JAK2与CD95对结直肠癌细胞增殖、凋亡及上皮—间质转化作用的研究

发布时间：2018-03-22 00:32

  本文选题：结直肠癌　切入点：CD95　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：目的 本研究探讨介导炎症反应的JAK2和介导免疫反应的CD95与结直肠癌预后的相关性；探讨JAK2和CD95受体在结直肠癌组织及细胞株中的表达水平，分析二者的相关性；通过抑制或增强JAK2的活化，探讨大肠癌细胞在增殖能力、细胞表型（上皮型/间质型）以及CD95活性方面的变化，从细胞和分子水平证实JAK2介导的炎症信号通路与大肠癌细胞的免疫功能之间的关系及其作用机制。 方法 1.利用生物信息学分析的方法：借助荷兰医学生物数据平台，分析来源于荷兰8家医院的大肠癌数据库中CD95和JAK2的表达水平、CD95和JAK2的表达与生存期的Kaplan-Meier曲线，借助平台中的基因热图（geneheatmap），将与CD95关系密切的基因呈现出来，以形象地呈现出CD95表达相关的功能基因；利用STRING在线软件分析预测CD95相关蛋白及其功能；利用基因文氏图（Gvenn）分析CD95相关基因与JAK2相关基因的重叠程度，并预测数据库中CD95与JAK2的相关性。利用在线meta软件计算3个数据库中CD95与JAK2总体相关性系数（r值、p值）。 2.结直肠癌组织切片和细胞的获取：2011年10月-2012年5月在荷兰Utrecht大学医学中心胃肠外科经手术切除的结直肠癌组织，经福尔马林固定、石蜡包埋后制成结直肠癌组织切片，从中选取10例石蜡包埋组织切片；取同期手术切除的结直肠癌新鲜组织进行细胞建株并稳定传代获得结直肠癌（CRC）细胞，本实验选取其中9株用于研究。 3. CD95和JAK2相关性分析：通过免疫组化染色检测CD95和JAK2在结直肠癌组织切片中的表达水平，通过免疫印迹法检测二者在CRC细胞中的表达水平，并分析二者的相关性。 4.结直肠癌细胞中JAK2/STAT3的阻断：在细胞培养体系中加入JAK2特异性抑制剂AZD1480和CEP33779，通过western blot检测AZD1480和CEP33779在不同浓度（0.5μM~5μM）和不同作用时间（2h~24h）下JAK2、STAT3和pSTAT3水平，对照组为同一细胞株在相应浓度DMSO和作用时间下JAK2、STAT3和pSTAT3水平，阳性对照采用同一细胞株在IL-6（10ng/ml，30min）刺激后JAK2、STAT3和pSTAT3的水平。 5.结直肠癌细胞的克隆形成能力检测：结直肠癌细胞团被解离成单个细胞后，以1000个细胞/100μl基质胶的浓度接种到12孔板中，待基质胶凝固后每孔添加2ml培养基，实验组及对照组试剂添加到培养基中。2天换1次液，12天后观察细胞团形成数量。 6.流式细胞技术检测细胞凋亡：结直肠癌细胞分别经过一定浓度的JAK2特异性抑制剂AZD1480、CEP33779作用2天后，以等剂量的DMSO处理CRC细胞2天作为对照，采用荧光染料碘化吡啶（PI）染色（4℃）固定，4h后流式细胞仪检测细胞凋亡的百分率。 7.免疫荧光和实时荧光定量PCR检测：JAK2特异性抑制剂CEP33779抑制JAK2/STAT3通路活化，对参与上皮-间质转化（EMT）的相关蛋白进行免疫荧光检测，对EMT标志物HIF-1、ZEB-1、ZEB-2、FABP、Snail-1、Snail-2、vimentin等转录因子的mRNA进行逆转录后荧光定量PCR检测。 8. FasL刺激下细胞分泌细胞因子的检测：实验组培养体系FasL终浓度20ng/ml，对照组为同株无FasL的细胞，12h后收集上清液，经层析、浓缩后，通过细胞因子芯片检测结直肠癌细胞分泌细胞因子的变化。 9. CD95-黄色荧光蛋白报告基因（YFP-CD95）的转染及CRC细胞的共聚焦显微镜检查：分别构建pWPT-CD95-YFP，同时构建pWPT-YFP作为对照。采用磷酸钙法转染，，使用FuGene转染试剂将慢病毒包装载体和pWPT-CD95-YFP或pWPT-YFP转染进293T细胞。