ADAM22通过激活Integrin β1促进胃癌多药耐药和耐药相关侵袭转移的机制研究

发布时间：2018-05-08 23:16

  本文选题：胃癌 + 耐药　； 参考：《第四军医大学》2017年博士论文

【摘要】：【背景】胃癌是最常见的消化道恶性肿瘤,化疗在其综合治疗中发挥着极其重要的作用。但是,在临床上存在着化疗药物可以使肿瘤迅速缩小,但对患者生存期延长不明显的悖论。造成这一现象的重要原因是肿瘤细胞的多药耐药及其导致的肿瘤细胞恶性表型的进展。既往有研究发现化疗药物可以通过诱导肿瘤细胞发生EMT、改变肿瘤微环境以及富集肿瘤干细胞等途径促进多种肿瘤的侵袭转移。但是,化疗在促进胃癌细胞侵袭转移中的作用研究尚不充分。分泌蛋白质组学是以分泌和脱落的细胞膜蛋白为主要研究对象的蛋白质组学重要分支,其在肿瘤研究中的应用极大的推动新型肿瘤标志物的发现,也为阐明肿瘤发生发展的机制提供了新的技术手段。【目的】通过分泌蛋白质组学方法筛选促进胃癌多药耐药细胞侵袭转移的候选分子并明确其作用机制【方法】1.采用Transwell和裸鼠尾静脉注射肺部转移实验检测胃癌多药耐药和亲本细胞侵袭转移能力的差别;采用非标定量蛋白质谱技术筛选胃癌耐药和亲本细胞无血清条件培养基中差异分泌蛋白,并与既往基因芯片筛选结果对比分析取交集;采用q RT-PCR和Western blot技术对筛选结果在胃癌及其他肿瘤的耐药细胞系中进行验证;采用公共数据库查询候选分子在肿瘤中表达的临床意义;在胃癌细胞系和PDX裸鼠模型中检测化疗药物能否诱导候选分子表达。2.采用Transwell和尾静脉注射体内转移实验在耐药和亲本胃癌细胞中检测下调和过表达ADAM22对细胞侵袭转移能力的影响。3.采用q RT-PCR和Western blot技术检测EMT经典标志物的表达;采用体外黏附实验检测干预ADAM22表达对细胞对细胞外基质Fibronectin和CollagenⅠ黏附能力的变化并采用免疫荧光技术检测黏着斑标志物和F-actin纤维形成情况;采用尾静脉注射荧光标记肿瘤细胞技术检测干预ADAM22对肿瘤细胞在裸鼠肺内驻留能力的影响;采用体外穿内皮实验检测干预ADAM22表达对肿瘤细胞穿过内皮细胞能力的影响;采用Matrigel-on-Top(Mo T)3D培养技术检测干预ADAM22对单个肿瘤细胞克隆形成及丝状伪足样凸起(FLPs)形成能力的影响。4.采用Western blot技术检测下调ADAM22表达对黏附相关分子和Integrinβ1(ITGB1)表达和活性的影响;采用GST-pulldown联合Western blot技术检测下调ADAM22表达对胃癌耐药细胞中Rac1和Cdc42活性的影响;在下调ADAM22表达的耐药细胞中分别检测ITGB1激活抗体对细胞黏附、体内外侵袭迁移能力的影响;采用免疫共沉淀联合Western blot技术检测野生及突变型ADAM22与ITGB1的相互作用;采用针对ADAM22 m RNA 5’-UTR的si RNA下调内源性ADAM22表达后,验证野生和突变型ADAM22对耐药细胞黏附、迁移和3D克隆形成能力的补救作用。5.采用MTT法检测下调ADAM22、Rac1及Cdc42后耐药细胞针对多种化疗药物IC50的变化;采用q RT-PCR和Western blot技术检测检测下调ADAM22、Rac1和Cdc42后HIF1α和ABCB1 m RNA及其编码蛋白表达水平变化。6.采用免疫组织化学染色技术检测ADAM22和HUTS4在胃癌组织原发灶和转移灶中的表达情况;采用非参数统计方法分析ADAM22在胃癌原发灶和转移灶中表达强度的变化;采用Spearman秩相关分析ADAM22和HUTS4在胃癌组织中表达的相关性;采用Kaplan-Meier生存分析和Cox回归模型分析ADAM22和HUTS4在胃癌组织中表达的临床意义。【结果】1.Transwell和尾静脉注射实验结果表明胃癌耐药细胞SGC7901/ADR和SGC7901/VCR的体内外侵袭转移能力明显增强;同时,在尾静脉转移实验中我们发现,耐药细胞不仅成瘤率高,而且转移灶的数目和体积也较亲本细胞明显升高。非标定量质谱鉴定了91个在2株耐药细胞无血清条件培养基中含量明显升高的分泌蛋白,通过与既往基因芯片结果对比发现其中23个m RNA水平也明显升高。q RT-PCR和Western blot验证结果证实了ADAM22在2株胃癌耐药细胞以及K562和口腔鳞癌等多种肿瘤的耐药细胞中表达升高。通过查询Kaplan-Meier Plot数据库我们发现ADAM22高表达与胃癌患者预后不良相关。q RT-PCR和免疫组化结果表明多种化疗药物可以诱导胃癌细胞系和PDX肿瘤组织表达ADAM22。2.Transwell和尾静脉注射实验结果表明下调ADAM22表达后胃癌耐药细胞SGC7901/ADR和SGC7901/VCR的体内外侵袭转移能力受到明显抑制。特别需要指出的是在尾静脉注射实验中耐药细胞不仅成瘤率明显下降,肿瘤转移灶的数量和尺寸也明显下降。3.q RT-PCR和Western blot结果表明EMT经典标志物E-cadherin和Vimentin表达不受ADAM22影响。下调ADAM22后耐药细胞SGC7901/ADR和SGC7901/VCR对细胞外基质蛋白Fibronectin和CollagenⅠ的黏附能力明显下降,在肺部驻留能力也受到显著抑制。免疫荧光结果表明下调ADAM22后耐药细胞黏着斑和F-actin纤维形成受到明显抑制。同时,耐药细胞的体外内皮穿过能力和Mo T培养条件下的克隆形成能力和FLPs形成数量也明显下降。4.下调ADAM22表达后胃癌耐药细胞中黏附相关分子FAK以及Paxillin的磷酸化水平受到了明显抑制。GST-pulldown联合Western blot结果表明干扰ADAM22表达后耐药细胞中Rho GTP酶Rac1和Cdc42的活性也明显下降。针对ITGB1活化表位HUTS4的Western blot结果显示,ADAM22表达下降后ITGB1的活性也随之下降。功能实验表明激活ITGB1可以逆转下调ADAM22对耐药细胞黏附和体内外侵袭迁移能力的抑制。免疫共沉淀结果表明ITGB1可以与野生型ADAM22相互作用,但不能与Disintegrin缺失的突变型ADAM22相互作用。野生型而非突变型ADAM22可以补救下调内源性ADAM22对胃癌耐药细胞黏附、迁移和3D克隆形成能力的抑制。5.在胃癌耐药细胞中下调ADAM22、Rac1或者Cdc42均可以显著降低SGC7901/ADR和SGC7901/VCR对多种化疗药物的IC50。q RT-PCR和Western blot结果显示在胃癌耐药细胞中下调ADAM22或者Rac1和Cdc42的表达可以显著降低HIF1α和ABCB1及其编码蛋白的表达。6.ADAM22在胃癌转移灶中的表达水平明显高于其相应的原发灶组织。相关性分析表明ADAM22在胃癌组织中的表达与HUTS4的表达呈正相关。Kaplan-Meier生存分析显示胃癌组织中ADAM22和HUTS4高表达与患者预后差相关。Cox多因素回归分析发现ADAM22或HUTS4高表达及肿瘤的N分期和患者年龄是患者预后不良的独立风险预测因子。【结论】本课题筛选并阐明了一条促进胃癌耐药细胞侵袭转移的信号通路:化疗药物诱导胃癌细胞高表达ADAM22,后者通过Disintegrin结构激活ITGB1增加肿瘤细胞对细胞外基质的黏附和内皮穿透能力,并赋予转移肿瘤细胞在转移灶微环境的克隆增殖优势,从而促进耐药细胞的侵袭转移。同时,该通路通过影响HIF1α和ABCB1及其编码蛋白的表达促进胃癌细胞的多药耐药。该研究加深了对胃癌细胞耐药和转移关系的认识,并为开发同时针对胃癌耐药和转移的治疗策略提供了靶点。
