胃癌腹膜转移相关蛋白Myosin-9促进胃癌的侵袭和转移
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摘要
胃癌是消化道常见恶性肿瘤之一,近年来随着早期诊断、胃癌根治术和新辅助放化疗等的发展,其预后有所改善,但其死亡率仍居恶性肿瘤死亡率的前列。影响胃癌预后的因素有很多,其中腹膜转移在胃癌的复发和转移中十分常见。胃癌腹膜转移常合并有顽固性腹水和粘连性肠梗阻等症状,促使患者短期内死亡。因此,如何有效的预测与控制腹膜转移是目前提高进展期胃癌疗效的关键问题之一。
目前临床上用于胃癌腹膜转移早期诊断的方法主要包括腹腔液细胞学检查和分子标志物检查等。腹腔液细胞学检查是目前胃癌腹膜亚临床转移早期预测的最常用方法和金标准,但是其敏感性较低,因为并非所有浸透浆膜的进展期胃癌均能检测出腹膜脱落癌细胞。目前用于检测的分子标志物指标包括CEA、CA125、E-钙粘素、β1整合素、MMPs、VEGF等,但是目前国内外基础研究尚未找到一种公认的早期预测进展期胃癌腹膜转移的分子标志物。因此如何找到有效的胃癌腹膜转移早期预测的分子标志物,对于提高胃癌腹膜转移的预防和治疗,改善患者的预后具有重要现实意义。近年来开展的蛋白质组学技术为揭示肿瘤转移机制带来了新的希望。
蛋白质组学(proteomics)及其相关技术是近年来新兴的分子生物学分支,是以基因编码的所有蛋白质组为研究对象,了解蛋白质之间的相互作用与联系,从而揭示蛋白质功能以及基因表达调控机制的一个新的研究领域,其核心技术是以双相凝胶电泳(Two-dimensional polyacry-lamide gel electrophoresis,2-DE)为主的蛋白质分离技术和以质谱(mass spec-trometry, MS)、生物信息学(Bioinformatics)为主的蛋白质鉴定技术。目前已经有许多新的差异蛋白质组分析方法用于实践,其中荧光差异凝胶电泳(difference gel electrophoresis, DIGE)技术由于继承了二维凝胶电泳(two-dimensional electrophoresis,2-DE)的高分辨率,同时又具备高重现性、高灵敏度、高通量和高动态范围等优势,使得DIGE日益受到关注,成为最受欢迎的定量蛋白质组学研究手段之一。DIGE技术通过对用不同的荧光染料对不同的蛋白样品进行标记,在同一块双向凝胶中可同时分离多种蛋白样品,并且每块胶上均引入了内标,内标可以进一步增加可信度,确保结果能反映出真实的生物学差异,避免了系统误差对实验结果的影响。目前,蛋白质组学技术已在多种肿瘤中用于筛选和鉴定同早期诊断、预后以及化疗敏感性等相关的肿瘤标记物。
综上所述,本课题主要关注的是寻找同胃癌腹膜转移相关的蛋白质,并选择其中一个差异蛋白进行进一步的功能验证和机制的初步研究。实验的第一部分使用2D-DIGE分离结合MALDITOF/TOF质谱对胃癌组织和相应腹膜转移组织的差异表达蛋白质进行筛选鉴定,共鉴定出27种蛋白质。其中在胃癌腹膜转移组织中表达上调的有23个蛋白质,表达下调的有4个蛋白质,这为今后研究奠定了基础。然后利用生物信息学分析发现在上述这些差异蛋白中,Myosin-9蛋白可能在肌动蛋白细胞骨架重组、肌动蛋白纤维运动、血管生成、细胞-细胞间黏附以及蛋白转运等生物过程中发挥重要作用,根据以上结果我们推测Myosin-9可能参与了胃癌的侵袭和转移过程。因此第二和第三部分我们对Myosin-9进行了更深入的研究,结果发现抑制Myosin-9的表达后,细胞的迁移和侵袭能力减弱,而粘附能力增加,裸鼠腹腔播散肿瘤结节形成数目也明显减少。然后进一步检测上皮间质转化(epithelial-mesenchymal transition,EMT)相关标志物发现,抑制Myosin-9的表达后,胃癌细胞EMT过程受到抑制。基于以上结果,我们认为Myosin-9能够促进胃癌细胞的侵袭和转移能力,此过程可能是通过促进胃癌细胞EMT而实现的。
虽然本实验证实Myosin-9影响了胃癌细胞的侵袭转移能力,但是恶性肿瘤的发生和发展是多种基因和蛋白质相互作用的结果,任何单个基因或蛋白质都很难全面说明肿瘤复杂的生物学行为,且Myosin-9在胃癌中更深层次的机制仍需深入研究。在今后的研究中,我们将对更多前期筛选出的差异蛋白质进行分析,通过研究对比蛋白之间的相互关系来阐明胃癌腹膜转移发生发展的机制。此外,研究对象也将不只限于组织和细胞,胃癌腹膜转移患者的血液、体液均可以进行研究,以期能够找到一种有效的进展期胃癌腹膜转移早期预测的分子标志物,提高临床诊断水平,指导胃癌亚临床转移的合理防治。
本课题三部分的具体内容如下：
第一章胃癌腹膜转移相关差异蛋白质组的分离、鉴定与生物信息学分析
目的：将胃癌组织和相应腹膜转移组织蛋白进行分离,找到差异点并进行鉴定,结合生物信息学技术分析,以寻找潜在的预测指标,并为进一步的功能和机制研究提供理论依据。另外本实验还可验证2D-DIGE技术在胃癌研究中的实用价值。
方法：以胃癌组织和相应腹膜转移组织为研究对象,将纯化后的蛋白质应用双向荧光差异凝胶电泳(2D-DIGE)进行蛋白质分离,根据DIGE的差异图谱并结合Decyder分析软件进行差异蛋白点分析,差异倍数上调2倍或下调2倍以上的列为差异蛋白点,利用全自动切胶仪Spot picker挖取这些差异蛋白胶粒,然后进行样品胶内酶解,使用ABI4800MALDI TOF/TOF质谱仪进行分析,通过Mascot软件检索Uniprot数据库鉴定蛋白,并结合Western blot进行验证。最后利用String蛋白相互作用数据库对蛋白质问的相互作用进行预测和分析。
结果：1.根据2D-DIGE的差异图谱并结合Decyder分析软件进行差异蛋白点分析,共找到了32个差异蛋白点,然后通过Mascot软件检索Uniprot数据库鉴定蛋白,去除鉴定结果相同的蛋白及未鉴定出来的蛋白点,共鉴定出27种蛋白质。其中在胃癌腹膜转移组织中表达上调的有23个蛋白质,表达下调的有4个蛋白质。2.通过Uniprot数据库分析提示在上述这些差异蛋白中,Myosin-9蛋白可能在肌动蛋白细胞骨架重组、肌动蛋白纤维运动、血管生成、细胞-细胞间黏附以及蛋白转运等生物过程中发挥重要作用,进一步利用String蛋白相互作用数据库分析发现Myosin-9同ACE, RHOB, RHOA, ACTB, SVIL, CDK5, ACTG1,ROCK2,CALD1和S100A4等蛋白构成相互作用网络。基于以上结果分析,我们选取Myosin-9蛋白进行后续功能研究。3.应用Western blot和免疫组化方法对胃癌组织和相应腹膜转移组织中Myosin-9蛋白的表达水平进行验证,结果显示Myosin-9蛋白在腹膜转移组织中表达增强,与蛋白质组学结果一致。
第二章Myosin-9在胃癌侵袭转移中的功能研究
目的：探讨Myosin-9对胃癌细胞侵袭转移能力的影响。
方法：利用qRT-PCR和western-blot方法分别在mRNA水平和蛋白水平上检测Myosin-9在胃癌细胞株中的表达；合成针对Myosin-9的siRNA,利用慢病毒载体感染细胞株,构建稳定低表达Myosin-9蛋白的细胞系；利用纤维链接蛋白实验检测Myosin-9对胃癌细胞粘附能力的影响,Transwell小室迁移和侵袭实验检测Myosin-9对胃癌细胞迁移和侵袭能力的影响；构建裸鼠腹腔播散种植模型,观察Myosin-9对胃癌细胞腹膜转移能力的影响。
