类似结构的分子[8]。目前对SARM的功能研究发现,SARM是TLR信号通路中依赖 TRIF (TIR domain-containing adaptor inducing IFN- β , TRIF)的负性调控蛋白,在天然免疫应答和炎症发应中发挥重要作用[9-11];同时,有研究发现,SARM在压力条件下能促进小鼠神经元细胞凋亡[12]。但是目前尚无关于SARM在肿瘤发生、发展作用的研究报道,对SARM在肾细胞癌中的表达水平、是否参与调控肾细胞癌的发生、发展及其分子机制目前仍尚不清楚。因此对SARM在肾癌细胞中的功能及其分子机制的研究将是非常有意义的工作,其研究结果将为肾癌的分子靶向治疗提供新的靶点。研究目的本研究将通过体外体内实验,以肾癌细胞株、BALB/C-nu裸鼠移植瘤和肾癌临床组织标本为研究模型,检验SARM在肾癌组织和肾癌细胞株与癌旁正常肾组织和肾小管上皮细胞的表达水平有无差异,探讨SARM在肾癌细胞的生长的作用功能、诱导细胞自噬发生的作用,同时观察分子靶向药物舒尼替尼与SARM的相互作用,并从分子水平初步探讨SARM调控肾癌细胞生长的可能机制。以期明确SARM对肾癌细胞的生长的调控作用及其可能分子机制,为肾癌的分子靶向治疗药物的研究提供新的靶点和新的思路。研究方法一、临床标本和细胞株研究:提取肾透明细胞癌组织和癌旁正常肾组织、正常肾小管上皮细胞(HK-2)和肾癌细胞株(786-O、OS-RC-2)的蛋白和RNA,利用免疫印迹法和半定量PCR技术观察SARM在正常肾组织和细胞与肾透明细胞癌组织和细胞中的蛋白水平和RNA表达水平的差异。二、体外实验研究:本研究采用细胞转染和慢病毒包装感染方法构建了786-O和OS-RC-2稳定表达SARM的肾癌细胞株,通过细胞计数法、平板克隆形成实验检测了稳定表达SARM对肾癌细胞生长和增殖的影响;采用PI染色和Annexin V-PE/7AAD双染色、利用流式细胞仪观察稳定表达SARM对肾癌细胞周期进行和细胞凋亡的影响;应用分子靶向药物舒尼替尼处理肾癌细胞,观察分子靶向药物与SARM的相互作用;采用无血清饥饿方法诱导稳定表达SARM肾癌细胞发生自噬,探讨SARM与自噬的关系。三、体内试验研究:通过OS-RC-2稳定表达SARM肾癌细胞构建肾癌BALB/C-nu裸鼠移植瘤模型,检测稳定表达SARM后对肾癌裸鼠移植瘤生长曲线及荷瘤体积有无影响。四、分子机制的初步探讨:利用免疫印迹法研究稳定表达SARM对PI3K/Akt/mTOR信号通路及ERK/MAPK信号通路影响,探讨稳定表达SARM对肾癌细胞生长、增殖、周期进行、凋亡的调控及诱导肾癌细胞自噬发生的可能分子机制。五、统计学方法:每组实验均重复三次,用平均数土标准差进行统计学描述,根据实验设计分别采用两独立样本t检验(Independent sample t test)、重复测量数据的方差分析、析因设计资料的方差分析进行组间差异比较。实验数据采用IBM SPSS19.0软件进行上述统计学分析,P 0.05则表示为具有统计学意义。研究结果一、SARM在肾透明细胞癌组织蛋白表达水平降低肾透明细胞癌组织和肾癌细胞株中SARM的RNA水、平较癌旁正常肾组织和肾小管上皮细胞显著升高,但是在蛋白表达水平上较癌旁正常肾组织和细胞株显著减少,采用蛋白酶体抑制剂MG132抑制泛素发生后,SARM的蛋白水平明显升高,说明SARM在从RNA翻译到蛋白合成可能由于发生泛素化修饰,导致其在蛋白水平表达低下。二、成功构建稳定表达SARM细胞株通过X-tremeGENE HP DNA Transfection Reagen转染和慢病毒包装感染方法、流式细胞仪分选单克隆细胞成功构建了稳定表达SARM的786-0和OS-RC-2的细胞株,免疫印迹法对GFP-SARM蛋白表达水平进行验证。三、稳定表达SARM抑制了肾癌细胞的生长和增殖细胞计数法结果显示,与GFP对照组相比,稳定表达SARM后显著抑制了肾癌细胞的生长(P0.05),同时平板克隆形成实验结果发现稳定表达SARM对肾癌细胞的增殖有显著抑制作用(P0.05)。四、稳定表达SARM阻滞细胞周期进行和促进细胞死亡流式细胞仪检测发现,与GFP对照组相比,稳定表达SARM能够阻滞肾癌细胞从G1期进入S期,同时稳定表达SARM的肾癌细胞凋亡增多,说明SARM能阻滞细胞周期的进行、促进肾癌细胞死亡(P0.05)。五、SARM可促进舒尼替尼诱导肾癌细胞发生自噬免疫印迹法检测发现舒尼替尼可以增强SARM在肾癌细胞株的表达水平和诱导肾癌细胞自噬发生。稳定表达SARM可以促进肾癌细胞发生自噬,抑制SARM后可减弱舒尼替尼诱导肾癌细胞自噬发生,说明SARM在调控舒尼替尼诱导肾癌细胞自噬的发生中发挥重要作用。六、稳定表达SARM可抑制肾癌裸鼠移植瘤的生长将稳定表达SARM的OS-RC-2细胞株接种于4周龄的雌性BALB/C-nu裸鼠皮下,观察裸鼠移植瘤生长曲线,荷瘤生长21天后处死裸鼠收获荷瘤,发现稳定表达SARM抑制了裸鼠移植瘤的生长,其瘤块体积较GFP对照组显著减小(P0.05)。七、稳定表达SARM诱导肾癌细胞发生自噬通过无血清饥饿法诱导稳定表达SARM细胞株发生自噬,免疫印迹法检测LC-3和p62蛋白表达水平。与对照组相比,稳定表达SARM后LC-3蛋白表达水平显著升高,p62蛋白表达显著降低。相反,干扰SARM后,LC-3蛋白水平显著降低。说明SARM能诱导肾癌细胞发生自噬。八、稳定表达SARM抑制肾癌细胞PI3K/Akt/mTOR信号通路免疫印迹法检测发现,稳定表达SARM后,Akt、mTOR和p70S6K的磷酸化水平降低,说明稳定表达SARM能抑制PI3K/Akt/mTOR信号通路。九、稳定表达SARM抑制ERK信号通路免疫印迹法检测发现稳定表达SARM后,与对照组相比,磷酸化ERK1/2的表达水平显著降低,表明稳定表达SARM能抑制ERK信号通路。结论本研究首次研究SARM在肿瘤组织和细胞中的表达水平,首次研究并发现SARM对肾癌细胞生长的抑制作用及SARM可诱导肾癌细胞发生自噬,并初步探讨了 SARM调控肾癌生长和诱导肾癌细胞发生自噬的初步分子机制:SARM可能通过抑制P13K/Akt/mTOR信号通路和抑制ERK信号通路参与调控肾细胞癌的生长,SARM诱导的肾细胞癌细胞的自噬可能通过PI3K/Akt/mTOR信号通路。同时发现SARM可能是多靶点酪氨酸激酶抑制剂舒尼替尼的作用靶点,SARM参与调控舒尼替尼诱导肾癌细胞的自噬。SARM可作为肾癌分子靶向治疗的一个新的靶点,为肾癌分子靶向治疗研究提供了一个新的靶点和新的思路。
[Abstract]:Renal cell carcinoma (RCC) is a malignant tumor originating from renal tubular epithelium, accounting for 80% - 90% of renal malignancies. It is one of the common malignant tumors in adult urinary system. About 1/3 of the patients with RCC had metastasis at the time of their first visit and were unable to undergo radical surgery. About 1/3 of the patients with localized RCC still had distant metastasis after radical surgery. For localized RCC patients, radical nephrectomy or nephron-sparing surgery was performed, and about 90% of them had distant metastasis. At the same time, metastatic renal cell carcinoma (mRCC) is insensitive to conventional radiotherapy and chemotherapy, and its 5-year survival rate is less than 10%[4]. In recent years, molecular targeted drug therapy has become a hotspot in tumor therapy, and has made great progress in the treatment of many kinds of tumors. It has become an important part of anti-tumor therapy [5]. Hypoxia-inducible factor (HIF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (p) have