PRRX1下调对非小细胞肺癌细胞增殖和凋亡的作用及机制的研究

发布时间：2018-04-30 21:28

本文选题：配对相关同源框蛋白1 + A549细胞　；参考：《安徽医科大学》2017年博士论文

【摘要】：第一部分 PRRX1在非小细胞肺癌中的组织学表达特征及临床意义目的:通过检测PRRX1在非小细胞肺癌患者中的表达情况,结合患者临床资料,了解PRRX1在肺癌中临床价值。方法:利用免疫组化法检测52例非小细胞肺癌组织标本和28例癌旁正常肺组织标本中PRRX1表达情况,结合患者的临床病理资料和预后情况,应用Kaplan-Meier生存分析法和COX比例风险回归模型分析PRRX1与患者预后的关系。结果:1.免疫组化结果显示PRRX1在癌旁正常肺组织中呈现稳定中度表达;在非小细胞肺癌组织中PRRX1高度表达存在高分化的肺癌组织中,PRRX1中低表达存在中度分化的肺癌组织中,PRRX1低表达或者表达缺失存在于低分化的肺癌组织中。2.低表达PRRX1与患者的肿瘤大小分期,疾病分期和肿瘤分化程度相关(P=0.002,P=0.019和P=0.001)。3.COX比例风险回归模型多因素分析显示淋巴结分期、疾病分期和PRRX1表达水平是患者预后的独立因素(Cox回归分析:P=0.048,P=0.008和P=0.035)。4.PRRX1低表达患者生存周期较高表达者缩短(log-rank检验,P=0.010)。结论:PRRX1的表达情况与非小细胞肺癌患者的疾病分期、细胞分化、预后相关。低表达的PRRX1提示患者预后差。第二部分沉默PRRX1诱导A549细胞经历EMT和获得CSCs特性目的:构建PRRX1基因沉默的PRRX1-shRNA-A549细胞模型,进一步研究在A549细胞中沉默PRRX1与EMT和CSCs特性的关系。方法:通过shRNA技术沉默A549细胞中PRRX1表达,构建PRRX1-shRNA-A549细胞模型。Western blot实验验证模型构建情况,应用MTT和软琼脂克隆生长实验观察模型细胞的增殖能力,相差显微镜观察细胞形态学变化。Transwell体外侵袭、入侵实验和创伤愈合实验检测模型细胞的侵袭和移动能力。Western blot实验和免疫荧光实验观察模型细胞的上皮和间质标志物表达情况。流式细胞仪分选模型细胞上肺癌CSCs标志物比例。结果:1.Western blot检测结果显示A549细胞中PRRX1为高表达,可以应用基因沉默技术构建细胞模型。2.成功构建PRRX1-shRNA-A549、Mock-shRNA-A549细胞模型,Western blot实验显示PRRX1表达明显抑制在基因沉默细胞模型中。3.MTT和软琼脂克隆生长实验结果显示,沉默PRRX1后,细胞贴壁和非贴壁增殖情况明显受到增强。4.相差显微镜下可见,沉默PRRX1后,A549细胞形态向间质细胞表型特征转化。5.Transwell体外侵袭、入侵实验和创伤愈合实验显示,沉默PRRX1后,细胞的移动和侵袭能力增强。6.沉默PRRX1后,A549细胞的间质细胞标志物Vimentin和N-cadherin表达上调,上皮细胞标志物E-cadherin下调,并且免疫荧光实验结果得到相一致的结果。7.流式细胞仪检测结果,细胞沉默PRRX1后,肺癌干细胞标准物(CD133、CD44、ALDH1)细胞比例提高,分别为77.6%、4.1%、1.5%。结论:敲除PRRX1诱导肺腺癌细胞经历EMT,获得CSCs特性,增强了细胞增殖、侵袭的能力。第三部分沉默PRRX1通过干扰caspase-3凋亡通道激活抑制顺铂诱导的A549细胞凋亡目的:通过已经建立的PRRX1-shRNA-A549细胞模型,观察PRRX1沉默后,顺铂诱导的A549细胞凋亡和凋亡通道的变化,探讨PRRX1在顺铂诱导A549细胞凋亡中的作用机制。方法:通过MTT实验检测PRRX1沉默后顺铂对于细胞增殖抑制情况,并选择最佳顺铂实验浓度。流式细胞仪检测细胞周期和细胞凋亡情况。线粒体膜电位检测线粒体体外膜电位的变化情况。Western blot实验检测凋亡相关蛋白(caspase-3,caspase-9,cytochrome C和Apaf-1)的表达情况,判断PRRX1沉默后细胞凋亡通道。MTT实验和细胞凋亡实验中添加caspase-3激活剂PAC-1,反向验证PRRX1沉默干扰的细胞凋亡通道。结果:1.MTT实验结果显示,PRRX1沉默后阻止了顺铂诱导的A549细胞增殖抑制,顺铂浓度为20μg/ml是后续实验最佳浓度。2.流式细胞仪检测细胞凋亡实验结果显示,PRRX1沉默后,细胞凋亡明显受到抑制。3.流式细胞仪检测细胞周期实验结果显示,PRRX1沉默后,细胞周期阻滞在G2期。4.线粒体膜电位检测结果显示,PRRX1沉默后,顺铂诱导的线粒体膜电位下降受到抑制。5.PRRX1沉默后凋亡相关蛋白caspase-3,caspase-9,cytochrome C和Apaf-1表达下降。6.添加PAC-1的MTT试验和细胞凋亡实验结果显示,细胞增殖受到明显抑制,细胞凋亡明显增加。结论:沉默PRRX1通过抑制线粒体膜电位下降和干扰caspase-3凋亡通道的激活,抑制顺铂诱导的A549细胞凋亡。
[Abstract]:Part 1 the histological features and clinical significance of PRRX1 in non-small cell lung cancer: by detecting the expression of PRRX1 in patients with non-small cell lung cancer and combining the clinical data of the patients, the clinical value of PRRX1 in lung cancer was understood. Methods: 52 cases of non-small cell lung cancer were detected by immunohistochemistry and 28 cases of para cancer were detected by immunohistochemistry. PRRX1 expression in normal lung tissue specimens, combined with the patients' clinicopathological data and prognosis, Kaplan-Meier survival analysis and COX proportional risk regression model were used to analyze