结果：1.相对于人胎儿胃粘膜细胞株GES-1, Myosin-9在其余3株胃癌细胞株AGS、BGC823和SGC7901中均高表达,其中AGS和BGC-823细胞株都来源于胃癌组织(低分化),SGC7901细胞株来源于淋巴结转移组织(中分化),我们选择AGS和SGC7901进行后续实验。2.通过多次预实验,我们确定了细胞接种密度为50%,MOI值为10,Polybrene为4ug/ml的最佳优化条件,western-blot结果显示慢病毒序列1的干扰效果最佳,按照上述条件我们成功构建了稳定低表达Myosin-9蛋白的细胞系SGC7901-siRNA和AGS-siRNA。3.干扰Myosin-9后,细胞粘附实验结果显示细胞粘附能力增强,细胞迁移和侵袭实验结果显示细胞迁移和侵袭能力减弱,以上各组间差异均有统计学意义,P<0.01。4.干扰Myosin-9后,相对于阴性对照组,干扰组腹腔内肿瘤结节形成数目明显减少,两者间差异有统计学意义,P<0.01。
第三章Myosin-9在胃癌侵袭和转移中作用机制的初步研究
目的：探讨Myosin-9在胃癌侵袭和转移中的作用机制。
方法：稳定沉默Myosin-9表达后,利用western-blot方法检测AGS和SGC7901细胞EMT相关标记物E-cadhern、N-cadhern、vimentin和β-catenin的表达情况。
结果：相对于阴性对照组和空白对照组,干扰组中E-cadherin表达上调,而N-cadherin、vimentin和β-catenin均表达下调。
总之,本研究利用基于2D-DIGE分离-MALDI TOF/TOF质谱鉴定-生物信息学分析的差异蛋白质组学技术平台,成功分离并鉴定出胃癌腹膜转移相关的蛋白质,并对其中的一个差异蛋白Myosin-9进行了功能验证和初步的机制研究。
通过研究,我们得出以下结论：
1.抑制Myosin-9的表达后,胃癌细胞的迁移和侵袭能力减弱,而粘附能力增强,此外裸鼠腹腔肿瘤播散转移能力下降,提示Myosin-9能够促进胃癌的侵袭和转移。
2.Myosin-9促进胃癌的侵袭和转移可能是通过促进胃癌细胞EMT过程而发挥作用的,确切的机制仍需进一步研究。
本研究的创新处在于：
1.建立了胃癌腹膜转移组织差异蛋白图谱；
2.证实了胃癌腹膜转移相关蛋白Myosin-9能够促进胃癌的侵袭和转移,并发现Myosin-9表达下调能抑制胃癌细胞EMT过程。
Gastric cancer is the most common malignant tumor of alimentary tract, despite improved prognosis as result of early diagnosis, radical operation and the development of adjuvant therapy, the mortality rate is still ranked among the top of malignant tumor mortality. There are many factors that can affect the prognosis of gastric cancer, in which the peritoneal metastasis are common in recurrence and metastasis of gastric cancer. Peritoneal dissemination usually leads to the terminal stage of advanced GC and diminishes the quality of life by refractory ascites and intestinal obstruction. As a result, how to effectively forecast and control of peritoneal metastasis is one of the key issues on improving the curative effect of advanced gastric cancer currently.
The current clinical method for early diagnosis of peritoneal metastasis from gastric cancer including detection of peritoneal lavage cytology and molecular marker. Peritoneal lavage cytology detection is the most commonly used method and the gold standard in early prediction of peritoneal subclinical metastasis from gastric cancer, but it lacks high sensitivity, since not all the advanced gastric cancer that soaked serous could be found the exfoliated cancer cells. Currently the molecular markers for the detection are CEA, CA125, E-cadherin, β1-integrin, MMPs and VEGF. However, basic researchers at home and abroad have not yet found an accepted molecular marker for early detection of peritoneal metastasis from advanced gastric cancer. At present, most of the research associated with metastasis so far has been focused on the genetic changes of related molecules or single or few proteins without systematicstudy. But little is known about the key factors to trigger tumorigenic cells to initiatefurther invasion and metastasis facing with so many regulators up to now.
