【作者】 黄波；

【导师】 张燕捷； 姜斌； 刘峰；

【作者基本信息】 上海交通大学 ， 内科学， 2018， 硕士

【摘要】 研究目的探讨Rab1A蛋白在胃腺癌细胞恶性生物学行为中的作用及机制。研究方法1.应用组织芯片(HStm-Ade180Sur-01)技术,收集80例胃腺癌患者手术标本,每个病例包括一处胃腺癌组织和一处癌旁粘膜组织。运用免疫组化技术检测Rab1A蛋白表达水平,评估胃腺癌组织和癌旁粘膜组织的Rab1A蛋白表达水平的H-Score评分,并分析Rab1A蛋白表达水平在胃腺癌组织和癌旁粘膜组织的统计学差异,分析淋巴结转移阳性病例和淋巴结转移阴性病例的胃腺癌组织Rab1A表达的统计学差异。收集胃黏膜上皮细胞系和8种胃腺癌细胞系,应用Western blot检测胃黏膜上皮细胞系和胃腺癌细胞系Rab1A蛋白的表达情况。收集上述80例胃腺癌病人的随访信息,根据Rab1A蛋白的H-Score评分,按照中位数分为Rab1A蛋白高表达组和低表达组,并绘制生存曲线,分析Rab1A蛋白表达水平与胃腺癌病人生存期的关系。2.应用组织芯片(HStm-Ade076Met-01)技术,收集28个胃腺癌患者手术标本,其中13个病例包括一处胃腺癌原发灶、一处癌旁粘膜组织和一处转移灶,4个病例包括一处胃腺癌原发灶和两处转移灶,11个病例包括一处胃腺癌原发灶和一处转移灶。运用免疫组化技术检测病例原发灶和转移灶的Rab1A蛋白表达水平,评估Rab1A蛋白的H-Score评分,分析Rab1A蛋白表达水平在原发灶和转移灶的统计学差异。选取Rab1A蛋白表达水平较低的胃腺癌细胞系AGS和BGC823,构建稳定过表达Rab1A蛋白的人胃腺癌细胞系AGS-rab1a和BGC823-rab1a。选取Rab1A蛋白表达水平较高的胃腺癌细胞系MKN45和MKN28,构建稳定knockdown Rab1A蛋白的人胃腺癌细胞系MKN45-rab1a-kd 和MKN28-rab1a-kd。应用Transwell迁移实验、Matrigel侵袭实验和软琼脂集落形成实验分别检测过表达Rab1A蛋白和knockdown Rab1A蛋白对胃腺癌细胞迁移侵袭能力和锚定非依赖条件下克隆形成能力的影响,并计数穿透到下层的细胞数和形成的克隆数,分析统计学差异。3.在Rab1A表达水平较高的胃腺癌细胞系MKN45中,转染Rab1A si RNA,转染48h后提取总蛋白,应用信号通路磷酸化抗体芯片筛选活性变化明显的信号通路,并应用生物信息学技术筛选出与Rab1A蛋白关联密切的信号通路。选取稳定过表达Rab1A蛋白的胃腺癌细胞系AGS-rab1a,BGC823-rab1a和稳定knockdown Rab1A蛋白的胃腺癌细胞系MKN45-rab1a-kd,MKN28-rab1a-kd,应用Western blot技术验证Rab1A对信号通路活性的调控。为进一步验证Rab1A通过活化ERK MAPK信号通路促进胃腺癌细胞的恶性生物学行为,我们在稳定过表达Rab1A的稳转细胞株BGC823-rab1a给予MEK抑制剂和DMSO作为对照,观察细胞的恶性生物学行为变化,并用Western blot技术检测细胞ERK MAPK信号通路活性的变化。在稳定过表达Rab1A蛋白的胃腺癌细胞系BGC823-rab1a,应用免疫共沉淀技术pull down可能和Rab1A蛋白相互结合的蛋白分子,再应用蛋白电泳和银染选取出有差异的条带,通过蛋白质谱分析技术鉴定出目的条带中与Rab1A结合的蛋白分子,并进一步探讨与Rab1A调控胃腺癌细胞恶性生物学行为有关的分子机制。研究结果1.在胃腺癌组织与癌旁粘膜组织的免疫组化结果对比,胃腺癌组织Rab1A蛋白表达水平明显高于癌旁粘膜组织Rab1A蛋白表达水平,并具有统计学差异。淋巴结转移阳性病例Rab1A蛋白高表达率明显多于淋巴结转移阴性病例,并具有统计学差异。对比胃粘膜上皮细胞系和不同胃腺癌细胞系的Rab1A蛋白表达情况,胃腺癌细胞的Rab1A蛋白表达水平明显高于胃粘膜上皮细胞系。根据免疫组化结果及随访信息,绘制生存曲线,得出Rab1A蛋白高表达病例的生存期明显短于Rab1A蛋白低表达病例的生存期。2.在胃腺癌组织原发灶与转移灶的免疫组化结果对比中,转移灶Rab1A蛋白表达水平明显高于原发灶Rab1A蛋白表达水平,并具有统计学差异。稳定过表达Rab1A蛋白后,与转染空载质粒细胞相比,人胃腺癌细胞系AGS-rab1a,BGC823-rab1a的恶性生物学行为明显增强,并具有统计学差异。稳定knockdown Rab1A蛋白后,与转染空载质粒细胞相比,人胃腺癌细胞系MKN45-rab1a-kd,MKN28-rab1a-kd的恶性生物学行为明显减弱,并具有统计学差异。3.应用信号通路磷酸化抗体芯片筛选活性变化明显的信号通路,再通过生物信息学技术检索出与Rab1A蛋白可能有关联的信号通路是PI3K/AKT信号通路,MAPK信号通路和Apoptosis信号通路。我们发现Rab1A蛋白过表达增强ERK MAPK信号通路的活性,knockdown Rab1A蛋白抑制ERK MAPK信号通路的活性。通过PD98059阻断ERK MAPK信号通路,在给予DMSO的对照组,过表达Rab1A组的细胞迁移和侵袭到下室的细胞数明显增多,形成克隆数明显多于表达vector control的细胞株。在给予MEK抑制剂的实验组,过表达Rab1A细胞株和表达vector control的细胞株迁移和侵袭到下室的细胞均明显减少,形成的克隆数相对于DMSO对照组也均明显减少。Western blot显示在过表达Rab1A的稳转细胞株给予MEK抑制剂后,ERK MAPK信号通路的活性被完全抑制。在胃腺癌细胞BGC823-rab1a提取的总蛋白,通过蛋白质谱鉴定免疫共沉淀pull down的所有蛋白分子,排除角蛋白等非特异性蛋白后,确定一种与Rab1A直接结合的蛋白分子是e EF1A。经资料显示e EF1A可以被ERK MAPK信号通路上游的Raf磷酸化,同时Rab1A与e EF1A的相互结合导致e EF1A锚定在Raf的临近位置,使得Raf更容易磷酸化e EF1A,活化后的e EF1A进一步促进Raf的合成,最终活化ERK MAPK信号通路。提示我们Rab1A可能通过结合e EF1A的方式参与对ERK MAPK信号通路的活化。研究结论Rab1A蛋白在胃腺癌组织高表达,与胃腺癌的局部转移和远处转移相关,并提示预后不良。过表达Rab1A可以促进胃腺癌细胞的恶性生物学行为,knockdown Rab1A可以明显抑制胃腺癌细胞的恶性生物学行为。Rab1A蛋白可以活化ERK MAPK信号通路,可能的相关机制是通过直接结合e EF1A参与调控ERK MAPK信号通路的活性。更多还原