been found in the study. Latlet-derived growth factor (PDGF), epidermal growth factor (EGF) and its receptor (EGFR), mammalian target of rapamycin (mTOR) are all important target molecules for molecular targeted therapy. Abnormal expression of these molecules and the prognosis of renal cell carcinoma, especially clear cell renal cell carcinoma (RCC) are important. The most important molecular signaling pathways in RCC molecular targeted therapy include VEGF pathway and mTOR pathway. Vascular density in tumor tissues was significantly higher than that in normal kidney tissues [6,7]. Vascular growth factor binding to its receptor activates intracellular signal transduction pathways, including PI3K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase (MAPKK)/extracellular signal-regulated kinase (extrace). Llular signal regulated kinase (ERK) and other signaling pathways promote the proliferation and differentiation of endothelial cells, and thus participate in the growth, proliferation and differentiation of renal cell carcinoma. SARM (Sterile-alpha and HEAT/Armadillo motif containing protein) is a TLR (Toll-like receptor) signaling pathway containing TIR (Toll/interleukin-l receptor) structure. One of the five adaptor proteins of the TLR domain, which is evolutionarily conserved, has similar structure molecules from nematodes, fruit flies, amphioxus to mammals [8]. Answer and inflammatory response play an important role [9-11]; at the same time, some studies have found that SARM can promote the apoptosis of neurons in mice under pressure [12]. However, there are no reports about the role of SARM in tumorigenesis and development. The molecular mechanism of SARM in renal cell carcinoma is still unclear. Therefore, it is very meaningful to study the function and molecular mechanism of SARM in renal cell carcinoma. The results will provide a new target for molecular targeted therapy of renal cell carcinoma. To study the expression of SARM in renal cell carcinoma tissue and renal cell line, normal renal tissue and renal tubular epithelial cells adjacent to cancer, and to explore the role of SARM in the growth of renal cell carcinoma cells and the role of inducing autophagy, and to observe the interaction of molecular targeted drug sunitinib with SARM. In order to clarify the regulation of SARM on the growth of renal cell carcinoma and its possible molecular mechanism, and to provide new targets and new ideas for the research of molecular targeted therapy drugs for renal cell carcinoma. Protein and RNA expression in normal renal tissues and adjacent normal renal tissues, normal renal tubular epithelial cells (HK-2) and renal carcinoma cell lines (786-O, OS-RC-2) were detected by immunoblotting and semi-quantitative PCR. 2. In vitro Experimental study: In this study, 786-O and OS-RC-2 renal cell lines stably expressing SARM were constructed by cell transfection and lentiviral package infection. The effects of stably expressing SARM on the growth and proliferation of renal cell carcinoma cells were detected by cell counting and plate cloning assay. PI staining and Annexin V-PE/7AAD double staining were used, and flow cytometry was used. The effects of stable expression of SARM on cell cycle progression and apoptosis of renal cell carcinoma cells were observed. Molecular targeting drug sunitinib was used to treat renal cell carcinoma cells and the interaction between molecular targeting drugs and SARM was observed. Serum-free starvation was used to induce autophagy of renal cell carcinoma cells stably expressing SARM, and the relationship between SARM and autophagy was discussed. Study: The BALB/C-nu nude mice model of renal cell carcinoma was established by OS-RC-2 stably expressing SARM cells. The effects of stable expression of SARM on the growth curve and tumor-bearing volume of transplanted renal cell carcinoma in nude mice were detected. Fourthly, the preliminary study of molecular mechanism: The PI3K/Akt/mTOR signaling pathway and ERK/MAPK signaling were studied by Western blot. To explore the possible molecular mechanism of stably expressing SARM on the growth, proliferation, cycle progression, apoptosis regulation and autophagy of renal cell carcinoma. 