CCR7基因对食管鳞状细胞癌新生血管的作用及机制研究

发布时间：2018-05-25 20:05

本文选题：食管鳞状细胞癌 + VEGF-A　；参考：《第三军医大学》2016年博士论文

【摘要】：研究背景食管癌(Esophageal cancer,EC)是一种常见的消化系统恶性肿瘤。在我国食管癌发病率和死亡率均居世界首位,全球范围内因该病造成的死亡患者有一半发生在我国。其死亡率占据第二位,仅次于胃癌。食管鳞癌(esophageal squamous cell carcinoma,ESCC)占EC的90%以上,因此本研究主要针对ESCC进行探讨。虽然通过早期手术切除及后续的放疗、化疗等策略,可以使ESCC患者的生存率有所提高,然而这些治疗方式难以达到根治效果,且同时该类患者还伴有不同程度的区域淋巴结转移或远处转移。肿瘤的发生、发展、侵犯、转移是一个多基因、多分子及多系统共同参与的复杂的病理过程。其中肿瘤新血管的生成(angiogenesis)是重要因素之一。新生血管与肿瘤的生长、侵袭和淋巴结转移等过程紧密相关。趋化因子受体(chemokine receptor)是G蛋白偶联受体超家族的成员,是一类表达在某些特定的细胞膜表面的跨膜受体。近年来的研究表明,趋化因子通过与受体结合,参与新生血管生成,进一步影响肿瘤生成和转移。不同结构的趋化因子在血管生成中起不同作用,可以作为肿瘤治疗的新靶点。最近的研究已证实,趋化因子受体7(chemokine receptor,CCR7)在多种肿瘤细胞中存在过度表达,这提示肿瘤的发生发展、浸润和转移与CCR7有着密不可分的关系。血管内皮生长因子(vascular endothelial growth factor,VEGF),最早亦称作血管通透因子,其与内皮细胞的增殖、迁移和管腔形成密切相关。VEGF家族包括VEGF-A、VEGF-B、VEGF-C及VEGF-D等几种同类物。酪氨酸激酶在VEGF与其受体VEGFR结合后被激活,继发一系列信号通路级联反应,随后激发肿瘤血管内皮发生增殖、迁移和新生血管管腔形成。核转录因子-κB(nuclear transcription factor Kappa B,NF-ΚB),最早是从成熟的B淋巴细胞中分离提取出来的蛋白,为真核细胞转录调节因子,存在于几乎所有的动物细胞中。NF-κB具有广泛的生物学活性,是炎性反应与肿瘤相互联系的重要分子蛋白,在炎症、代谢、人体免疫以及肿瘤的发生、发展、侵犯、转移等方面发挥着关键作用。大量研究表明,NF-κB与肿瘤有密切关系,可以通过调节各种不同靶基因,参与多种信号通路,诱导细胞抑制凋亡并促进细胞的增殖、侵袭、转移,促进肿瘤细胞血管生成、与致癌作用密切相关。目前国内外有较多的临床资料显示,CCR7的高表达与ESCC的发生、浸润及转移密切相关。但是,对于CCR7在ESCC细胞株中的表达,和ESCC血管形成中的作用,及其与NF-κB、VEGF等分子的相互作用关系目前尚不清楚。目标在本研究中,我们首先检测CCR7在ESCC细胞株中的表达,进而通过体内外实验,观察CCR7的异常表达与ESCC新生血管形成的关系,以及CCR7与VEGF、NF-κB及其下游基因的相互作用关系,再进一步探讨其中的分子机制。力图为阐释食管癌转移、复发的机制,以及基于CCR7的靶向治疗药物开发奠定基础。第一部分CCR7在ESCC中表达情况方法1、应用生物信息学技术方法回顾性整理并研究了不同报道中ESCC患者肿瘤组织中CCR7表达情况;2、应用逆转录-聚合酶链反应(RT-PCR)及免疫印迹(WB)方法联合检测CCR7在多种不同种系的ESCC细胞中的表达情况。结果1、针对CCR7基因的表达情况在GEO PROFILE数据库(https://www.ncbi.nlm.nih.gov/gds/)中进行搜索分析,结果发现ESCC中的CCR7的表达较正常组织中CCR7的水平显著提高。2、Western Blot结果显示,所有6株ESCC细胞系(TE-1,TE-2,TE-3,TE-8,TE-12和ECA-109)CCR7蛋白表达均显著高于正常食管上皮细胞株HEEC。q RT-PCR结果显示在m RNA水平上,ESCC细胞系中的CCR7表达显著高于HECC细胞。结果提示,CCR7在食管鳞状细胞癌基因及蛋白水平均具有高表达的特性。第二部分CCR7在Eca109细胞增殖、迁移和诱导新生血管形成中的作用方法1、建立CCR7稳定过表达和沉默的Eca109细胞系。2、应用MMT法检测过表达和沉默CCR7对Eca109细胞的活性影响。3、采用人脐静脉内皮细胞增殖、迁移实验和CAM实验检测CCR7表达改变对Eca109细胞增殖、迁移及诱导血管生成能力的影响。4、采用Western Blot实验检测VEGF-A和VEGF-C在CCR7过表达和沉默的Eca109细胞系中的表达情况。结果1、成功建立CCR7稳定过表达和沉默的Eca109细胞系。2、CCR7过表达Eca109细胞增殖、迁移和诱导血管生成能力显著增强;而CCR7沉默Eca109细胞上述能力显著减弱。3、CCR7过表达Eca109细胞VEGF-A和VEGF-C蛋白表达显著增高;而CCR7沉默Eca109细胞上述蛋白表达显著降低。结果提示,过表达CCR7增强Eca109细胞的细胞存活率,增加内皮细胞的迁移,增强血管形成能力,并上调VEGF-A和VEGF-C蛋白的表达,提高食管鳞癌细胞在体外诱导新血管形成能力。相反,沉默抑制CCR7表达则得到相反的结果。这些结果证实,CCR7能促进食管鳞状细胞癌的血管生成能力。第三部分CCR7对NF-k B信号通路的调节作用方法1、荧光素酶报告分析验证过表达CCR7和沉默CCR7对NF-k B通路的影响。2、通过Western blot、RT-PCR和ELISA等实验检测过表达和沉默CCR7对NF-k B信号通路的影响。结果1、CCR7蛋白的过表达能使NF-k B报告基因的转录活性增强。2、通过Wetern blotting实验结果证实ECA109中过表达CCR7能够显著提高磷酸化的IKK和磷酸化的Ik Bα的表达水平,但不能显著改变IKK和Ik Bα的总蛋白表达水平。3、Western botting实验和ELISA实验的结果显示多种NF-k B靶基因(包括TNF-α、IL-6、IL-8和TGF-β)在过表达CCR7的Eca109细胞中表达上调;而在CCR7沉默的Eca109细胞中表达下调。结果显示,过表达CCR7增强了NF-k B的报告基因的转录活性,而抑制CCR7表达则抑制了NF-k B的转录活性。CCR7的表达增加,增强了磷酸化IKK和磷酸化Ik Bα的水平,却没有显著改变IKK或Ik Bα总蛋白水平。此外,包括TNF-α,IL-6,IL-8,TGF-β在内的NF-k B相关的靶基因水平数,在上调CCR7的表达和下调细胞CCR7沉默食管癌细胞分别被上调和抑制了。表明CCR7对NF-k B信号通路起促进作用。第四部分CCR7下调对Eca109裸鼠移植瘤生长的影响及机制方法1、建立裸小鼠皮下ESCC移植瘤模型。分组注射1×107个control-si RNA和CCR7-si RNA ECA109细胞后观察肿瘤的生长。2、Western botting实验分析了肿瘤中VEGF-A、VEGF-C、TNF-α、IL-6、IL-8、TGF-β和NF-k B通路中磷酸化IKK和磷酸化Ik Bα及IKK或Ik Bα总蛋白的表达水平。结果1、下调CCR7蛋白的表达,ESCC移植瘤体积显著减小2、抑制CCR7表达后,VEGF-A、VEGF-C、TNF-α、IL-6、IL-8、TGF-β和NF-κB的蛋白表达水平显著降低,IKK和Ik Bα的磷酸化水平降低,而IKK和Ik Bα总蛋白水平无显著改变。结果提示,si RNA沉默CCR7在Eca109裸鼠移植瘤模型的抑制肿瘤疗效非常显著,其可能是通过抑制NF-k B通路来抑制ESCC细胞生长。全文结论1、CCR7在ESCC细胞中表达增高。2、通过构建稳定CCR7过表达和沉默的Eca109细胞系,明确了CCR7对Eca109肿瘤新生血管的促进作用。表明CCR7可能对ESCC的发生发展和血管生成发挥重要作用,可能作为ESCC新的治疗靶点。3、CCR7蛋白的过表达能使NF-k B报告基因的转录活性增强;CCR7能够显著提高IKK和Ik Bα的磷酸化水平;且多种NF-k B靶基因(包括TNF-α、IL-6、IL-8和TGF-β)在过表达CCR7的Eca109细胞中表达上调,而在CCR7沉默的Eca109细胞中表达下调。表明CCR7对NF-k B信号通路起促进作用。4、通过体内实验证实注射CCR7沉默组Eca109细胞裸鼠的肿瘤平均体积比对照组裸鼠明显减少。且移植瘤中VEGF-A、VEGF-C、TNF-α、IL-6、IL-8和TGF-β的蛋白表达水平显著降低,IKK和Ik Bα的磷酸化水平降低,而IKK和Ik Bα总蛋白水平无显著改变。进一步证实CCR7可能通过NF-k B信号通路对ESCC发挥促进作用。
