ALX1诱导EMT促进肺癌侵袭和迁移的机制以及β-榄香烯的逆转作用研究

发布时间：2018-01-26 06:53

  本文关键词： ALX1 Snail 肺癌 EMT 侵袭 迁移 β-榄香烯　出处：《大连医科大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分ALX1在肺癌中的表达研究目的:比较ALX1在肺癌组织中表达差异情况,揭示ALX1表达水平与肺癌恶性程度以及预后的关系。方法:通过Real time-PCR实验考察47例肺癌组织和癌旁组织样本中ALX1m RNA的表达水平;利用免疫组织化学实验考察发生淋巴结转移的肺癌组织和未转移的肺癌组织中ALX1蛋白的表达水平;利用Kaplan-Meier生存分析研究ALX1的表达水平与肺癌病人的预后情况。结果:1.47例肺癌组织均表达ALX1,且显著高于癌旁组织样本中ALX1 m RNA的表达水平。2.47例肺癌组织中,发生淋巴结转移的肺癌组织的ALX1 m RNA的表达水平显著高于未转移的肺癌组织。3.免疫组化分析显示,发生淋巴结转移的肺癌组织中ALX1阳性细胞比例要显著高于未转移的肺癌组织中ALX1阳性细胞的比例。4.ALX1表达水平较低的肺癌患者的生存期显著长于ALX1高表达组。结论:ALX1在肺癌组织中特异性高表达,发生淋巴结转移的肺癌组织中表达水平更高。ALX1高表达缩短肺癌患者的生存期。第二部分构建ALX1过表达和基因沉默细胞系目的:构建ALX1稳定转染和基因沉默的细胞模型,为ALX1调控肺癌细胞侵袭和迁移的分子机制研究提供工具。方法:通过Real time-PCR实验考察8种肺癌细胞系中ALX1 m RNA的表达水平。应用逆转录病毒转导构建过表达人ALX1的H1975细胞模型,应用sh RNA技术沉默H460细胞中ALX1的表达。通过Western blot实验验证模型构建情况,采用MTT实验观察构建细胞模型的增殖能力,并观察模型细胞的形态变化。通过Western blot实验和免疫荧光实验考察ALX1诱导肺癌细胞EMT情况。结果:1.8种肺癌细胞系中ALX1表达水平各异,H1975细胞表达较低,而H460细胞表达较高,适用于细胞转染研究。2.成功构建了ALX1-p Babe-H1975细胞模型和ALX1-si RNA-H460细胞模型,Western blot实验显示过表达ALX1后,H1975细胞ALX1蛋白表达水平显著高于mock-H1975和p Babe-H1975细胞;三组ALX1si RNA转染细胞中,ALX1蛋白表达水平显著低于对照细胞。3.MTT结果表明,过表达ALX1后,ALX1-p Babe-H1975细胞的增殖显著快于对照细胞;沉默ALX1后,ALX1-si RNA-H460细胞的增殖能力下降。4.过表达ALX1后,ALX1-p Babe-H1975细胞呈现间质细胞样改变;沉默ALX1后,ALX1-si RNA-H460细胞呈现上皮样细胞形态。5.过表达ALX1后,ALX1-p Babe-H1975细胞上皮细胞标志蛋白E-cadherin和α-catenin表达降低,间质细胞标志蛋白Vimentin和N-cadherin表达上调;沉默ALX1后,ALX1-si RNA-H460细胞E-cadherin和α-catenin表达上调,而Vimentin和N-cadherin表达降低。免疫荧光实验结果与Western blot实验结果一致。结论:成功构建了ALX1-p Babe-H1975和ALX1-si RNA-H460细胞模型,并成功运用于ALX1诱导肺癌细胞EMT的研究。第三部分ALX1诱导肺癌细胞EMT的机制研究目的:分析ALX1诱导肺癌细胞EMT的分子机制,揭示ALX1诱导EMT对肺癌细胞侵袭和迁移的影响。方法:利用构建的ALX1高表达和基因沉默细胞模型,通过Transwell和基质胶侵袭实验、Real time-PCR实验、Western blot实验和荧光素酶实验考察ALX1诱导肺癌细胞EMT的分子机制。结果:1.过表达ALX1增加ALX1-p Babe-H1975细胞通过Transwell膜和基质胶的数量,侵袭和迁移能力增强。沉默ALX1后,ALX1-si RNA-H460细胞侵袭和迁移能力减弱。2.过表达ALX1不影响ALX1-p Babe-H1975细胞中ZEB1、Slug和Twist的m RNA和蛋白表达,显著提高Snail的m RNA和蛋白表达水平。沉默ALX1后,ALX1-si RNA-H460细胞Snail的m RNA和蛋白表达水平显著下调。3.过表达ALX1显著提高H1975细胞中荧光素的强度。沉默ALX1后,ALX1-si RNA-H460细胞荧光素强度显著下降。4.沉默Snail后,ALX1-p Babe-H1975细胞中低水平的E-cadherin和α-catenin表达恢复,而高水平的Vimentin和N-cadherin表达下降;穿透基质胶和Transwell膜的细胞个数减少。结论:ALX1通过Snail诱导肺癌细胞EMT,增强肺癌细胞的侵袭和迁移能力。这些发现为ALX1作为潜在治疗靶点的进一步研究奠定了基础。第四部分β-榄香烯逆转ALX1诱导肺癌细胞EMT的机制研究目的:考察β-榄香烯对ALX1诱导的EMT的影响,揭示β-榄香烯逆转肺癌EMT的分子机制。方法:利用构建的ALX1高表达细胞模型,通过MTT、Transwell侵袭、Transwell迁移实验和Western blot实验、免疫荧光实验考察β-榄香烯对ALX1诱导肺癌细胞EMT的影响和机制。结果:1.β-榄香烯处理后,ALX1过表达的ALX1-p Babe-H1975细胞形态呈现上皮样改变,增殖速度减慢。2.β-榄香烯处理后,通过Transwell膜和基质胶的ALX1-p Babe-H1975细胞数量减少,ALX1-p Babe-H1975细胞侵袭和迁移能力减弱。3.Western blot实验表明β-榄香烯上调ALX1过表达的ALX1-p Babe-H1975细胞的E-cadherin和α-catenin,下调Vimentin和N-cadherin。免疫荧光分析发现E-cadherin荧光强度增强,而N-cadherin强度减弱。4.β-榄香烯降低ALX1-p Babe-H1975细胞Snail的蛋白水平。结论:β-榄香烯抑制Snail的表达,阻断ALX1诱导的肺癌细胞EMT,减慢增殖速率,减弱细胞侵袭和迁移能力,β-榄香烯可以作为ALX1过表达的肺癌的潜在治疗药物加以开发利用。