5. Statistical methods: Each group of experiments were repeated three times, and were statistically described by mean soil standard deviation. According to the experimental design, two independent sample t test was used (In The experimental data were analyzed by IBM SPSS 19.0 software, and P 0.05 was statistically significant. Results 1. The expression of SARM protein in clear cell carcinoma of kidney decreased renal dialysis. The level of SARM protein in clear cell carcinoma and renal cell lines was significantly higher than that in normal renal tissues and renal tubular epithelial cells, but the level of protein expression was significantly lower than that in normal renal tissues and renal tubular epithelial cells. NA translation to protein synthesis may be due to ubiquitination modification, resulting in its low expression at protein level. 2. A stable expression of SARM cell line was successfully constructed by X-tremeGENE HP DNA Transfection Reagen transfection and lentiviral packaging infection. Flow cytometry sorted monoclonal cells successfully constructed 786-0 and OS-RC stably expressing SARM. The expression level of GFP-SARM protein was verified by Western blot. 3. The stable expression of SARM inhibited the growth and proliferation of renal cell carcinoma cells. Compared with the GFP control group, the stable expression of SARM significantly inhibited the growth of renal cell carcinoma cells (P 0.05). At the same time, the results of plate cloning showed stable expression of S. ARM significantly inhibited the proliferation of renal cell carcinoma cells (P 0.05). 4. Steady expression of SARM inhibited cell cycle and promoted cell death. Compared with GFP control group, stable expression of SARM inhibited the progression of renal cell carcinoma cells from G1 phase to S phase, and increased apoptosis of renal cell carcinoma cells stably expressing SARM, suggesting that SARM could block the apoptosis of renal cell carcinoma cells. Cell cycle progression, promote renal cell death (P 0.05). Fifthly, SARM can promote sunitinib-induced renal cell autophagy Western blot assay found that sunitinib can enhance the expression of SARM in renal cell lines and induce renal cell autophagy. Stable expression of SARM can promote renal cell autophagy, inhibit SARM. SARM plays an important role in the regulation of Sunitinib-induced autophagy. 6. Stable expression of SARM can inhibit the growth of renal cell carcinoma xenografts in nude mice. OS-RC-2 cell line stably expressing SARM was inoculated subcutaneously in 4-week-old female BALB/C-nu mice to observe the effect of SARM on the autophagy. The growth curve of transplanted tumor was observed. The stable expression of SARM inhibited the growth of transplanted tumor in nude mice, and the tumor volume was significantly smaller than that of GFP control group (P 0.05). 