【作者】 谢远杰；

【导师】 贺修胜；

【作者基本信息】 南华大学 ， 基础医学（病理学与病理生理学）， 2015， 博士

【摘要】 [目的]本研究运用sh RNA技术,敲低永生化人鼻咽黏膜上皮NP69细胞中STGC3基因的表达,构建STGC3稳定低表达NP69细胞,分析STGC3基因表达降低对其生长、增殖、侵袭、迁移及裸鼠成瘤能力等生物学行为的影响;应用蛋白质组学技术,分析STGC3基因低表达对NP69细胞蛋白质表达谱的影响,筛选与鉴定差异表达蛋白质分子,实验验证与分析差异表达蛋白质分子的功能,明确STGC3基因在鼻咽黏膜上皮细胞恶性转化过程可能发挥的作用及其分子机制,为全面揭示STGC3基因的功能提供实验依据。[方法]根据碱基序列设计靶向干扰STGC3基因的sh RNA,构建真核表达干扰载体,应用脂质体2000将重组质粒及对照质粒分别转染NP69细胞,经不同浓度G418筛选,建立STGC3稳定低表达的NP69细胞系,运用MTT法、流式细胞术及细胞集落形成实验,分析观察其生长、增殖及细胞周期的变化;同时分别采用Transwell小室及裸鼠成瘤实验,分析探究其迁移、侵袭能力及裸鼠成瘤能力的变化;再运用蛋白质组学技术筛选、鉴定其差异表达蛋白质分子,分析其生物学功能;并运用IPA(Ingenuity Pathways Analysis)分析差异蛋白质分子之间的相互作用。随后运用RT-PCR、Western-Blotting验证部分差异蛋白质分子的表达,并检测其在鼻咽癌细胞及鼻咽癌组织中的表达;在此基础上进一步干预差异表达蛋白质分子的功能,研究其对STGC3基因低表达NP69细胞系及鼻咽癌细胞生长、增殖、侵袭与迁移能力的影响,同时检测其相关信号通路蛋白质分子的表达。[结果]经菌落PCR、双酶切及测序鉴定,确定成功构建了STGC3真核表达干扰载体并命名为p RNAi-U6.1/sh RNA/STGC3。将其与对照质粒p RNAi-U6.1/scramble分别转染NP69细胞并经400μg/ml G418筛选后,荧光显微镜下能见到转染细胞内的绿色荧光,RT-PCR检测结果显示,p RNAi-U6.1/sh RNA/STGC3干扰载体转染NP69细胞后,STGC3 m RNA水平显著低于对照组,表明该干扰载体能成功敲低NP69细胞中STGC3基因的表达,稳定低表达STGC3的NP69细胞(p RNAi-U6.1/sh RNA/STGC3/NP69细胞)建立成功。细胞生长曲线及细胞集落形成实验结果显示,从第4天开始,p RNAi-U6.1/sh RNA/STGC3/NP69细胞生长增殖速度明显快于对照组;其所形成的细胞集落数目明显多于对照组,集落体积也大于对照组(p<0.05),表明STGC3基因低表达后能促进NP69细胞生长增殖,增强其克隆形成能力。FCM分析结果表明,p RNAi-U6.1/sh RNA/STGC3/NP69细胞处于(G2+S)期比例明显高于对照组(p<0.05),表明STGC3基因低表达后能影响细胞周期进程,提高细胞增殖速度及克隆形成能力。Transwell小室实验结果显示,未包被Matrigel时,p RNAi-U6.1/sh RNA/STGC3/NP69细胞其迁移细胞数(320.42±28.54)明显多于p RNAi-U6.1/scramble/NP69细胞(206.26±15.40)及未转染的NP69细胞(180.72±16.03);包被Matrigel时,p RNAi-U6.1/sh RNA/STGC3/NP69细胞其迁移细胞数(116.33±3.55)也明显多于对照组[分别为(43.00±2.00)、(40.67±2.52)],差异均有显著性意义(p<0.05),表明STGC3基因低表达后能增强NP69细胞的迁移、侵袭能力;将p RNAi-U6.1/sh RNA/STGC3/NP69细胞及对照组细胞分别接种于裸鼠皮下,经8周饲养并观察,但均未见肿瘤的形成。i TRAQ同位素标记及质谱技术分析结果显示,与对照组比较,p RNAi-U6.1/sh RNA/STGC3/NP69细胞中蛋白质表达谱发生改变,共鉴定出表达差异明显的蛋白质分子83种,其中表达上调的蛋白质有45种,包括m TOR、TPM2、Cofilin-1、ANXA3、Musashi-2、Kindlin-2等;表达下调的蛋白质有38种,包括Beclin-1、TSC-22、AMOTL2等,这些差异蛋白主要参与调控细胞生长增殖、蛋白质合成、物质代谢、细胞骨架、信号传导及自噬等细胞生命活动过程。IPA蛋白质相互作用分析软件表明,这些差异表达蛋白之间形成复杂的相互作用网络,影响p RNAi-U6.1/sh RNA/STGC3/NP69细胞的功能。经过RT-PCR、Western-Blotting及免疫组化等检测发现,与对照组比较,p RNAi-U6.1/sh RNA/STGC3/NP69细胞中m TOR表达增强、Beclin-1表达降低,证实了蛋白质组学检测结果的准确性和可信性;同时检测发现在CNE1、CNE2鼻咽癌细胞及鼻咽癌组织中也存在m TOR表达增强、Beclin-1表达降低,且鼻咽癌组织中m TOR蛋白的表达与Beclin-1蛋白表达呈负相关;临床Ⅲ、Ⅳ期患者及有淋巴结转移的鼻咽癌组织中m TOR蛋白阳性表达率分别高于临床Ⅱ期患者及无淋巴结转移者,差异具有显著性意义(p<0.05)。应用不同浓度(0、1、2、4、8μmol/L)的雷帕霉素处理p RNAi-U6.1/sh RNA/STGC3/NP69、CNE1、CNE2细胞后,雷帕霉素能显著抑制p RNAi-U6.1/sh RNA/STGC3/NP69、CNE2细胞的生长增殖,且变化呈浓度依赖性,而不同浓度雷帕霉素作用不同时间后均对CNE1细胞无明显抑制作用。同一浓度雷帕霉素处理24h后,对p RNAi-U6.1/sh RNA/STGC3/NP69、CNE2细胞的抑制率低于处理48h和72h的抑制率,差异有统计学意义(P<0.05),而处理48h和72h的抑制率之间差异无统计学意义。8μmol/L雷帕霉素处理48h后,p RNAi-U6.1/sh RNA/STGC3/NP69、CNE2细胞迁移、侵袭能力均明显降低,差异有统计学意义(P<0.05),但雷帕霉素对CNE1细胞迁移、侵袭能力无明显影响。Western blot检测结果显示,雷帕霉素处理48h后,p RNAi-U6.1/sh RNA/STGC3/NP69细胞、CNE2细胞中P-p70S6K和cyclin D1的表达降低,但m TOR、p70S6K蛋白的表达无明显变化;而8μmol/L雷帕霉素处理48h后,CNE1细胞中m TOR、p70S6K、P-p70S6K和cyclin D1的表达较处理前均无明显变化;这些结果表明雷帕霉素能抑制p RNAi-U6.1/sh RNA/STGC3/NP69和CNE2细胞生长增殖及迁移、侵袭能力,可能与抑制其P-p70S6K和cyclin D1的表达有关。[结论]1)STGC3表达降低可导致NP69细胞呈现出部分恶性表型,同时伴随m TOR、Beclin-1等83种蛋白质表达发生改变。2)STGC3低表达导致NP69细胞呈现部分恶性特征,其机制可能与m TOR信号通路密切相关。更多还原