细胞培养24-48h后收集上清液获得病毒。CRC29和L145细胞培养6-16h后进行解离，用聚凝胺进行慢病毒上清转染24h。转染后的细胞根据YFP-CD95表达高低经流式细胞分选，从而获得纯度较高的CD95高表达细胞株。利用FasL和/或CEP33779作用CRC细胞1h、2h后，在共聚焦荧光显微镜下观察细胞表面YFP-CD95荧光信号的变化。 结果 1. CD95与JAK2在结直肠癌中的相关性 （1）生物信息学分析CD95和JAK2与结直肠癌患者预后呈正相关CD95和JAK2在8个结直肠癌数据库共1293例结直肠癌病人组织中每组内部表达水平差异较大，而各数据库间无显著性差异；CD95和JAK2高表达，病人生存期较短。 （2）CD95与JAK2在结直肠癌中的表达及功能相关性 STRING分析显示CD95与结直肠癌的免疫反应、炎症反应与炎症因子关系密切。Gvenn分析显示CD95相关基因与JAK2高度相关。在线meta分析发现CD95相关基因中JAK2与其相关性最好。与CD95同时表达的基因，能够调控炎症反应、JAK2/STAT3信号通路、上皮-间质转化（EMT）。对10例结直肠癌石蜡切片的免疫组化检查发现CD95高表达的肿瘤组织同时存在JAK2高表达，反之亦然，二者呈正相关。 （3）CD95和JAK2在结直肠癌细胞中表达的相关性 经细胞建株并稳定传代的结直肠癌细胞株，包括来源于结直肠癌原发肿瘤的细胞株CRC26、CRC29、CR9、CR16、CRC48、CRC47，来源于肝转移灶的细胞株为L145、L167、L169。JAK2表达水平CRC29、CR9、CR16最高，其余依次为L169、CRC47、CRC48、L167、L145、CRC26，CD95表达水平由高到低依次为CRC29、L145、CRC47、CR16、CRC48、CRC26、L169、CR9、L167。9株CRC细胞株中CD95与JAK2的表达具有一定的相关性（r2=0.5087，p=0.0470），而来源结直肠的原发肿瘤细胞CD95与JAK2的表达相关性较好（r2=0.7360，p=0.0289）。 2. JAK2对结直肠癌细胞增殖、凋亡、EMT的影响 （1）JAK2活化抑制后结直肠癌细胞的克隆增殖能力变化结直肠癌细胞在基质胶中培养，对照组用等浓度的DMSO处理，实验组利用JAK2抑制剂阻断JAK2活化，其克隆增殖能力明显降低。 （2）JAK2活化后结直肠癌细胞的克隆增殖能力变化结直肠癌细胞在基质胶中培养，实验组利用合适浓度的IL-6活化JAK2，其克隆增殖能力较对照组明显增强。不同IL-6浓度刺激下结直肠癌细胞的克隆增殖无明显差异。 （3）JAK2阻断后CRC细胞凋亡率变化 对于悬浮培养的结直肠癌细胞CRC29，用不同作用浓度的JAK2特异性抑制剂AZD1480和CEP33779分别阻断JAK2活化，抑制剂作用2天后，采用碘化吡啶（Propidium Iodide，PI）染色法检测CRC细胞凋亡。与对照组相比，JAK2特异性抑制后CRC细胞凋亡无明显增加（p0.05）。 （4）JAK2阻断下FasL诱导细胞凋亡率变化 对于悬浮培养的结直肠癌细胞CRC29，JAK2特异性抑制AZD1480和CEP33779阻断JAK2活化，2h后在培养体系中加入杀伤剂量（20ng/ml）的FasL，作用2天后检测CRC细胞凋亡。AZD1480和CEP33779两组中CRC29细胞凋亡率分别为33.52%、38.76%，AZD1480预先抑制JAK2活化后FasL诱导CRC29细胞凋亡增至41.20%、45.19%，CEP33779预先抑制JAK2活化后FasL诱导CRC29细胞凋亡增至62.34%、62.65%。 （5）JAK2抑制后与EMT相关的蛋白表达及转录因子水平发生变化 ①CRC29细胞系在悬浮培养条件下，与IL-6刺激的CRC29细胞系相比，受到JAK2抑制的CRC29细胞系Caveolin-1表达减低，E-cadherin表达升高，共聚焦显微镜下可见到实验组细胞－细胞连接处Caveolin-1荧光信号减弱，E-cadherin荧光信号明显增强。