7. The stable expression of SARM induced autophagy of renal cancer cells. The stable expression of SARM induced autophagy of renal cancer cells by serum-free starvation induced stable expression of SARM cell lines. The expression of LC-3 and p62 protein was detected by Western blot. Compared with the control group, the expression of LC-3 protein was significantly increased and that of p62 protein was significantly decreased after stably expressing SARM. On the contrary, the expression of LC-3 protein was significantly decreased after interfering with SARM. The phosphorylation levels of Akt, mTOR and p70S6K decreased after stably expressing SARM, indicating that stably expressing SARM could inhibit PI3K/Akt/mTOR signaling pathway. Ninth, stably expressing SARM inhibiting ERK signaling pathway was detected by immunoblotting. After stably expressing SARM, the phosphorylated ERK1/2 expression level was significantly lower than that of the control group. Conclusion This study is the first to study the expression level of SARM in tumor tissues and cells. It is the first to find that SARM can inhibit the growth of renal cell carcinoma cells and induce autophagy of renal cell carcinoma cells. Initial Molecular Mechanisms: SARM may be involved in regulating the growth of renal cell carcinoma by inhibiting P13K/Akt/mTOR signaling pathway and ERK signaling pathway. SARM-induced autophagy of renal cell carcinoma cells may be mediated by PI3K/Akt/mTOR signaling pathway. SARM may be the target of Sunitinib, a multi-target tyrosine kinase inhibitor. SARM can be used as a new target for molecular targeted therapy of renal cell carcinoma, which provides a new target and a new idea for molecular targeted therapy of renal cell carcinoma.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11
本文编号：2211560
[Abstract]:Renal cell carcinoma (RCC) is a malignant tumor originating from renal tubular epithelium, accounting for 80% - 90% of renal malignancies. It is one of the common malignant tumors in adult urinary system. About 1/3 of the patients with RCC had metastasis at the time of their first visit and were unable to undergo radical surgery. About 1/3 of the patients with localized RCC still had distant metastasis after radical surgery. For localized RCC patients, radical nephrectomy or nephron-sparing surgery was performed, and about 90% of them had distant metastasis. At the same time, metastatic renal cell carcinoma (mRCC) is insensitive to conventional radiotherapy and chemotherapy, and its 5-year survival rate is less than 10%[4]. In recent years, molecular targeted drug therapy has become a hotspot in tumor therapy, and has made great progress in the treatment of many kinds of tumors. It has become an important part of anti-tumor therapy [5]. Hypoxia-inducible factor (HIF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (p) have been found in the study. Latlet-derived growth factor (PDGF), epidermal growth factor (EGF) and its receptor (EGFR), mammalian target of rapamycin (mTOR) are all important target molecules for molecular targeted therapy. Abnormal expression of these molecules and the prognosis of renal cell carcinoma, especially clear cell renal cell carcinoma (RCC) are important. The most important molecular signaling pathways in RCC molecular targeted therapy include VEGF pathway and mTOR pathway. Vascular density in tumor tissues was significantly higher than that in normal kidney tissues [6,7]. Vascular growth factor binding to its receptor activates intracellular signal transduction pathways, including PI3K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase (MAPKK)/extracellular signal-regulated kinase (extrace). Llular signal regulated kinase (ERK) and other signaling pathways promote the proliferation and differentiation of endothelial cells, and thus participate in the growth, proliferation and differentiation of renal cell carcinoma. SARM (Sterile-alpha and HEAT/Armadillo motif containing protein) is a TLR (Toll-like receptor) signaling pathway containing TIR (Toll/interleukin-l receptor) structure. One of the five adaptor proteins of the TLR domain, which is evolutionarily conserved, has similar structure molecules from nematodes, fruit flies, amphioxus