Proteomics along with its related technologies, an emerging branch of molecular biology in recent years, focuses on studies of the proteome encoded by genes, and makes complete analysis of the composition, expression levels and modification state of the intracellular proteins with dynamic changes, and understands the interactions and associations among proteins, thus revealing protein function and its relationship with laws of the cellular activities as well as regulation mechanisms of gene expression as a new research field. Its core technology is protein separation techniques based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification technology based on mass spectrometry (MS) and bioinformatics. At present there are many novel differential proteomics analysis methods, among which the difference gel electrophoresis (difference gel electrophoresis, DIGE) technology becomes one of the most popular one, because it's not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. DIGE is a method which can label protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the real biological differences, while avoid influence of systematic errors on experimental results. To date,2D-DIGE technologies have been used to identify novel candidate biomarkers associated with early diagnosis, differential diagnosis, prognosis, and response to chemotherapy in various diseases.
Above all, this topic mostly focused on screening and identificating peritoneal metastasis related proteins of gastric cancer, and then selecting one of interesting protein for further functional verification and mechanism of a preliminary study. In the first part of experiment, two-dimensional fluorescence difference gel electropho-resis (2D-DIGE) was employed together with MALDI TOF/TOF mass spectrometry to screen and identify differentially expressed proteins between gastric cancer and corresponding peritoneal metastasis tissues.27differential proteins were identified,23proteins were significantly up-regulated and4were down-regulated in peritoneal metastatic foci. This result laid the foundation for future research. After analysis with Uniprot, Myosin-9was predicted to play important roles in the bioprocesses of actin cytoskeleton reorganization, actin filament-based movement, angiogenesis, cell-cell adhesion, cytokinesis, protein transport and regulation of cell shape. Above of all, we speculated that Myosin-9might be involved in the process of invasion and metastasis of gastric cancer. Hence the second and third parts were carried on to further study. The results showed that RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, promote the adhesion of gastric cancer cell, and notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. Myosin-9promotes the invasion and metastasis of gastric cancer probably by facilitating the EMT process of gastric cancer.
Although the present study demonstrated that Myosin-9expression impacted invasion and metastasis ability of the gastric carcinoma cells, but the development and progression of malignant tumors were the results of interactions between a variety of genes and proteins and it was difficult to fully explain the complex tumor biological behavior with a single gene. In the future research, we will analyze other differential proteins and contrast the relationships between these proteins, and eventually clarify the potential mechanism of peritoneal metastasis from gastric cancer. Besides, the study will be not only on tissues and cells but also on bold and body fluids of patients with peritoneal metastasis. We are looking forward to find an effective molecular marker to predict the peritoneal metastasis from gastric cancer.