[Abstract]:Background Esophageal cancer (EC) is a common malignant tumor of the digestive system. The incidence and mortality of esophageal cancer in China are the first in the world, and half of the deaths in the world are in China. The mortality rate is second, only inferior to gastric cancer. Esophageal squamous cell car Cinoma, ESCC) accounts for more than 90% of EC, so this study focuses on ESCC. Although early surgical resection and follow-up radiotherapy, chemotherapy and other strategies can improve the survival rate of patients with ESCC, however, these treatments are difficult to achieve radical effect, and at the same time the patients are accompanied by different degrees of regional lymph node metastasis. Tumor occurrence, development, invasion and metastasis are complex pathological processes involving multiple genes, multiple molecules and multiple systems. The formation of neovascularization (angiogenesis) is one of the important factors. Neovascularization is closely related to tumor growth, invasion and lymph node metastasis. Chemokine receptor (chemokin) E receptor) is a member of the G protein coupling receptor superfamily, a class of transmembrane receptors expressed on certain specific cell membrane surfaces. Recent studies have shown that chemokines are involved in the formation of neovascularization through binding with the receptors and further affect the formation and metastasis of tumors. Chemokines of different structures play a different role in angiogenesis. It can be used as a new target for cancer treatment. Recent studies have shown that chemokine receptor (CCR7) is overexpressed in a variety of tumor cells, which suggests the development of tumor, infiltration and metastasis are inseparable from CCR7. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF), the most Early also known as vascular permeability factor, which is closely related to the proliferation, migration and lumen formation of endothelial cells,.VEGF families include VEGF-A, VEGF-B, VEGF-C and VEGF-D. Tyrosine kinase is activated by VEGF and its receptor VEGFR, secondary to a series of signal pathway cascade reactions, followed by stimulating the proliferation of tumor vascular endothelial cells. Migration and neovascular lumen formation. Nuclear transcription factor - kappa B (nuclear transcription factor Kappa B, NF- B), the earliest proteins isolated from mature B lymphocytes, are eukaryotic cell transcriptional regulators, which exist in almost all animal cells with extensive biological activity in.NF- kappa B, and are inflammatory reactions and tumors. Important molecular proteins linked to each other play a key role in inflammation, metabolism, human immunity and the occurrence, development, invasion and metastasis of cancer. A large number of studies have shown that NF- kappa B is closely related to tumors. It can regulate various target genes and participate in multiple signaling pathways, inducing cells to inhibit apoptosis and promote cell proliferation. Invasion, metastasis, metastasis and promoting angiogenesis of tumor cells is closely related to carcinogenesis. At present, there are many clinical data at home and abroad that the high expression of CCR7 is closely related to