to mammals [8]. Answer and inflammatory response play an important role [9-11]; at the same time, some studies have found that SARM can promote the apoptosis of neurons in mice under pressure [12]. However, there are no reports about the role of SARM in tumorigenesis and development. The molecular mechanism of SARM in renal cell carcinoma is still unclear. Therefore, it is very meaningful to study the function and molecular mechanism of SARM in renal cell carcinoma. The results will provide a new target for molecular targeted therapy of renal cell carcinoma. To study the expression of SARM in renal cell carcinoma tissue and renal cell line, normal renal tissue and renal tubular epithelial cells adjacent to cancer, and to explore the role of SARM in the growth of renal cell carcinoma cells and the role of inducing autophagy, and to observe the interaction of molecular targeted drug sunitinib with SARM. In order to clarify the regulation of SARM on the growth of renal cell carcinoma and its possible molecular mechanism, and to provide new targets and new ideas for the research of molecular targeted therapy drugs for renal cell carcinoma. Protein and RNA expression in normal renal tissues and adjacent normal renal tissues, normal renal tubular epithelial cells (HK-2) and renal carcinoma cell lines (786-O, OS-RC-2) were detected by immunoblotting and semi-quantitative PCR. 2. In vitro Experimental study: In this study, 786-O and OS-RC-2 renal cell lines stably expressing SARM were constructed by cell transfection and lentiviral package infection. The effects of stably expressing SARM on the growth and proliferation of renal cell carcinoma cells were detected by cell counting and plate cloning assay. PI staining and Annexin V-PE/7AAD double staining were used, and flow cytometry was used. The effects of stable expression of SARM on cell cycle progression and apoptosis of renal cell carcinoma cells were observed. Molecular targeting drug sunitinib was used to treat renal cell carcinoma cells and the interaction between molecular targeting drugs and SARM was observed. Serum-free starvation was used to induce autophagy of renal cell carcinoma cells stably expressing SARM, and the relationship between SARM and autophagy was discussed. Study: The BALB/C-nu nude mice model of renal cell carcinoma was established by OS-RC-2 stably expressing SARM cells. The effects of stable expression of SARM on the growth curve and tumor-bearing volume of transplanted renal cell carcinoma in nude mice were detected. Fourthly, the preliminary study of molecular mechanism: The PI3K/Akt/mTOR signaling pathway and ERK/MAPK signaling were studied by Western blot. To explore the possible molecular mechanism of stably expressing SARM on the growth, proliferation, cycle progression, apoptosis regulation and autophagy of renal cell carcinoma. 5. Statistical methods: Each group of experiments were repeated three times, and were statistically described by mean soil standard deviation. According to the experimental design, two independent sample t test was used (In The experimental data were analyzed by IBM SPSS 19.0 software, and P 0.05 was statistically significant. Results 1. The expression of SARM protein in clear cell carcinoma of kidney decreased renal dialysis. The level of SARM protein in clear cell carcinoma and renal cell lines was significantly higher than that in normal renal tissues and renal tubular epithelial cells, but the level of protein expression was significantly lower than that in normal renal tissues and renal tubular epithelial cells. NA translation to protein synthesis may be due to ubiquitination modification, resulting in its low expression at protein level. 