【Abstract】 [Objectives] To explore the roles of STGC3 gene in carcinogenesis of nasopharyngeal carcinoma and its molecular mechanisms, effects of knocked-down STGC3 by sh RNA were investigated on cell growth and proliferation, malignant abilities of migration, invasion and tumorigenicity of immortalized nasopharyngeal epithelial NP69 cell line in vitro and in vivo, and the differentially expressed proteins were analysed with i TRAQ coupled with mass spectrometry in NP69 cell line with stable low expression of STGC3 gene.After verification of some differentially expressed proteins, the expression and function of them were detected in nasopharyngeal carcinoma cell lines and tissues.[Methods] sh RNA targeting STGC was designed to establish expression plasmid which was confirmed by double enzyme digestion and DNA sequencing, and then transfected into NP69 cell line and selected by G418 to establish a stable cell line with low expression of STGC3. After that, the cell cycle, cell growth and proliferation were tested by flow cytometry and MTT, respectively.The changes in abilities of colongenic, invasion, migration and tumorigenicity were measured by cell colony formation assay, transwell experiments and inoculation in nude mice, respectively. Subsequently, the total proteins were extracted and quantified from the NP69 cells with low expression of STGC3 and the control cells respectively and information of abundance about protein expression were obtained by i TRAQ coupled with mass spectrometry after proteolysis, and then the differentially expressed proteins were screeninged by searching related database and the interactional networks among proteins were constructed by bioinformatics IPA( Ingenuity Pathways Analysis).Then the important protein molecules with different abundance were verified by RT-PCR and Western Blotting in p RNAi-U6.1/sh RNA/STGC3/NP69 cells, and their expression in nasopharyngeal carcinoma cell lines and tissues were detected. After intervention of function of the differentially-expressed proteins, effects on cell growth, proliferation, invasion and migration ability of nasopharyngeal carcinoma CNE1, CNE2 cells and NP69 cells with low expression of STGC3 were observed, and the expression of protein molecules in signal pathways related to differentially-expressed proteins were detected at the same time.[Results] The recombinant vector of p RNAi-U6.1/sh RNA/STGC3 targeting to STGC3 was successfully constructed and verified by bacteria liquid PCR, double enzyme digestion and DNA sequencing. The expression of STGC3 m RNA in NP69 cell line was significantly decreased and verified by RT-PCR after transfection with this recombinant vector, and this cell line with STGC3 low expression was named p RNAi-U6.1/sh RNA/STGC3/NP69, and the cell line transfected with p RNAi-U6.1/scramble plasmid was accordingly named p RNAi-U6.1/scramble/NP69.According to the results from MTT assay cell growth curve showed that proliferation of p RNAi-U6.1/sh RNA/STGC3 /NP69 cells was faster than that of NP69 and p RNAi-U6.1/scramble /NP69 cells on the 4th day.Numbers and volume of cell colonies of p RNAi-U6.1/sh RNA/STGC3/NP69 cell line were much more than that of the controls showed by colongenic experiment(p < 0.05). Numbers of p RNAi-U6.1/ sh RNA/STGC3/NP69 cells in(G2+S) phase were notably higher than that of control group which was showed by FCM analysis, indicating that low expression of STGC3 gene can promote cell proliferation and improve colongenic ability of NP69 cells by affecting cell cycle progression.Numbers of migrated p RNAi-U6.1/ sh RNA/ STGC3/NP69 cells were much more than that of p RNAi-U6.1/ scramble/NP69 and NP69 cell line which were showed by Transwell with package of matrigel or not,suggesting that lower expression of STGC3 gene in NP69 cell line significantly enhanced its capability of cell migration and invasion.However, during the period of 8 weeks the xenografted