[Abstract]:Objective to study the expression of ALX1 in lung cancer in the first part: the difference of ALX1 expression in lung cancer tissues, revealing the expression level of ALX1 and the degree of malignancy of lung cancer and the prognosis. Methods: To investigate the expression level of 47 cases of lung cancer tissues and paracancerous tissue samples in ALX1m RNA by Real time-PCR by immunohistochemical experiment; experimental studying lymph node metastasis of lung cancer and the expression level of ALX1 protein in non metastatic lung carcinoma; prognosis analysis to study the expression of ALX1 and Kaplan-Meier in lung cancer patients with survival. Results: ALX1 was expressed in 1.47 cases of lung cancer, and was significantly higher than that in adjacent tissue samples in ALX1 m RNA.2.47 expression level in lung cancer patients the expression level of ALX1, m lymph node metastasis of RNA lung cancer was significantly higher than that in lung cancer without metastasis of.3. immunohistochemical analysis showed that the occurrence of lymph node Metastasis of lung cancer tissues was significantly higher than the proportion of ALX1 positive cells and ALX1 positive cells without metastasis in lung cancer survival ratio of.4.ALX1 expressed low levels of lung cancer patients was significantly longer than that of ALX1 high expression group. Conclusion: high expression of ALX1 in lung cancer tissue specific, higher expression level occurred in lung cancer lymph node metastasis in the high expression of.ALX1 reduced survival of patients with lung cancer. The second part is the construction of ALX1 overexpression and gene silencing cell line Objective: cell model was constructed and stably transfected with ALX1 gene silencing, provide a tool for the study of molecular mechanism of invasion and migration of ALX1 regulation of lung cancer cell. Methods: the effects of 8 kinds of lung cancer cell lines ALX1 m RNA expression the level of Real by time-PCR experiment. Application of retroviral transduction to construct H1975 cell model expressing human ALX1, the expression of ALX1 using SH RNA technology to silence H460 cells by Wes. The construction of tern blot experiment model, MTT to observe the construction of cell proliferation model, and to observe the morphological changes of model cells. By Western blot assay and immunofluorescence experiments to investigate ALX1 induced lung cancer cell EMT. Results: 1.8 different levels of expression of ALX1 in lung cancer cell lines, H1975 cells and H460 expression is low. Expression is high, suitable for cell transfection