the relationship between PRRX1 and patients' prognosis. Results: 1. the results of immunohistochemistry showed that PRRX1 showed stable medium expression in normal lung tissue adjacent to cancer; in non small cell lung cancer. PRRX1 highly expressed in highly differentiated lung cancer tissues, low expression of PRRX1 in moderately differentiated lung cancer tissues, PRRX1 low expression or expression deletion existed in low differentiated lung cancer tissues with.2. low expression PRRX1 and tumor size and staging of patients, disease staging and degree of swelling differentiation (P=0.002, P=0.019 and P=0.001).3. Multifactor analysis of the COX proportional risk regression model showed that lymph node staging, disease staging and PRRX1 expression were independent factors for patients' prognosis (Cox regression analysis: P=0.048, P=0.008 and P=0.035) with low expression of.4.PRRX1 in patients with low expression of.4.PRRX1 (log-rank test, P=0.010). Conclusion: the expression of PRRX1 and non small cell lung cancer The patient's disease staging, cell differentiation and prognosis are related. Low expression of PRRX1 suggests that the patient has a poor prognosis. Second silent PRRX1 induces A549 cells to undergo EMT and obtain CSCs characteristics: to construct a PRRX1-shRNA-A549 cell model of PRRX1 gene silencing, and to further study the relationship between the silence of PRRX1 and EMT and CSCs in A549 cells. Methods: The expression of PRRX1 in A549 cells was silenced by shRNA technique, and the construction of PRRX1-shRNA-A549 cell model.Western blot was constructed. The proliferation ability of the model cells was observed by MTT and soft agar clone growth experiments. The phase microscope observation of cell morphology changes of cell morphology changes in.Transwell in vitro, invasion experiment and wound healing test test. Measurement of cell invasion and mobility of the model cells.Western blot experiment and immunofluorescence test to observe the expression of epithelial and interstitial markers in the model cells. Flow cytometry was used to determine the proportion of CSCs markers on the lung cancer cells on the model cells. Results: the results of 1.Western blot detection showed that PRRX1 in A549 cells was highly expressed, and gene silencing technique could be applied. The cell model.2. was constructed successfully to construct PRRX1-shRNA-A549, Mock-shRNA-A549 cell model, and Western blot experiment showed that the expression of PRRX1 expression was obviously inhibited in the gene silencing cell model, the results of.3.MTT and soft agar clonal growth showed that the cell adherence and non adherent colonization of the cells were obviously enhanced by the enhanced.4. phase microscope after the silencing of PRRX1. After the silence of PRRX1, the phenotype of A549 cells transformed into.5.Transwell