The specific content of this topic is as follows:
Part1Isolation, identification and bioinformatics analysis of peritoneal metastasis related proteins of gastric cancer.
Purpose:To separate proteins in gastric cancer and corresponding peritoneal metastasis tissues, find and identify the differences; combined the technology of bioinformatics analysis, to search for the predictive indicators and ulteriorly provide a theoretical basis for tumor mechanism study. In addition, through this experiment the practical value of2D-DIGE technology in the gastric carcinoma study will be verified.
Methods:gastric cancer and corresponding peritoneal metastasis tissues served as the subjects,2D-DIGE method was applied to the purified protein for protein separation, According to the analysis of differential protein spots combining different maps of DIGE and Decyder software, we set these which was up-regulated or down-regulated2.0times as differential protein spots. Protein spots of interest were excised by automatic Spot picker, and then the picked spots were carried out with in-gel digestion, followed by using ABI4800MALDI TOF/TOF mass spectrometry. For understanding the relationship among proteins, we use the "String" protein interaction databases.
Results:(1).According to the analysis of differential protein spots combining different maps of DIGE and Decyder software,32differential protein spots were found. Retrieving SWISSPROT database through the mascot software, getting rid of the same protein within appraisal result and unidentified protein spots,27differential proteins were identified.23proteins were significantly up-regulated and4were down-regulated in peritoneal metastatic foci.(2) After analysis with Uniprot, myosin-9was predicted to play important roles in the bioprocesses of actin cytoskeleton reorganization, actin filament-based movement, angiogenesis, cell-cell adhesion, cytokinesis, protein transport and regulation of cell shape. Furthermore, single protein analysis by the String9.0program indicated that myosin-9was a key functional partner with ACE, RHOB, RHOA, ACTB, SVIL, CDK5, ACTG1, ROCK2, CALD1, and S100A4.Based on the above results, we selected Myosin-9for subsequent function research.(3) Western blot and immunocytochemistry were used to further verify Myosin-9in order to ensure the reliability of the proteome results. The western blot and immunocytochemistry results showed that Myosin-9was higher in peritoneal metastasis tissues, compared to that in gastric cancer tissues. Evidently, western blot results were consistent with proteome analysis.
Part2Studies on function of Myosin-9in the invasion and metastasis of gastric cancer.
Purpose:To explore the effects of Myosin-9on invasive and metastatic ability of gastric cancer cells.
Methods:The expression of Myosin-9mRNA was detected in gastric cancer cells by quantitate real-time PCR. The level of Myosin-9protein was detected by western blot. We transfected the lentivirus RNAi vectors into the gastric cancer cells to silence Myosin-9. Cell adhesion assay was used to analyze the effect of decreased Myosin-9on cell adhesion ability. Transwell assay was used to assess the effect of decreased Myosin-9on cell migration and invasion ability. The animal model with gastric cancer metastasized to peritoneal cavity was used to assess the effect of decreased Myosin-9on peritoneal metastasis ability of gastric cancer.
Results:(1) Compared with GES-1cells (gastric epithelial cells), enhanced signals were found in AGS and BGC823cells (derived from the primary lesion), especially in SGC7901cells (derived from the metastatic lymph node).Western blot analysis confirmed high Myosin-9protein expression in AGS, BGC823, and SGC7901cells. We choose the AGS and SGC7901for subsequent experiments.(2)After several preliminary experiments, we determined the best optimization conditions:cell inoculation density=50%, MOI=10, Polygrene=4μg/ml. Western blot showed that the lenti-virus sequence1had the best interference effect.(3) RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, and promote the adhesion of gastric cancer cell. The distinction between the groups was significantly different (P<0.01).(4) RNAi Myosin-9could notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. The distinction between the groups was significantly different (P<0.01).
Part3The preliminary research on the mechanism of Myosin-9in invasion and metastasis of gastric cancer.
Purpose:To explore to how that Myosin-9was involved in the invasion and metastasis of gastric cancer.
Methods:The expression of EMT related markers E-cadherin, N-cadherin, vimentin and β-catenin were detected by western blot between interference and negative control groups.
Results:RNAi Myosin-9could up-regulate the expression of E-cadherin, and down-regulate the expression of N-cadherin, vimentin and3-catenin.
After all, we have used a technology platform based on difference gel electrophoresis, MALDI TOF/TOF mass spectrometry and bioinformatics analysis to find and identify the proteins associated with peritoneal metastasis from gastric cancer. And then we conducted the initial functional verification and mechanism study of a differential protein.
After the research, four conclusions are drawn as follows:
1. RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, promote the adhesion of gastric cancer cell, and notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. These results suggest that Myosin-9can promote the invasion and metastasis of gastric cancer.
2. Myosin-9promotes the invasion and metastasis of gastric cancer probably by facilitating the EMT process of gastric cancer, the exact mechanisms still needs further study.