2. A stable expression of SARM cell line was successfully constructed by X-tremeGENE HP DNA Transfection Reagen transfection and lentiviral packaging infection. Flow cytometry sorted monoclonal cells successfully constructed 786-0 and OS-RC stably expressing SARM. The expression level of GFP-SARM protein was verified by Western blot. 3. The stable expression of SARM inhibited the growth and proliferation of renal cell carcinoma cells. Compared with the GFP control group, the stable expression of SARM significantly inhibited the growth of renal cell carcinoma cells (P 0.05). At the same time, the results of plate cloning showed stable expression of S. ARM significantly inhibited the proliferation of renal cell carcinoma cells (P 0.05). 4. Steady expression of SARM inhibited cell cycle and promoted cell death. Compared with GFP control group, stable expression of SARM inhibited the progression of renal cell carcinoma cells from G1 phase to S phase, and increased apoptosis of renal cell carcinoma cells stably expressing SARM, suggesting that SARM could block the apoptosis of renal cell carcinoma cells. Cell cycle progression, promote renal cell death (P 0.05). Fifthly, SARM can promote sunitinib-induced renal cell autophagy Western blot assay found that sunitinib can enhance the expression of SARM in renal cell lines and induce renal cell autophagy. Stable expression of SARM can promote renal cell autophagy, inhibit SARM. SARM plays an important role in the regulation of Sunitinib-induced autophagy. 6. Stable expression of SARM can inhibit the growth of renal cell carcinoma xenografts in nude mice. OS-RC-2 cell line stably expressing SARM was inoculated subcutaneously in 4-week-old female BALB/C-nu mice to observe the effect of SARM on the autophagy. The growth curve of transplanted tumor was observed. The stable expression of SARM inhibited the growth of transplanted tumor in nude mice, and the tumor volume was significantly smaller than that of GFP control group (P 0.05). 7. The stable expression of SARM induced autophagy of renal cancer cells. The stable expression of SARM induced autophagy of renal cancer cells by serum-free starvation induced stable expression of SARM cell lines. The expression of LC-3 and p62 protein was detected by Western blot. Compared with the control group, the expression of LC-3 protein was significantly increased and that of p62 protein was significantly decreased after stably expressing SARM. On the contrary, the expression of LC-3 protein was significantly decreased after interfering with SARM. The phosphorylation levels of Akt, mTOR and p70S6K decreased after stably expressing SARM, indicating that stably expressing SARM could inhibit PI3K/Akt/mTOR signaling pathway. Ninth, stably expressing SARM inhibiting ERK signaling pathway was detected by immunoblotting. After stably expressing SARM, the phosphorylated ERK1/2 expression level was significantly lower than that of the control group. Conclusion This study is the first to study the expression level of SARM in tumor tissues and cells. It is the first to find that SARM can inhibit the growth of renal cell carcinoma cells and induce autophagy of renal cell carcinoma cells. Initial Molecular Mechanisms: SARM may be involved in regulating the growth of renal cell carcinoma by inhibiting P13K/Akt/mTOR signaling pathway and ERK signaling pathway. SARM-induced autophagy of renal cell carcinoma cells may be mediated by PI3K/Akt/mTOR signaling pathway. SARM may be the target of Sunitinib, a multi-target tyrosine kinase inhibitor. SARM can be used as a new target for molecular targeted therapy of renal cell carcinoma, which provides a new target and a new idea for molecular targeted therapy of renal cell carcinoma.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11
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