[Abstract]:[background] gastric cancer is the most common malignant tumor of the digestive tract. Chemotherapy plays an extremely important role in its comprehensive treatment. However, there is a clinical presence of chemotherapeutic drugs that can make the tumor reduce rapidly, but the paradox of prolonging the life period is not obvious. The important reason for this phenomenon is the multidrug resistance and its guidance of the tumor cells. Progress in the malignant phenotype of tumor cells. Previous studies have found that chemotherapeutic agents can promote the invasion and metastasis of various tumors by inducing EMT, changing tumor microenvironment and enriching tumor stem cells. However, the role of chemotherapy in promoting invasion and metastasis of gastric cancer cells is not sufficient. Learning is an important branch of proteomics, which is the main research object of secretory and exudate cell membrane proteins. Its application in cancer research greatly promotes the discovery of new tumor markers and provides new technical means to elucidate the mechanism of tumor development. The candidate molecules of the invasion and metastasis of cancer multidrug resistant cells and the mechanism of its action [method] 1. Transwell and nude mice tail vein were injected into the lungs to detect the difference in the ability of multidrug resistance and the invasion and metastasis of the parent cells. The non standard quantitative protein mass spectrometry was used to screen the gastric cancer resistance and the serum-free conditional culture of the parent cells. The differentially secreted proteins in the substrate were compared with the previous gene chip screening results. Q RT-PCR and Western blot were used to verify the screening results in the drug-resistant cell lines of gastric cancer and other tumors; the clinical significance of the candidate molecules in the tumor was inquired by the public database, and the gastric cancer cell line and the PDX nude mice were used. Whether or not the chemotherapeutic drugs can induce the expression of the candidate molecules to the expression of.2., the effect of down regulation and overexpression of ADAM22 on the invasion and metastasis of cells in the drug-resistant and parent gastric cancer cells was detected by Transwell and caudal vein injection..3. was used to detect the expression of the classical EMT markers using Q RT-PCR and Western blot technology. The effect of ADAM22 expression on the adhesion ability of cell to extracellular matrix Fibronectin and Collagen I was detected by experimental detection, and immunofluorescence technique was used to detect the formation of plaque marker and F-actin fiber. Tail vein injection of fluorescence labeled tumor cell technique was used to detect the ability of ADAM22 to reside in the lung of nude mice. The effect of ADAM22 expression on the ability of tumor cells passing through endothelial cells in vitro, Matrigel-on-Top (Mo T) 3D culture technique was used to detect the influence of intervention ADAM22 on the formation of single tumor cell clone formation and the influence of filamopopfoot like protruding (FLPs) formation energy,.4. using Western blot