the occurrence of ESCC, infiltration and metastasis. However, the expression of CCR7 in the ESCC cell line, the role of ESCC in the formation of blood tube, and the interaction with NF- kappa B, VEGF and other molecules In this study, we first detected the expression of CCR7 in the ESCC cell line, and then we observed the relationship between the abnormal expression of CCR7 and the angiogenesis of ESCC, as well as the interaction between CCR7 and VEGF, NF- kappa B and its downstream genes, and further explored the molecular mechanism of the molecular mechanism. To illustrate the mechanism of esophageal cancer metastasis, recurrence, and the development of target therapy based on CCR7. The first part is the expression of CCR7 in ESCC 1. The expression of CCR7 in the tumor tissues of ESCC patients in different reports is reviewed and studied by bioinformatics technique; and 2, the use of reverse transcription polymerase chain reaction (RT PCR) (RT-PCR) and immunoblotting (WB) method combined to detect the expression of CCR7 in ESCC cells of various different lines. Results 1, the expression of CCR7 gene was searched and analyzed in GEO PROFILE database (https://www.ncbi.nlm.nih.gov/gds/). The results showed that the expression of CCR7 in ESCC was significantly higher than that of CCR7 in the normal tissue. The results of.2, Western Blot showed that all 6 ESCC cell lines (TE-1, TE-2, TE-3, TE-8, TE-12 and ECA-109) were significantly higher than the normal esophageal epithelial cell lines, and the expression in the cell line was significantly higher than that of the cells. Second CCR7 in the proliferation, migration and induction of Eca109 cells in the proliferation, migration and induction of neovascularization of Eca109 cells, the establishment of a CCR7 stable overexpressed and silent Eca109 cell line.2, the use of MMT to detect and silence the activity of CCR7 to Eca109 cells, and to use human umbilical vein endothelial cells to proliferate and migrate. The effects of CCR7 expression on the proliferation, migration and angiogenesis of Eca109 cells were detected by the test and CAM test..4 was used to detect the expression of VEGF-A and VEGF-C in the CCR7 overexpressed and silent Eca109 cell lines by Western Blot. Results 1, the CCR7 stable overexpression and silent Eca109 cell lines were successfully established. Cell proliferation, migration and induction of angiogenesis significantly increased, while the above ability of CCR7 silenced Eca109 cells significantly weakened.3, CCR7 overexpressed Eca109 cells, VEGF-A and VEGF-C protein expression increased significantly, while CCR7 silenced Eca109 cells expressed a significant decrease in the expression of the above protein. The results suggested that overexpression of CCR7 enhanced the cell survival rate of Eca109 cells, and increased the cell survival rate of Eca109 cells. The migration of endothelial cells enhanced angiogenesis, increased the expression of VEGF-A and VEGF-C proteins and enhanced the ability to induce neovascularization in esophageal squamous cell carcinoma cells in vitro. On the contrary, silence inhibition of CCR7 expression was the opposite. These results confirmed that CCR7 could promote the angiogenesis of squamous cell carcinoma of the esophagus. Third part CCR7 pairs The regulation of NF-k B signaling pathway 1, luciferase report analysis verified the effect of overexpression of CCR7 and silent CCR7 on the NF-k B pathway. The effect of expression and silence CCR7 on the signaling