in vitro, and the invasion experiment and wound healing experiment showed that after the silence of PRRX1, the cell movement and invasion ability enhanced.6. silent PRRX1, the expression of Vimentin and N-cadherin in the interstitial cell markers of A549 cells was up, and the epithelial marker E-cadher was E-cadher. In was down, and the results of immunofluorescence test were consistent with the results of.7. flow cytometry. After cell silencing of PRRX1, the proportion of lung cancer stem cell standard (CD133, CD44, ALDH1) cells increased, respectively, 77.6%, 4.1%, 1.5%. conclusion: knockout of PRRX1 induced lung adenocarcinoma cells experienced EMT, acquired CSCs characteristics, enhanced cell proliferation, invasion. Ability. Third silence PRRX1 inhibits the apoptosis of A549 cells induced by cisplatin by interfering with caspase-3 apoptosis pathway: through the established PRRX1-shRNA-A549 cell model, the changes in apoptosis and apoptosis of A549 cells induced by cisplatin are observed after PRRX1 silencing, and the role of PRRX1 in the apoptosis of A549 cells induced by cisplatin is discussed. Methods: MTT test was used to detect the proliferation inhibition of cisplatin after PRRX1 silencing, and the optimal concentration of cisplatin was selected. Flow cytometry was used to detect cell cycle and apoptosis. Mitochondrial membrane potential was used to detect the changes in the epicardial potential of mitochondria..Western blot was used to detect apoptosis related protein (caspase-3, caspase-9,) The expression of cytochrome C and Apaf-1, the caspase-3 activator PAC-1 was added to the apoptosis channel.MTT experiment and the apoptosis experiment after PRRX1 silence, and the apoptosis channel of PRRX1 silencing interference was reversed. The results of 1.MTT experiment showed that the PRRX1 silence prevented the inhibition of cisplatin induced A549 cell proliferation, and the concentration of cisplatin was 2. 0 mu g/ml was the best concentration of.2. flow cytometry to detect cell apoptosis. After PRRX1 silencing, apoptosis was obviously inhibited by.3. flow cytometry. After PRRX1 silencing, cell cycle arrest in G2 phase.4. mitochondrial membrane potential detection results showed that after PRRX1 was silent, cisplatin was induced. The decrease of mitochondrial membrane potential was inhibited by inhibition of.5.PRRX1 silencing, apoptosis related protein Caspase-3, caspase-9, cytochrome C and Apaf-1 expression decreased.6. adding PAC-1 in MTT test and apoptosis experiment results showed that cell proliferation was obviously inhibited and cell apoptosis increased significantly. And interfere with the activation of Caspase-3 apoptosis channel, inhibit cisplatin induced apoptosis in A549 cells.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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