【作者】 夏振坤；

【导师】 尹邦良；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 研究背景及目的食管癌的死亡率在我国恶性肿瘤中居第四位,超过90%的食管癌患者是鳞状细胞癌。由于大多数食管癌患者就诊时已处于晚期,因此采用手术切除为主的综合治疗,其5年生存率仍然很低。研究表明,酪氨酸激酶信号通路在多种肿瘤中表达异常,但是由于酪氨酸磷酸化蛋白质在组织中的丰度很低,目前尚无对食管鳞癌及癌旁正常食管组织(简称癌旁组织,下同)进行酪氨酸磷酸化蛋白质组学的研究。本实验采用新的酪氨酸磷酸化蛋白质组分析方法,对食管鳞癌及癌旁组织进行差异性酪氨酸磷酸化蛋白质组学研究,为揭示酪氨酸激酶信号通路在食管鳞癌发生、发展中的作用提供依据,同时为食管鳞癌的治疗及早期诊断研究开辟新的思路。方法在第一章中我们收集了28对食管鳞癌及癌旁组织标本,采用酪氨酸磷酸化抗体免疫沉淀富集酪氨酸磷酸化多肽,并用LTQ Orbitrap质谱仪进行鉴定,将鉴定结果导入Genepattern软件进行分析,筛选出差异明显的酪氨酸磷酸化蛋白质,其中酪氨酸磷酸化差异最为明显的MYH9(编码NMIIA蛋白),其与DDR1关系密切。在第二章中我们提取了MYH9磷酸化水平最高的10例食管鳞癌组织基因组DNA,经PCR和测序检测了MYH9基因是否存在相关位点突变,western blot验证了NMⅡA蛋白在28对食管鳞癌和癌旁组织中的表达差异,采用免疫组织化学的方法研究了50例食管鳞癌组织和30例癌旁组织中NMⅡA和DDR1的表达,并与临床病理资料进行相关性分析。在第三章中我们检测了3株食管鳞癌细胞株KYSE-140、KYSE-150、KYSE-510的酪氨酸磷酸化蛋白质谱,比较了人食管鳞癌组织和鳞癌细胞株酪氨酸磷酸化蛋白质表达的差异,同时为第四章进行NMⅡA蛋白的功能学研究提供细胞依据。在第四章中,我们应用SiRNA沉默MYH9基因,qRT-PCR和westernblot检测SiRNA对NMⅡA蛋白的沉默水平,细胞计数明确NMⅡA蛋白沉默对细胞增殖能力的影响,细胞粘附实验检测NMⅡA蛋白沉默对细胞粘附能力的影响,划痕试验和transwell实验评价NMⅡA蛋白沉默对细胞的平面和3-D迁移能力的影响。结果1.在28对食管鳞癌及癌旁组织中共检测到1037种差异性酪氨酸磷酸化蛋白质,其中有477种在鳞癌组织中上调,560种在癌旁组织中上调。在肿瘤组织中酪氨酸磷酸化水平最高的是MYH9, palladin, APP, DDR1, K105种蛋白,在癌旁组织中酪氨酸磷酸化水平最高的是K23,K20, Sciellin, envoplakin, EphA15种蛋白。食管鳞癌及癌旁组织中总体酪氨酸磷酸化蛋白质无明显差异。MYH9的酪氨酸磷酸化位点是Tyr754和Tyr1408。经相关分析,MYH9与DDR1的关系密切。2.测序结果显示MYH9基因的1,10,16,24-26,30-32,35-39等14个外显子没有检测到常见突变位点。Western blot检测表明NMⅡA蛋白在28对肿瘤组织中的表达高于癌旁组织。免疫组化显示,NMⅡA蛋白在所有的食管鳞癌组织中表达,显著高于癌旁组织(100% VS 26.7%,P=O.000),其中强阳性表达25例(50%),NMⅡA的强阳性表达与肿瘤的侵润深度无关,与淋巴结转移个数、组织学分级相关,随着转移淋巴结个数增加,NMⅡA强阳性表达率明显增加(P=0.015<0.05),低分化组病例的强阳性表达率也显著高于高分化组(P=0.018<0.05)；DDR1蛋白在肿瘤组织中阳性率显著高于癌旁组织中的阳性率(86% VS30%,P=0.000),DDR1的表达仅与肿瘤的侵润深度有关(P=0.027<0.05),与转移淋巴结个数和肿瘤组织分化程度无关。3.食管鳞癌细胞株中的酪氨酸磷酸化蛋白质类型和磷酸化水平与鳞癌组织中的磷酸化类型和水平存在明显差异；在3株食管鳞癌细胞株中,只有KYSE-510发生了动力/收缩蛋白的酪氨酸磷酸化,并且磷酸化的动力/收缩蛋白只有KLC2, MYH9和dnnactin2三种。三株细胞株中均有NMⅡA蛋白的表达,KYSE-140的表达相对较低。4. Western blot检测结果显示基因沉默效率在72h SiRNA/转染试剂比例为75ng/1.5ul时最高,qRT-PCR检测提示MYH9 mRNA被SiRNA沉默了89.9%,应用MYH9 SiRNA沉默KYSE-510细胞NMⅡA蛋白表达后,与阴性转染组相比,细胞的增殖速度减慢,体外粘附实验显示细胞的粘附能力增强,细胞的平面和3-D迁移能力降低(P<0.01)。结论1.应用PhosphoScan技术对食管鳞癌及癌旁组织进行了酪氨酸磷酸化蛋白质组学分析,筛选出了多个在食管鳞癌中酪氨酸磷酸化异常增高的蛋白质,最明显的是动力/收缩蛋白NMⅡA和受体型酪氨酸激酶DDR1；2.食管鳞癌组织中NMⅡA的高酪氨酸磷酸化可能不是由MYH9基因突变引起,而是与组织中NMⅡA蛋白的表达量增高有关；3.食管鳞癌组织中NMⅡA的高表达与食管鳞癌组织的分化和转移有关；DDR1蛋白的高表达只与食管鳞癌组织的侵袭性有关。4. KYSE-510细胞表达NMⅡA蛋白和酪氨酸磷酸化NMⅡA；5.采用SiRNA沉默MYH9基因抑制细胞内NMⅡA的表达后抑制了KYSE-510细胞的迁移和增殖能力但增强了粘附能力。更多还原

【Abstract】 Background and purpose Esophageal cancer is the fourth most common malignancies in China and more than 90% patients are squamous cell carcinoma. Owing to the advanced stage when patients are examined, the 5-year survival rate is still low although they might receive comprehensive treatment including operation. Recently, abnormal expression of tyrosine kinase signaling pathway has been found in many tumors by some researchers, however, tyrosine phosphorylation is at low abundance in tissues. Hence there was no tyrosine