studies of.2. to construct ALX1-p model of Babe-H1975 cells and ALX1-si RNA-H460 cells Western model, blot assay showed that the overexpression of ALX1, H1975 cells ALX1 protein expression level was significantly higher than that of mock-H1975 and P in Babe-H1975 cells; three groups of ALX1si RNA in the transfected cells, the expression level of ALX1 protein was significantly lower than the control cells.3.MTT the results show that the overexpression of ALX1 and ALX1-p on the proliferation of Babe-H1975 cells significantly faster than control cells; after ALX1 was silenced, ALX1-si RNA-H460 Cell proliferation decreased expression of.4. ALX1, ALX1-p Babe-H1975 cells showed interstitial cells changed; after ALX1 was silenced, ALX1-si RNA-H460 cells showed epithelioid cell morphology after overexpression of ALX1.5., ALX1-p Babe-H1975 cell marker of epithelial cells and decrease the expression of protein E-cadherin and -catenin alpha, interstitial cell marker expression of protein Vimentin and N-cadherin; silence ALX1, ALX1-si RNA-H460 E-cadherin cells and alpha -catenin expression, while Vimentin and N-cadherin expression decreased. Immunofluorescence experiment results are consistent with the experimental results of Western blot. Conclusion: the successful construction of ALX1-p Babe-H1975 and ALX1-si RNA-H460 cell model, and successfully applied to ALX1 induced lung cancer cell EMT. The third part is the research objective mechanism of ALX1 induced lung cancer EMT cells: analysis of molecular mechanism of ALX1 induced lung cancer cell line EMT, ALX1 induced EMT of lung cancer revealed Effect of cell migration and invasion. Methods: the high expression of ALX1 and gene silencing cell model was constructed by Transwell, and Matrigel invasion assay Real time-PCR experiment, Western blot experiment and experimental study of molecular mechanism of ALX1 induced luciferase EMT lung cancer cells. Results: 1. overexpression of ALX1 increased the number of ALX1-p Babe-H1975 cells by Transwell and membrane Matrigel, enhance the invasion and migration ability. After ALX1 was silenced, ALX1-si RNA-H460 cell invasion and migration ability of.2. decreased expression of ALX1 ZEB1 ALX1-p does not affect Babe-H1975 cells, the expression of M protein and RNA Slug and Twist, significantly increased m RNA and protein expression level of Snail. After ALX1 was silenced, the expression level of M and RNA ALX1-si RNA-H460 protein Snail cells significantly reduced.3. expression of ALX1 increased significantly in H1975 cells. Fluorescence intensity of ALX1 silencing, ALX1-si RNA-H460 cells Guang Suqiang. 搴︽樉钁椾笅闄，

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