【Abstract】 Objectives To explore the role of Rab1 A in the malignant biological properties of gastric adenocarcinoma cells.Methods 1.Tissue chip(HStm-Ade160Sur-01)was used to detect expression level of Rab1 A protein in tissues-one gastric adenocarcinoma tissue and one paired adjacent mucosal tissue from 80 gastric adenocarcinoma cases.we assessed the H-Score of Rab1 A protein in gastric adenocarcinoma tissue and adjacent mucosal tissue and analyzed the statistical differences of Rab1 A expression between gastric adenocarcinoma tissue and adjacent mucosal tissue.Next,we analyzed positive rate of Rab1 A expression in metastatic lymph node cases and non-metastatic lymph node cases.We detected expression level of Rab1 A protein in gastric adenocarcinoma cell lines and gastric mucosa cell line by Western blot by collecting gastric mucosa epithelial cell line and eight gastric cancer cell lines.We collected follow-up information of those 80 gastric adenocarcinoma cases ended in August 2015.According to the H-Score of Rab1 A,80 cases were divided into two groups: low expression of Rab1 A and high expression of Rab1 A.We drew survival curve and analyzed the relationship between the expression level of Rab1 A and the prognosis of gastric adenocarcinoma patients.2.Tissue chip(HStm-Ade076Met-01)was used to obtain tissues from 27 gastric adenocarcinoma cases-13 cases with one primary gastric adenocarcinoma tissue,one paired adjacent mucosal tissue and one paired malignant tissue,3 cases with one primary gastric adenocarcinoma tissue and two malignant tissues,11 cases with one primary gastric adenocarcinoma tissue and one malignant tissue.We detected expression level of Rab1 A protein by immunohistochemical and assessed the H-Score of Rab1 A protein in primary gastric adenocarcinoma tissues and malignant tissues.Then we analyzed the statistical difference of Rab1 A expression between primary gastric adenocarcinoma tissues and malignant tissues.We selected gastric adenocarcinoma cell lines AGS,BGC823 with low Rab1 A expression and gastric adenocarcinoma cell lines MKN45,MKN28 with high Rab1 A expression to reconstruct stable Rab1 A overexpression cell lines AGS-rab1 a,BGC823-rab1 a and stable Rab1 A lower expression cell lines MKN45-rab1a-kd,MKN28-rab1a-kd.Transwell migration assay,Matrigel invasion assay and Soft Agar assay were applied to investigate the role of overexpression Rab1 A and knockdown Rab1 A to migration,invasion and colony formation of gastric adenocarcinoma cells.Cells of lower chamber and colonies were counted and we analyzed their statistical differences.3.Rab1 A si RNA was transfected into gastric cancer cell MKN45.Protein microarray screening with broad-spectrum phosphorylated signaling pathway was used to discover signaling pathways obviously changed on total proteins collected at 48 hours.Biological information technology was used to identify the most probably related signaling pathways.Western blot technology was used to verify the role of Rab1 A on the activity of cell signaling pathways in stable Rab1 A overexpression cell lines AGS-rab1 a,BGC823-rab1 a and stable Rab1 A lower expression cell lines MKN45-rab1a-kd,MKN28-rab1a-kd.For further exploring whether Rab1 A can promote the malignant biological properties of gastric adenocarcinoma by activating ERK MAPK signaling,stable Rab1 A overexpression cell lines BGC823-rab1 a,DMSO control and PD98059(inhibitor of MEK)were used to explore the malignant biological properties of gastric adenocarcinoma cells.Western blot was used to detect the protein expression changes of ERK MAPK signaling pathway.In stable Rab1 A overexpression cell lines BGC823-rab1 a,we pulled down proteins