phospho-proteomics research on esophageal cancer yet. Here, we investigated differential protein tyrosine phosphorylation between esophageal cancer and para-esophageal cancer normal tissues (simply, para-cancer tissues) with a new tyrosine phospho-proteomics method. This study would provide basic proof for understanding the role of tyrosine kinase signaling in the occurrence and development of esophageal cancer, and also offer new insights into the treatment and early diagnosis of esophageal cancer.Methods In the first chapter, we collected 28 cases of esophageal squamous cancer and corresponding para-cancer tissues. Tyrosine phosphorylated peptides were enriched by immunoaffinity purification, and analyzed by LTQ Orbitrap mass spectrometer. Differential phosphorylated proteins were analyzed by using Genepattern software. MYH9 (encoding NMⅡA) was the top phosphorylated protein between the cancer and para-cancer group and was closely correlated with DDR1.In the second chapter, we extracted genomic DNA from ten cancer tissues with highest MYH9 phosphorylation and sequenced PCR products to find any mutations of MYH9. Western blot was adopted to validate NMⅡA expression in the 28 pair specimen. Another 50 cancer specimen and 30 para-cancer normal specimen were chosen to investigate the expression of NMⅡA and DDR1 using immunohistochemistry (IHC) and correlations between their expressions and patients’clinical data were also analyzed.In chapter three, we cultured and profiled three esophageal squamous cancer cell lines(KYSE-140、KYSE-150、KYSE-510) by mass spectrometer. Comparison of primary human esophageal cancer and cancer cell lines was made and suggested that cell lines can be used as models to validate targets identified in primary tumors.In the fourth chapter, we silenced MYH9 with small interfering RNA; qRT-PCR and western blot were used to study the silence effect. We counted cell number to find any effect of decreased NMⅡA on cell proliferation. Cell adhesion assay was used to analyze the effect of decreased NMⅡA on cell adhesion ability. Wound closure assay and transwell assay were used to assess the effect of decreased NMⅡA on cell migration ability.Results1. In this study, we identified 1037 tyrosine phosphorylated proteins. 477 of them were enriched in cancers while 560 were enriched in para-cancer tissues. The top 5 tyrosine phosphorylated proteins were MYH9, palladin, APP, DDR1 and K10 in the cancer group and K23, K20, Sciellin, envoplakin and EphA1 in para-cancer group. No significant difference of tyrosine phosphorylated protein clusters was found between the two groups. The tyrosine phosophorylated sites of NMⅡA were Tyr754 and Tyr1408. Correlation analysis showed that MYH9 was closely related with DDR1. 