technique to detect down regulation ADAM22 The effect of expression on the expression and activity of adhesion related molecules and Integrin beta 1 (ITGB1); the effect of GST-pulldown combined with Western blot on the activity of Rac1 and Cdc42 in gastric cancer resistant cells by down regulation of ADAM22; detection of adhesion to cells by ITGB1 activated antibodies and invasion and migration in vivo and in vivo The interaction between wild and mutant ADAM22 and ITGB1 was detected by immunoprecipitation combined with Western blot, and Si RNA for ADAM22 m RNA 5 '-UTR down regulated endogenous ADAM22 expression was used to verify the remedial action of wild and mutant ADAM22 on the adhesion, migration and clone formation of drug-resistant cells. The changes in ADAM22, Rac1 and Cdc42 were measured against a variety of chemotherapeutic agents, IC50, and Q RT-PCR and Western blot techniques were used to detect the changes in ADAM22, Rac1 and Cdc42, and the changes in the expression level of HIF1 alpha and its encoded protein. The expression of ADAM22 in the primary and metastatic foci of gastric cancer was analyzed by non parametric statistical method. The correlation of expression of ADAM22 and HUTS4 in gastric cancer tissues was analyzed by Spearman rank correlation, and the expression of ADAM22 and HUTS4 in gastric cancer tissues was analyzed by Kaplan-Meier survival analysis and Cox regression model. Clinical significance. [results] the results of 1.Transwell and tail vein injection showed that the invasion and metastasis of gastric cancer cells SGC7901/ADR and SGC7901/VCR increased significantly in vivo and in vitro. At the same time, we found that in the tail vein metastasis experiment, the drug resistant cells not only have high tumor formation rate, but also the number and volume of the transfer focus are also significantly higher than those of the parent cells. Non standard quantitative mass spectrometry was used to identify 91 secretory proteins which were significantly elevated in the serum-free medium of 2 resistant cells. By comparison with previous gene chip results, 23 m RNA levels were also significantly increased by.Q RT-PCR and Western blot verification results confirmed that ADAM22 was in 2 gastric cancer resistant cells and K562 and oral squamous cell carcinoma. The expression of multidrug-resistant cells in a variety of tumors was elevated. By querying the Kaplan-Meier Plot database, we found that the high expression of ADAM22 was associated with poor prognosis in patients with gastric cancer,.Q RT-PCR and immunohistochemical results showed that a variety of chemotherapeutic drugs could induce the expression of ADAM22.2.Transwell in gastric cancer cell lines and PDX tumor tissue and the result of the tail vein injection. The invasion and metastasis of gastric cancer resistant cells SGC7901/ADR and SGC7901/VCR were obviously inhibited after the ADAM22 expression was down-regulation. It was particularly pointed out that the tumor rate decreased significantly in the caudal vein injection experiment, and the number and size of tumor metastasis also decreased significantly.3.q RT-PCR and Western blot results indicated EMT classics. The expression of E-cadherin and Vimentin