that may be combined with Rab1 A by CO-IP and identified the protein which combined with Rab1 A on selected different bands seen in protein electrophoresis and silver staining by protein mass spectrometry technology to explore the related molecular mechanisms of Rab1 A in the malignant biological properties of gastric adenocarcinoma cells.Results 1.The expression level of Rab1 A protein in gastric adenocarcinoma tissue was significantly higher than that in adjacent mucosal tissue and there was statistical difference between gastric adenocarcinoma tissue and adjacent mucosal tissue.The rate of Rab1 A high expression in metastatic lymph nodes cases was significantly higher than that in non-metastatic lymph nodes cases and there was statistical difference between metastatic lymph nodes cases and non-metastatic lymph nodes cases.The expression level of Rab1 A protein in gastric adenocarcinoma cells was significantly higher than that in gastric mucosal cells.It was concluded that the survival period of Rab1 A positive cases was significantly shorter than that of Rab1 A negative cases and there was statistical difference between high expression of Rab1 A cases and low expression of Rab1 A cases by survival curve.2.The expression level of Rab1 A protein in malignant tissues was significantly higher than that in primary gastric adenocarcinoma tissues and there was statistical difference between primary gastric adenocarcinoma tissues and malignant tissues.Compared with cells transfected by empty plasmid,the malignant biological properties of human gastric adenocarcinoma cell lines AGS-rab1 a and BGC823-rab1 a were significantly elevated and there was statistical difference between experimental group and control group.Compared with cells transfected by empty plasmid,the malignant biological properties of human gastric adenocarcinoma cell lines MKN45-rab1a-kd,MKN28-rab1a-kd were significantly decreased and there was statistical difference between experimental group and control group.3.The PI3K/AKT signaling pathway,MAPK signaling pathway and Apoptosis signaling pathway were identified to be associated with Rab1 A protein through protein chip and bioinformatics research.Activity of ERK MAPK signaling pathway was consistent with the expression level of Rab1 A protein in gastric adenocarcinoma cell lines.The malignant biological properties of gastric adenocarcinoma cell BGC823-rab1 a was significantly inhibited by PD98059 compared to the group treated by DMSO.It was reported that e EF1 A pulled down by CO-IP can be phosphorylated by Raf.The combination of Rab1 A and e EF1 A pulled e EF1 A close to Raf so that Raf was more likely to phosphorylate e EF1 A.In the end,ERK MAPK signaling was activated.It indicated that Rab1 A regulated the activity of ERK MAPK signaling via combinating with e EF1 A.Conclusions Rab1 A protein is highly expressed in gastric adenocarcinoma tissue and can promote the malignant biological properties of gastric adenocarcinoma tissue and aggravate the bad prognosis of gastric adenocarcinoma patients.Lower expression of Rab1 A protein can significantly inhibit the malignant biological properties of gastric adenocarcinoma.Rab1 A protein can active the ERK MAPK signaling through combinating with e EF1 A.更多还原