The innovation of this study:
1. Establishment of peritoneal metastasis of gastric cancer tissue protein finger prints.
2. Confirmed Myosin-9's role in invasion and metastasis of gastric cancer, and found that inhibition of Myosin-9could restrain the EMT process of gastric cancer.
目前临床上用于胃癌腹膜转移早期诊断的方法主要包括腹腔液细胞学检查和分子标志物检查等。腹腔液细胞学检查是目前胃癌腹膜亚临床转移早期预测的最常用方法和金标准,但是其敏感性较低,因为并非所有浸透浆膜的进展期胃癌均能检测出腹膜脱落癌细胞。目前用于检测的分子标志物指标包括CEA、CA125、E-钙粘素、β1整合素、MMPs、VEGF等,但是目前国内外基础研究尚未找到一种公认的早期预测进展期胃癌腹膜转移的分子标志物。因此如何找到有效的胃癌腹膜转移早期预测的分子标志物,对于提高胃癌腹膜转移的预防和治疗,改善患者的预后具有重要现实意义。近年来开展的蛋白质组学技术为揭示肿瘤转移机制带来了新的希望。
蛋白质组学(proteomics)及其相关技术是近年来新兴的分子生物学分支,是以基因编码的所有蛋白质组为研究对象,了解蛋白质之间的相互作用与联系,从而揭示蛋白质功能以及基因表达调控机制的一个新的研究领域,其核心技术是以双相凝胶电泳(Two-dimensional polyacry-lamide gel electrophoresis,2-DE)为主的蛋白质分离技术和以质谱(mass spec-trometry, MS)、生物信息学(Bioinformatics)为主的蛋白质鉴定技术。目前已经有许多新的差异蛋白质组分析方法用于实践,其中荧光差异凝胶电泳(difference gel electrophoresis, DIGE)技术由于继承了二维凝胶电泳(two-dimensional electrophoresis,2-DE)的高分辨率,同时又具备高重现性、高灵敏度、高通量和高动态范围等优势,使得DIGE日益受到关注,成为最受欢迎的定量蛋白质组学研究手段之一。DIGE技术通过对用不同的荧光染料对不同的蛋白样品进行标记,在同一块双向凝胶中可同时分离多种蛋白样品,并且每块胶上均引入了内标,内标可以进一步增加可信度,确保结果能反映出真实的生物学差异,避免了系统误差对实验结果的影响。目前,蛋白质组学技术已在多种肿瘤中用于筛选和鉴定同早期诊断、预后以及化疗敏感性等相关的肿瘤标记物。
综上所述,本课题主要关注的是寻找同胃癌腹膜转移相关的蛋白质,并选择其中一个差异蛋白进行进一步的功能验证和机制的初步研究。实验的第一部分使用2D-DIGE分离结合MALDITOF/TOF质谱对胃癌组织和相应腹膜转移组织的差异表达蛋白质进行筛选鉴定,共鉴定出27种蛋白质。其中在胃癌腹膜转移组织中表达上调的有23个蛋白质,表达下调的有4个蛋白质,这为今后研究奠定了基础。然后利用生物信息学分析发现在上述这些差异蛋白中,Myosin-9蛋白可能在肌动蛋白细胞骨架重组、肌动蛋白纤维运动、血管生成、细胞-细胞间黏附以及蛋白转运等生物过程中发挥重要作用,根据以上结果我们推测Myosin-9可能参与了胃癌的侵袭和转移过程。因此第二和第三部分我们对Myosin-9进行了更深入的研究,结果发现抑制Myosin-9的表达后,细胞的迁移和侵袭能力减弱,而粘附能力增加,裸鼠腹腔播散肿瘤结节形成数目也明显减少。然后进一步检测上皮间质转化(epithelial-mesenchymal transition,EMT)相关标志物发现,抑制Myosin-9的表达后,胃癌细胞EMT过程受到抑制。基于以上结果,我们认为Myosin-9能够促进胃癌细胞的侵袭和转移能力,此过程可能是通过促进胃癌细胞EMT而实现的。
虽然本实验证实Myosin-9影响了胃癌细胞的侵袭转移能力,但是恶性肿瘤的发生和发展是多种基因和蛋白质相互作用的结果,任何单个基因或蛋白质都很难全面说明肿瘤复杂的生物学行为,且Myosin-9在胃癌中更深层次的机制仍需深入研究。在今后的研究中,我们将对更多前期筛选出的差异蛋白质进行分析,通过研究对比蛋白之间的相互关系来阐明胃癌腹膜转移发生发展的机制。此外,研究对象也将不只限于组织和细胞,胃癌腹膜转移患者的血液、体液均可以进行研究,以期能够找到一种有效的进展期胃癌腹膜转移早期预测的分子标志物,提高临床诊断水平,指导胃癌亚临床转移的合理防治。
本课题三部分的具体内容如下：
第一章胃癌腹膜转移相关差异蛋白质组的分离、鉴定与生物信息学分析
目的：将胃癌组织和相应腹膜转移组织蛋白进行分离,找到差异点并进行鉴定,结合生物信息学技术分析,以寻找潜在的预测指标,并为进一步的功能和机制研究提供理论依据。另外本实验还可验证2D-DIGE技术在胃癌研究中的实用价值。
方法：以胃癌组织和相应腹膜转移组织为研究对象,将纯化后的蛋白质应用双向荧光差异凝胶电泳(2D-DIGE)进行蛋白质分离,根据DIGE的差异图谱并结合Decyder分析软件进行差异蛋白点分析,差异倍数上调2倍或下调2倍以上的列为差异蛋白点,利用全自动切胶仪Spot picker挖取这些差异蛋白胶粒,然后进行样品胶内酶解,使用ABI4800MALDI TOF/TOF质谱仪进行分析,通过Mascot软件检索Uniprot数据库鉴定蛋白,并结合Western blot进行验证。最后利用String蛋白相互作用数据库对蛋白质问的相互作用进行预测和分析。