was not affected by ADAM22. After the downregulation of ADAM22, the adhesion ability of SGC7901/ADR and SGC7901/VCR to the extracellular matrix protein Fibronectin and Collagen I was significantly decreased, and the ability to reside in the lungs was also significantly inhibited. The immunofluorescence results showed that the adhesion and F-acti of drug-resistant cells were down regulated after ADAM22. The formation of N fibers was significantly inhibited. At the same time, the ability of the endothelium in vitro and the Mo T culture, the ability of clonformation and the formation of FLPs to decrease the.4. down ADAM22 expression, the adhesion related molecule FAK and the phosphorylation level of Paxillin were significantly inhibited by.GST-pulldown combined Wester. The results of N blot showed that the activity of Rho GTP enzyme Rac1 and Cdc42 in drug-resistant cells decreased significantly after interference of ADAM22 expression. The Western blot results against ITGB1 activation epitope HUTS4 showed that the activity decreased after the ADAM22 expression decreased. Immune coprecipitation results show that ITGB1 can interact with wild type ADAM22 but can not interact with Disintegrin missing mutant ADAM22. Wild type and non mutant ADAM22 can remediate endogenous ADAM22 to inhibit the adhesion of gastric cancer resistant cells, migration and the ability of 3D cloning to inhibit the tolerance to gastric cancer The down-regulation of ADAM22, Rac1 or Cdc42 in the drug cells could significantly reduce the IC50.q RT-PCR and Western blot results of SGC7901/ADR and SGC7901/VCR to a variety of chemotherapeutic drugs. The expression of ADAM22 or Rac1 and Cdc42 in the drug resistant cells of gastric cancer could significantly reduce the expression of alpha and protein and its encoding protein in gastric cancer metastasis. The expression level of the ADAM22 was significantly higher than that of the corresponding primary tissue. The correlation analysis showed that the expression of ADAM22 in gastric cancer tissues was positively correlated with the expression of HUTS4, and the high expression of ADAM22 and HUTS4 in gastric cancer tissues was associated with the.Cox multiple regression analysis of the prognosis of the patients. The high expression of ADAM22 or HUTS4 and the tumor were found to be highly expressed and tumor. N staging and patient age are independent risk predictors of poor prognosis in patients. [Conclusion] this topic screening and clarifying a signal pathway to promote the invasion and metastasis of gastric cancer cells: chemotherapeutic drugs induce high expression of ADAM22 in gastric cancer cells, and the latter increases the adhesion of tumor cells to the extracellular matrix through Disintegrin structure stimulated by ITGB1 The penetration of HIF1 and ABCB1 and the expression of the encoded protein can promote the multidrug resistance of gastric cancer cells. This study deepens the recognition of the drug resistance and metastasis of gastric cancer cells. It also provides a target for developing strategies for gastric cancer resistance and metastasis.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2
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本文编号：1863514