tumor did not form after subcutaneous inoculation with these three kinds of cell lines in nude mice, respectively.After successfully transfection with p RNAi-U6.1/sh RNA/STGC3, a total of 83 kinds of proteins with different expression were showed by i TRAQ isotope labeling and mass spectrometry. Of them 45 kinds of proteins such as m TOR, TPM2, Cofilin-1, ANXA3, Musashi-2, Kindlin-2were up-regulated and 38 were down-regulated including Beclin-1,TSC-22, AMOTL2 and so on.The function of these 83 kinds of protein molecules was involved in protein synthesis, cell growth and proliferation,cytoskeleton, signal transduction and autophagy, and which formed the complex network with interaction among them.The enhanced expression of m TOR and reduced Beclin-1 were validated through RT-PCR, Western Blotting and immunohistochemical detection in p RNAi-U6.1/sh RNA/STGC3/NP69 cell line. The expression of m TOR and Beclin-1 were also detected in nasopharyngeal carcinoma tissues, CNE1 and CNE2 cell lines in which there were enhanced m TOR and reduced Beclin-1.The positive expression rate of m TOR protein in nasopharyngeal carcinoma tissues of patients with clinical Ⅲ, Ⅳstage and with lymph node metastasis were higher than that in patients with clinical Ⅱstage and without lymph node metastasis,respectively. The expression of Beclin-1 protein was negatively correlated with m TOR in nasopharyngeal carcinoma tissue.After that, effects of rapamycin with different concentrations(0, 1, 2,4 and 8umol/L) on cell growth, proliferation, invasion and migration of CNE1, CNE2 and p RNAi-U6.1/sh RNA/STGC3/NP69 cell lines were observed. The results showed that rapamycin can significantly inhibit cell growth, proliferation, invasion and migration of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines corresponding to the concentration gradient and time of rapamycin, but there were no obvious inhibitory effects on CNE1 cells after rapamycin treatment with different concentration and time. After treatment with rapamycin for 24 h at the same concentration, the inhibition rate on cell growth and proliferation of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines were markedly lower than that at 48 h and 72 h, but there was no statistical difference between the inhibition rate at 48 h and 72 h. After treatment with 8umol/L rapamycin for 48 h, migratory and invasive abilities of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines were significantly suppressed.The results of Western blot showed that the expression of P-p70S6 K and cyclin D1 in these two kinds of cell lines were reduced, but there were no obvious changes at expression of m TOR, and p70S6 K protein after treatment with 8μmol/L rapamycin for 48 h. These results suggested that rapamycin can inhibit cell proliferation, migratory and invasive abilities of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines through suppressing expression of P-p70s6 k and Cyclin D1, but there were no obvious effects on their expression and abilities of CNE1 cell line.[Conclusions]1) Knock-Down STGC3 by recombinant vector p RNAi-U6.1/sh RNA/STGC3 can lead to different expression of 83 kinds of proteins including enhanced m TOR and reduced Beclin-1, and promote growth and proliferation, enhance abilities of migration and invasion, and result in partly malignant transformation of NP69 cell line.2) Partly malignant transformation of NP69 cell line result from Knock-Down STGC3 maybe related to m TOR signal pathway.更多还原