结果：1.根据2D-DIGE的差异图谱并结合Decyder分析软件进行差异蛋白点分析,共找到了32个差异蛋白点,然后通过Mascot软件检索Uniprot数据库鉴定蛋白,去除鉴定结果相同的蛋白及未鉴定出来的蛋白点,共鉴定出27种蛋白质。其中在胃癌腹膜转移组织中表达上调的有23个蛋白质,表达下调的有4个蛋白质。2.通过Uniprot数据库分析提示在上述这些差异蛋白中,Myosin-9蛋白可能在肌动蛋白细胞骨架重组、肌动蛋白纤维运动、血管生成、细胞-细胞间黏附以及蛋白转运等生物过程中发挥重要作用,进一步利用String蛋白相互作用数据库分析发现Myosin-9同ACE, RHOB, RHOA, ACTB, SVIL, CDK5, ACTG1,ROCK2,CALD1和S100A4等蛋白构成相互作用网络。基于以上结果分析,我们选取Myosin-9蛋白进行后续功能研究。3.应用Western blot和免疫组化方法对胃癌组织和相应腹膜转移组织中Myosin-9蛋白的表达水平进行验证,结果显示Myosin-9蛋白在腹膜转移组织中表达增强,与蛋白质组学结果一致。
第二章Myosin-9在胃癌侵袭转移中的功能研究
目的：探讨Myosin-9对胃癌细胞侵袭转移能力的影响。
方法：利用qRT-PCR和western-blot方法分别在mRNA水平和蛋白水平上检测Myosin-9在胃癌细胞株中的表达；合成针对Myosin-9的siRNA,利用慢病毒载体感染细胞株,构建稳定低表达Myosin-9蛋白的细胞系；利用纤维链接蛋白实验检测Myosin-9对胃癌细胞粘附能力的影响,Transwell小室迁移和侵袭实验检测Myosin-9对胃癌细胞迁移和侵袭能力的影响；构建裸鼠腹腔播散种植模型,观察Myosin-9对胃癌细胞腹膜转移能力的影响。
结果：1.相对于人胎儿胃粘膜细胞株GES-1, Myosin-9在其余3株胃癌细胞株AGS、BGC823和SGC7901中均高表达,其中AGS和BGC-823细胞株都来源于胃癌组织(低分化),SGC7901细胞株来源于淋巴结转移组织(中分化),我们选择AGS和SGC7901进行后续实验。2.通过多次预实验,我们确定了细胞接种密度为50%,MOI值为10,Polybrene为4ug/ml的最佳优化条件,western-blot结果显示慢病毒序列1的干扰效果最佳,按照上述条件我们成功构建了稳定低表达Myosin-9蛋白的细胞系SGC7901-siRNA和AGS-siRNA。3.干扰Myosin-9后,细胞粘附实验结果显示细胞粘附能力增强,细胞迁移和侵袭实验结果显示细胞迁移和侵袭能力减弱,以上各组间差异均有统计学意义,P<0.01。4.干扰Myosin-9后,相对于阴性对照组,干扰组腹腔内肿瘤结节形成数目明显减少,两者间差异有统计学意义,P<0.01。
第三章Myosin-9在胃癌侵袭和转移中作用机制的初步研究
目的：探讨Myosin-9在胃癌侵袭和转移中的作用机制。
方法：稳定沉默Myosin-9表达后,利用western-blot方法检测AGS和SGC7901细胞EMT相关标记物E-cadhern、N-cadhern、vimentin和β-catenin的表达情况。
结果：相对于阴性对照组和空白对照组,干扰组中E-cadherin表达上调,而N-cadherin、vimentin和β-catenin均表达下调。
总之,本研究利用基于2D-DIGE分离-MALDI TOF/TOF质谱鉴定-生物信息学分析的差异蛋白质组学技术平台,成功分离并鉴定出胃癌腹膜转移相关的蛋白质,并对其中的一个差异蛋白Myosin-9进行了功能验证和初步的机制研究。
通过研究,我们得出以下结论：
1.抑制Myosin-9的表达后,胃癌细胞的迁移和侵袭能力减弱,而粘附能力增强,此外裸鼠腹腔肿瘤播散转移能力下降,提示Myosin-9能够促进胃癌的侵袭和转移。
2.Myosin-9促进胃癌的侵袭和转移可能是通过促进胃癌细胞EMT过程而发挥作用的,确切的机制仍需进一步研究。
本研究的创新处在于：
1.建立了胃癌腹膜转移组织差异蛋白图谱；
2.证实了胃癌腹膜转移相关蛋白Myosin-9能够促进胃癌的侵袭和转移,并发现Myosin-9表达下调能抑制胃癌细胞EMT过程。
Gastric cancer is the most common malignant tumor of alimentary tract, despite improved prognosis as result of early diagnosis, radical operation and the development of adjuvant therapy, the mortality rate is still ranked among the top of malignant tumor mortality. There are many factors that can affect the prognosis of gastric cancer, in which the peritoneal metastasis are common in recurrence and metastasis of gastric cancer. Peritoneal dissemination usually leads to the terminal stage of advanced GC and diminishes the quality of life by refractory ascites and intestinal obstruction. As a result, how to effectively forecast and control of peritoneal metastasis is one of the key issues on improving the curative effect of advanced gastric cancer currently.