pathway was detected by Western blot, RT-PCR and ELISA. Results 1, the overexpression of the protein could increase the transcriptional activity of the reporter gene. The strong.2, through the Wetern blotting experiment, confirmed that the overexpression of CCR7 in ECA109 could significantly increase the expression level of IKK and phosphorylated Ik B alpha, but could not significantly change the total protein expression level of IKK and Ik B alpha. The expression of beta expression was up regulated in the Eca109 cells overexpressing CCR7, and down regulated in the CCR7 silent Eca109 cells. The results showed that overexpression of CCR7 enhanced the transcriptional activity of the NF-k B reporter gene, while inhibition of CCR7 expression inhibited the increase of the expression of the NF-k B transcriptional activity.CCR7, and increased the level of phosphorylated IKK and phosphorylated Eca109. There was a significant change in the total protein level of IKK or Ik B alpha. In addition, the number of NF-k B related target genes, including TNF- alpha, IL-6, IL-8 and TGF- beta, was up-regulated and inhibited in the up regulation of CCR7 and down regulated cell CCR7 silent esophageal cancer cells. The effect of the growth of the tumor and the mechanism method 1, the subcutaneous ESCC tumor model of nude mice was established. The growth.2 of the tumor was observed after group injection of 1 x 107 control-si RNA and CCR7-si RNA ECA109 cells. Western botting experiment was used to analyze VEGF-A, VEGF-C, TNF- alpha and phosphorylation of phosphorylation and phosphorylation in the tumor. Or the expression level of Ik B alpha total protein. Results 1, down regulation of the expression of CCR7 protein, the volume of ESCC transplanted tumor significantly decreased by 2. After the inhibition of CCR7 expression, the protein expression level of VEGF-A, VEGF-C, TNF- a, IL-6, IL-8, TGF- beta and NF- kappa decreased significantly, but there was no significant change in the level of the total protein level. The inhibitory effect of RNA silencing CCR7 on the tumor model of Eca109 nude mice is very significant. It may be to inhibit the growth of ESCC cells by inhibiting the NF-k B pathway. Conclusion 1, CCR7 is expressed in.2 in ESCC cells. By constructing a Eca109 cell line that stable CCR7 over expression and silence, the promotion of CCR7 to neovascularization of tumor cells is clear. It shows that CCR7 may play an important role in the development and angiogenesis of ESCC, may be a new therapeutic target for ESCC,.3, and the overexpression of CCR7 protein can enhance the transcriptional activity of the NF-k B reporter gene, and CCR7 can significantly increase the phosphorylation level of IKK and Ik B alpha. The expression of overexpressed CCR7 Eca109 cells was up-regulated and down regulated in CCR7 silent Eca109 cells. It showed that CCR7 promoted.4 in NF-k B signaling pathway. Through in vivo experiments, the average volume of tumor in nude mice with Eca109 cells injected with CCR7 silencing group was significantly lower than that of the control group. The protein expression level of TGF- beta and IKK decreased significantly, and the phosphorylation level of IKK and Ik B alpha was decreased, while the total protein level of IKK and Ik B alpha was not significantly changed. It was further confirmed that CCR7 may play a role in ESCC through the NF-k B signaling pathway.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1

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