②CRC29细胞系在悬浮培养条件下，与IL-6刺激的CRC29细胞系相比，受到JAK2抑制的CRC29细胞系HIF-1、ZEB-1、FABP、Snail-1、等基因mRNA表达降低，其中ZEB-2、Snail-2和HIF-2变化最为明显，vimentin表达增加。 3.特异性阻断JAK2对CRC细胞CD95表型的影响 （1）FasL能够刺激CRC细胞分泌细胞因子变化 FasL作用24h后CRC细胞分泌IL-8、IL-15R alpha水平升高，而预先加入CEP33779抑制JAK2活化的CRC细胞分泌IL-8水平进一步升高。 （2）转染CD95-YFP细胞株对FasL及JAK2阻断的反应 对成功转染了CD95-YFP质粒并稳定表达CD95-YFP的结直肠癌细胞系CRC29，分别用FasL、CEP33779、CEP33779预处理1h而后FasL（CEP33779→FasL），作用1h后，给予固定，随后通过荧光显微镜观察细胞膜上带有黄色荧光信号的CD95的表型变化，即是否聚集成为活性形式，对照组以等浓度的DMSO作用于培养体系，结果发现在JAK2阻断1h后，细胞膜表面荧光强度明显高于对照组，而CEP33779→FasL处理组CRC细胞表面CD95表达明显耗减。 结论 1.对多个结直肠癌数据库的生存曲线荟萃分析得知，结直肠癌组织中CD95和JAK2高表达，患者的生存期较短。 2.结直肠癌数据库中CD95与JAK2在表达、功能相关通路等方面的相关性分析表明，CD95与JAK2在结直肠癌组织中的高度相关性，主要体现在肿瘤的免疫反应、炎症反应、JAK2/STAT3信号通路、上皮-间质转化（EMT）几个方面。 3.体外培养的结直肠癌细胞在增殖过程中，JAK2/STAT3处于较低水平的活化状态，特异性阻断JAK2/STAT3通路能够降低或完全抑制肿瘤细胞的克隆形成能力，提示JAK2在肿瘤细胞增殖方面具有关键作用；特异性阻断JAK2/STAT3通路在一定程度上抑制了肿瘤EMT，从而对肿瘤细胞的迁移可能产生一定的抑制作用；而JAK2/STAT3的过度活化则促进肿瘤细胞增殖。JAK2抑制不能诱导CRC细胞凋亡，而FasL可诱导肿瘤细胞凋亡增多。 4.通过转染CD95-YFP报告基因并使其稳定表达CD95的结直肠癌细胞系CRC29（CRC29CD95-YFP），抑制JAK2/STAT3通路有助于肿瘤细胞表面CD95单体寡聚化形成转变成活性形式，为介导细胞凋亡提供细胞表面结构条件，从而证明JAK2对CRC细胞膜上CD95具有一定的稳定作用。
[Abstract]:objective
This study was to investigate the relationship between CD95 and prognosis of colorectal cancer mediated inflammation and JAK2 mediated immune response; to investigate the expression of JAK2 and CD95 receptor in colorectal cancer tissues and cell lines, analysis of the relationship between the two; by inhibiting or enhancing the activation of JAK2 on colorectal cancer cell in proliferation, cell phenotype (epithelial / mesenchymal type) changes and the activity of CD95, confirmed the relationship between immune function and inflammatory signaling pathways in colorectal cancer cells mediated by JAK2 and the mechanism of the cellular and molecular level.