The current clinical method for early diagnosis of peritoneal metastasis from gastric cancer including detection of peritoneal lavage cytology and molecular marker. Peritoneal lavage cytology detection is the most commonly used method and the gold standard in early prediction of peritoneal subclinical metastasis from gastric cancer, but it lacks high sensitivity, since not all the advanced gastric cancer that soaked serous could be found the exfoliated cancer cells. Currently the molecular markers for the detection are CEA, CA125, E-cadherin, β1-integrin, MMPs and VEGF. However, basic researchers at home and abroad have not yet found an accepted molecular marker for early detection of peritoneal metastasis from advanced gastric cancer. At present, most of the research associated with metastasis so far has been focused on the genetic changes of related molecules or single or few proteins without systematicstudy. But little is known about the key factors to trigger tumorigenic cells to initiatefurther invasion and metastasis facing with so many regulators up to now.
Proteomics along with its related technologies, an emerging branch of molecular biology in recent years, focuses on studies of the proteome encoded by genes, and makes complete analysis of the composition, expression levels and modification state of the intracellular proteins with dynamic changes, and understands the interactions and associations among proteins, thus revealing protein function and its relationship with laws of the cellular activities as well as regulation mechanisms of gene expression as a new research field. Its core technology is protein separation techniques based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification technology based on mass spectrometry (MS) and bioinformatics. At present there are many novel differential proteomics analysis methods, among which the difference gel electrophoresis (difference gel electrophoresis, DIGE) technology becomes one of the most popular one, because it's not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. DIGE is a method which can label protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the real biological differences, while avoid influence of systematic errors on experimental results. To date,2D-DIGE technologies have been used to identify novel candidate biomarkers associated with early diagnosis, differential diagnosis, prognosis, and response to chemotherapy in various diseases.
Above all, this topic mostly focused on screening and identificating peritoneal metastasis related proteins of gastric cancer, and then selecting one of interesting protein for further functional verification and mechanism of a preliminary study. In the first part of experiment, two-dimensional fluorescence difference gel electropho-resis (2D-DIGE) was employed together with MALDI TOF/TOF mass spectrometry to screen and identify differentially expressed proteins between gastric cancer and corresponding peritoneal metastasis tissues.27differential proteins were identified,23proteins were significantly up-regulated and4were down-regulated in peritoneal metastatic foci. This result laid the foundation for future research. After analysis with Uniprot, Myosin-9was predicted to play important roles in the bioprocesses of actin cytoskeleton reorganization, actin filament-based movement, angiogenesis, cell-cell adhesion, cytokinesis, protein transport and regulation of cell shape. Above of all, we speculated that Myosin-9might be involved in the process of invasion and metastasis of gastric cancer. Hence the second and third parts were carried on to further study. The results showed that RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, promote the adhesion of gastric cancer cell, and notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. Myosin-9promotes the invasion and metastasis of gastric cancer probably by facilitating the EMT process of gastric cancer.
Although the present study demonstrated that Myosin-9expression impacted invasion and metastasis ability of the gastric carcinoma cells, but the development and progression of malignant tumors were the results of interactions between a variety of genes and proteins and it was difficult to fully explain the complex tumor biological behavior with a single gene. In the future research, we will analyze other differential proteins and contrast the relationships between these proteins, and eventually clarify the potential mechanism of peritoneal metastasis from gastric cancer. Besides, the study will be not only on tissues and cells but also on bold and body fluids of patients with peritoneal metastasis. We are looking forward to find an effective molecular marker to predict the peritoneal metastasis from gastric cancer.
The specific content of this topic is as follows:
Part1Isolation, identification and bioinformatics analysis of peritoneal metastasis related proteins of gastric cancer.