2. There was no mutation in exon 1,10,16,24-26,30-32 and 35-39 of MYH9. Western blot analysis found that the expression of NMⅡA was higher in cancer tissues than para-cancer tissues in the 28 pair tissues. In IHC analysis, NMⅡA was expressed in all cancer tissues, and were significantly higher than the expression in para-cancer tissues (100% VS 26.7%, P=0.000), strong positive expression of NMⅡA was found in 25 cancer specimen(50%) and was correlated with metastatic lymph nodes and the differentiation of the cancer but not with cancer invasive depth. Corresponding increased strong positive NMⅡA expression was correlated with the increasing metastatic lymph nodes (P=0.015<0.05) and the poorer differentiation (P=0.018<0.05). The expression of DDR1 was significantly higher in cancer tissues than para-cancer tissues (86% VS 30%, P=0.000). DDR1 expression was only correlated with the invasive depth (P=0.027<0.05), but not with metastatic lymph nodes or the differentiation of the cancer.3. Significant differences of tyrosine phosphorylated protein levels and clusters were found between the esophageal cancer tissues and cell lines. In three cell lines, phosphorylation of the motor/contractile protein was only found in KYSE-510 cell line, and there were only three phosphorylated proteins:KLC2, MYH9 and dnnactin2. The expression of NMⅡA protein was found in all three cancer cell lines with a relative lower expression in KYSE-140 cell line.4. The silence efficacy was highest when the ratio of SiRNA/transfection reagent was 75ng/1.5ul at 72h post-transfection with western blot analysis. MYH9 mRNA was silenced up to 89.9% demonstrated by qRT-PCR. When the expression of NMⅡA protein was almost eliminated by targeted MYH9 SiRNA, KYSE-510 cells exhibited decreased proliferation rate, planer and 3-D migration rate while with higher adhesion ability compared with cells transfected with negative SiRNA (P＜0.01).Conclusions1. PhosphoScan technology was successfully applicated in proteomic analysis of tyrosine phosphorylation in esophageal squamous cancer and para-cancer tissues; a number of over-expressed tyrosine phosphorylated proteins were screened in esophageal cancer tissues, of which the most obvious were the motor/contractile protein NMⅡA and the receptor tyrosine kinase DDR1;2. Increased tyrosine phosphorylation of NMⅡA might not be induced by genetic mutation of MYH9 gene, but might be caused by elevated total NMⅡA protein expression;3. NMⅡA expression correlated with esophageal squamous cancer differentiation and metastasis in cancer tissues; DDR1 expression only correlated with the cancer invasion depth.4. KYSE-510 cell line expressed NMⅡA protein and tyrosine phosphorylated NMⅡA protein;5. Decreased NMⅡA expression by targeted MYH9 SiRNA inhibited the migration and proliferation ability of KYSE-510 cell line but enhanced the adhesive ability.更多还原