Method
1. by bioinformatics analysis: with the Holland medical and biological data platform, the expression level of CD95 and JAK2 analysis from 8 hospitals in Holland of colorectal cancer database, CD95 and JAK2 expression and survival curve of Kaplan-Meier, with the help of gene thermography in the platform (geneheatmap), the close relationship with CD95 gene to show, vividly showing the expression of CD95 related functional genes; analysis and prediction of CD95 protein and its function using STRING online software; using gene Venn diagram (Gvenn) analysis of overlap CD95 related genes associated with JAK2 gene, and to predict the relationship between CD95 and JAK2 in the database. Using the online software meta 3 the database CD95 and JAK2 general correlation coefficient (r value, P value).
To obtain 2. colorectal cancer tissues and cells: October 2011 -2012 year in May in gastrointestinal surgery, Utrecht University Medical Center in Holland after surgical resection of colorectal cancer, formalin fixed, paraffin embedded tissue sections made of colorectal cancer, 10 cases of paraffin embedded tissue sections from; take over the same period surgical resection of colorectal cancer tissues cell lines stably and obtain colorectal cancer (CRC) cells, 9 of them were selected for this experiment.
3. CD95 and JAK2 correlation analysis: immunohistochemical staining was used to detect the expression level of CD95 and JAK2 in colorectal cancer tissue slice. The expression level of two in CRC cells was detected by Western blotting, and the correlation between the two was analyzed.
Blockade of JAK2/STAT3 4. in colorectal cancer cell: the JAK2 specific inhibitor AZD1480 and CEP33779 were added into the culture system, through the Western blot detection of AZD1480 and CEP33779 in different concentration (0.5 M~5 M) and different time (2h~24h) of JAK2, STAT3 and pSTAT3, the control group for the same cell lines in the corresponding the concentration of DMSO and reaction time JAK2, STAT3 and pSTAT3, positive control using the same cell line in IL-6 (10ng/ml, 30min) after the stimulation of JAK2, STAT3 and pSTAT3 levels.
The ability to detect the formation of clone 5. colorectal cancer cells: colorectal cancer cells were dissociated into single cells after inoculation of 1000 cells in a concentration of /100 l matrix to 12 Kong Banzhong, the matrix Plastic solidified per hole adding 2ml medium, the experimental group and the control group reagent was added to the medium.2 for 1 days liquid, 12 days after the observation of cell colony number.
6. flow cytometry: apoptosis of colorectal cancer cells respectively by JAK2 specific inhibitor AZD1480 in certain concentration, CEP33779 for 2 days, DMSO cells were treated with low-dose CRC for 2 days as control, using the fluorescent dye propidium iodide (PI) staining (4 C) fixed, the percentage of 4H cell apoptosis detection cell apparatus.
7. immunofluorescence and real-time fluorescence quantitative PCR detection of JAK2 specific inhibitor CEP33779 inhibits the activation of JAK2/STAT3 pathway, involved in epithelial mesenchymal transition (EMT) related proteins were detected by immunofluorescence, EMT markers HIF-1, ZEB-1, ZEB-2, FABP, Snail-1, Snail-2, vimentin and other transcription factors mRNA reverse transcription fluorescence quantitative detection of PCR.