Purpose:To separate proteins in gastric cancer and corresponding peritoneal metastasis tissues, find and identify the differences; combined the technology of bioinformatics analysis, to search for the predictive indicators and ulteriorly provide a theoretical basis for tumor mechanism study. In addition, through this experiment the practical value of2D-DIGE technology in the gastric carcinoma study will be verified.
Methods:gastric cancer and corresponding peritoneal metastasis tissues served as the subjects,2D-DIGE method was applied to the purified protein for protein separation, According to the analysis of differential protein spots combining different maps of DIGE and Decyder software, we set these which was up-regulated or down-regulated2.0times as differential protein spots. Protein spots of interest were excised by automatic Spot picker, and then the picked spots were carried out with in-gel digestion, followed by using ABI4800MALDI TOF/TOF mass spectrometry. For understanding the relationship among proteins, we use the "String" protein interaction databases.
Results:(1).According to the analysis of differential protein spots combining different maps of DIGE and Decyder software,32differential protein spots were found. Retrieving SWISSPROT database through the mascot software, getting rid of the same protein within appraisal result and unidentified protein spots,27differential proteins were identified.23proteins were significantly up-regulated and4were down-regulated in peritoneal metastatic foci.(2) After analysis with Uniprot, myosin-9was predicted to play important roles in the bioprocesses of actin cytoskeleton reorganization, actin filament-based movement, angiogenesis, cell-cell adhesion, cytokinesis, protein transport and regulation of cell shape. Furthermore, single protein analysis by the String9.0program indicated that myosin-9was a key functional partner with ACE, RHOB, RHOA, ACTB, SVIL, CDK5, ACTG1, ROCK2, CALD1, and S100A4.Based on the above results, we selected Myosin-9for subsequent function research.(3) Western blot and immunocytochemistry were used to further verify Myosin-9in order to ensure the reliability of the proteome results. The western blot and immunocytochemistry results showed that Myosin-9was higher in peritoneal metastasis tissues, compared to that in gastric cancer tissues. Evidently, western blot results were consistent with proteome analysis.
Part2Studies on function of Myosin-9in the invasion and metastasis of gastric cancer.
Purpose:To explore the effects of Myosin-9on invasive and metastatic ability of gastric cancer cells.
Methods:The expression of Myosin-9mRNA was detected in gastric cancer cells by quantitate real-time PCR. The level of Myosin-9protein was detected by western blot. We transfected the lentivirus RNAi vectors into the gastric cancer cells to silence Myosin-9. Cell adhesion assay was used to analyze the effect of decreased Myosin-9on cell adhesion ability. Transwell assay was used to assess the effect of decreased Myosin-9on cell migration and invasion ability. The animal model with gastric cancer metastasized to peritoneal cavity was used to assess the effect of decreased Myosin-9on peritoneal metastasis ability of gastric cancer.
Results:(1) Compared with GES-1cells (gastric epithelial cells), enhanced signals were found in AGS and BGC823cells (derived from the primary lesion), especially in SGC7901cells (derived from the metastatic lymph node).Western blot analysis confirmed high Myosin-9protein expression in AGS, BGC823, and SGC7901cells. We choose the AGS and SGC7901for subsequent experiments.(2)After several preliminary experiments, we determined the best optimization conditions:cell inoculation density=50%, MOI=10, Polygrene=4μg/ml. Western blot showed that the lenti-virus sequence1had the best interference effect.(3) RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, and promote the adhesion of gastric cancer cell. The distinction between the groups was significantly different (P<0.01).(4) RNAi Myosin-9could notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. The distinction between the groups was significantly different (P<0.01).
Part3The preliminary research on the mechanism of Myosin-9in invasion and metastasis of gastric cancer.
Purpose:To explore to how that Myosin-9was involved in the invasion and metastasis of gastric cancer.
Methods:The expression of EMT related markers E-cadherin, N-cadherin, vimentin and β-catenin were detected by western blot between interference and negative control groups.
Results:RNAi Myosin-9could up-regulate the expression of E-cadherin, and down-regulate the expression of N-cadherin, vimentin and3-catenin.
After all, we have used a technology platform based on difference gel electrophoresis, MALDI TOF/TOF mass spectrometry and bioinformatics analysis to find and identify the proteins associated with peritoneal metastasis from gastric cancer. And then we conducted the initial functional verification and mechanism study of a differential protein.
After the research, four conclusions are drawn as follows:
1. RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, promote the adhesion of gastric cancer cell, and notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. These results suggest that Myosin-9can promote the invasion and metastasis of gastric cancer.
2. Myosin-9promotes the invasion and metastasis of gastric cancer probably by facilitating the EMT process of gastric cancer, the exact mechanisms still needs further study.
The innovation of this study:
1. Establishment of peritoneal metastasis of gastric cancer tissue protein finger prints.
2. Confirmed Myosin-9's role in invasion and metastasis of gastric cancer, and found that inhibition of Myosin-9could restrain the EMT process of gastric cancer.
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