8. FasL stimulated the secretion of cytokines detection cell culture system: experimental group FasL final concentration of 20ng/ml, the control group without monoecious FasL cells, 12h cell culture supernatants were collected by chromatography, concentration, the changes of cytokine microarray for detection of colorectal cancer cells to secrete cytokines.
9. CD95- yellow fluorescent protein (YFP-CD95) and confocal microscopy and transfection of CRC cells: pWPT-CD95-YFP were constructed, pWPT-YFP was constructed as control. Using calcium phosphate transfection method, using FuGene transfection reagent package lentivirus vector and pWPT-CD95-YFP or pWPT-YFP and transfected into 293T cells. The cell culture supernatant was collected after dissociation of the 24-48h were obtained.CRC29 virus and L145 cell culture after 6-16h was transfected into 24h. by lentivirus supernatant of transfected cells after Polybrene according to the expression level of YFP-CD95 by flow cytometry, so as to obtain a high degree of purity of CD95 high expression cell line. Using FasL and / or CEP33779 CRC cells 1H, 2h, observe the changes of cell surface YFP-CD95 the fluorescence signal in the confocal fluorescence microscope.
Result
Correlation between 1. CD95 and JAK2 in colorectal cancer
(1) bioinformatics analysis of CD95 and JAK2 in colorectal cancer and the prognosis of the patients was positively related to CD95 and JAK2 within each in 8 colorectal cancer database of 1293 cases of colorectal cancer patients the expression level differences, but no significant difference between each database; high expression of CD95 and JAK2, the survival time is short.
(2) expression and function correlation of CD95 and JAK2 in colorectal cancer
STRING analysis showed that the immune response of CD95 and colorectal cancer, the relationship between inflammatory response and inflammatory factors in close.Gvenn analysis showed that CD95 gene is highly correlated with JAK2. The online meta analysis showed that CD95 related gene JAK2 and its expression in the best correlation. At the same time with the CD95 gene, can control inflammation, JAK2/STAT3 signaling pathway, epithelial mesenchymal transition (EMT). Immunohistochemistry in 10 cases of colorectal cancer paraffin section examination found high expression of CD95 in tumor tissue and high expression of JAK2, and vice versa, two were positively correlated.
(3) the correlation between CD95 and JAK2 expression in colorectal cancer cells
The cell lines and stable passage of the colorectal cancer cell lines, including from the primary colorectal tumor cell lines CRC26, CRC29, CR9, CR16, CRC48, CRC47, derived from liver metastasis cell lines L145, L167, L169.JAK2 expression level of CRC29, CR9, CR16 is the highest, followed by the remaining L169 CRC47, CRC48, L167, L145, CRC26, CD95 expression level from high to low was CRC29, L145, CRC47, CR16, CRC48, CRC26, L169, CR9, CD95 and JAK2 expression of CRC cell lines in L167.9 has a certain correlation (r2=0.5087, p=0.0470), and the primary source of colorectal CD95 and the good correlation between the expression of JAK2 tumor cells (r2=0.7360, p=0.0289).
Effect of 2. JAK2 on the proliferation, apoptosis and EMT of colorectal cancer cells
(1) the clonal proliferation ability of colorectal cancer cells was inhibited after JAK2 activation inhibition. The colorectal cancer cells were cultured in matrix glue. The control group was treated with equal concentration of DMSO. The experimental group used JAK2 inhibitor to block JAK2 activation, and the clonal proliferation ability of colorectal cancer cells was significantly reduced.
(2) the proliferation changes of JAK2 activation after cloning of colorectal cancer cells of colorectal cancer cells cultured on Matrigel, the experimental group using the appropriate concentration of IL-6 activated JAK2, the clone proliferation ability were markedly increased compared to control group. Cloning stimulation under different IL-6 concentration in colorectal cancer cell proliferation had no significant difference.
(3) changes in the apoptosis rate of CRC cells after JAK2 blockage
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