转录因子KLF5在缺氧性肺动脉高压中的作用

发布时间：2018-03-10 14:43

本文选题：缺氧　切入点：KLF5　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分转录因子KLF5在缺氧性肺动脉高压中的作用目的：缺氧性肺动脉高压是一种复杂的系统性疾病,肺小动脉重构是其主要的病理特征。KLF5(Kruppel-like factor5)作为一种锌指结构的转录因子,参与了肿瘤细胞的增殖、凋亡、分化以及迁移过程。它在肺动脉高压病人的肺动脉平滑肌细胞中高表达。研究发现KLF5参与了心血管重构的形成过程。然而,目前KLF5在缺氧性肺动脉高压中的作用及分子机制尚不清楚。方法：实验采用成年雄性SD大鼠(体重为200-250g),分别置于常氧或缺氧环境(10%氧浓度)中培养。‘检测大鼠血流动力学指标,评估其肺动脉中膜厚度和右心室肥厚程度,行肺组织HE染色观测大鼠血管重构情况。在常氧或缺氧(5%氧浓度)培养箱中培养人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells, HPASMC)。在HPASMC中行CCK-8、BrdU标记、PI单染、Annexin-V/PI双染、transwell迁移实验检测细胞活力、细胞增殖、细胞周期、细胞凋亡和细胞迁移能力。行免疫印迹、免疫组织化学、免疫荧光染色实验检测目的蛋白水平。行实时荧光定量聚合酶链反应(polymerase chain reaction, PCR)检测目的基因mRNA水平。提取暴露于常氧和缺氧环境的HPASMC裂解产物,通过免疫共沉淀实验明确KLF5和缺氧诱导因子-1α(hypoxia inducible factor-la, HIF-1α)之间是否存在关联。采用小干扰RNA (small interfering RNA, siRNA)下调HPASMC中KLF5和HIF-1α的表达。为上调KLF5水平,建立过表达KLF5基因的慢病毒载体并感染HPASMC。构建KLF5基因短发卡结构RNA (short hairpin RNA, shRNA)慢病毒载体,通过尾静脉注射入大鼠体内以下调KLF5表达。结果：缺氧组大鼠的平均肺动脉压力显著升高,伴随远端肺小动脉中膜增厚及右心室肥厚。缺氧引起大鼠肺动脉中膜层PCNA、Ki67阳性细胞增多,TUNEL阳性细胞减少。缺氧HPASMC的增殖和迁移能力增强,细胞凋亡减少。沉默KLF5或HIF-1α可以逆转这些生物学改变。在平滑肌细胞中,沉默KLF5后HIF-1α蛋白水平下调,过表达KLF5可以上调HIF-1α水平,而沉默HIF-1α后KLF5表达无显著改变。免疫共沉淀实验提示KLF5和HIF-1α存在直接结合,缺氧刺激下沉淀的目的蛋白增多。细胞周期蛋白cyclin B1、cyclin D1和凋亡相关蛋白bax、bc-2、survivin、caspase-3和caspase-9参与了KLF5-HIF-1α轴对HPASMC生长的调控。KLF5沉默大鼠的肺动脉中HIF-1α表达显著下调,平均肺动脉压和肺动脉重构情况显著改善,与细胞实验的结果一致。结论：缺氧促进肺动脉平滑肌细胞增殖、迁移增加,凋亡减少,血管重构形成。本研究从细胞水平和在体水平验证了KLF5-HIF-1α轴存在于缺氧大鼠的肺动脉平滑肌细胞,并且在缺氧性肺血管重构中发挥了重要作用。KLF5是缺氧性肺动脉高压治疗的潜在靶点。第二部分转录因子KLF5在在缺氧对非小细胞肺癌细胞增殖和凋亡中的作用目的：转录因子KLF5在肿瘤组织中广泛表达,且是肿瘤的一个预后因子。然后,KLF5在非小细胞肺癌中的作用尚未阐明。缺氧通过HIF-1α在肿瘤的发展中起重要作用。本研究旨在探讨KLF5在缺氧性肺癌细胞生长中的作用以及KLF5和HIF-1α的潜在关联。方法：我们通过对暴露于常氧(20%氧浓度)或缺氧(1%氧浓度)环境的A549细胞进行CCK-8实验、克隆形成实验、CFSE标记实验、Annexin-V/PI实验检测细胞的活力、克隆形成能力、‘增殖和凋亡。通过合成小干扰RNA来下调KLF5和HIF-1α的表达。采用PCR和免疫印迹的方法检测mRNA和蛋白的表达水平。结果：研究显示缺氧可以增强A549细胞活力、克隆形成能力,促进增殖,抑制细胞凋亡。缺氧上调HIF-1α和KLF5的表达,呈时间依赖性。沉默KLF5和HIF-1α可以通过下调cyclin B1、survivin,上调caspase-3逆转缺氧引起的细胞活力增加和凋亡减少。免疫共沉淀实验提示KLF5和HIF-1α存在直接关联,且缺氧环境中沉淀的目的蛋白更多。此外,沉默KLF5可以降低HIF-1α的水平,而KLF5表达不受HIF-1α的影响,确定了KLF5是HIF-1α的上游调控因子。结论：本研究证实缺氧是一个促癌因素,在肺癌细胞中通过激活KLF5→HIF-1α→cyclin B1/survivin/caspase-3发挥作用。
[Abstract]:Part one
The role of transcription factor KLF5 in hypoxic pulmonary hypertension
Objective: hypoxic pulmonary hypertension is a complex systemic disease, pulmonary vascular remodeling is the main pathological features of.KLF5 (Kruppel-like factor5) is a zinc finger transcription factor structure, involved in tumor cell proliferation, apoptosis, differentiation and migration process. Its high expression in patients with pulmonary hypertension lung arterial smooth muscle cells. The study found that KLF5 is involved in the formation process of cardiovascular remodeling. However, the role of KLF5 in hypoxic pulmonary hypertension and its molecular mechanism is unclear.
Methods: adult male SD rats (weight 200-250g), respectively in normoxia or hypoxia environment (10% oxygen) in vitro. "Dynamic detection indexes of rats to evaluate the pulmonary artery blood flow, film thickness and right ventricular hypertrophy, lung tissue HE staining observation of rat vascular remodeling. Normoxia or hypoxia (5% oxygen) in cultured human pulmonary artery smooth muscle cells in the box (human pulmonary artery smooth muscle cells, HPASMC). Mark BrdU in HPASMC CCK-8, Bank of China, PI staining, Annexin-V/PI staining, Transwell migration assay cell viability, cell proliferation, cell cycle, apoptosis and cell migration the ability for. Western blotting, immunohistochemistry and immunofluorescence staining experiments to detect protein level. For real-time fluorescence quantitative polymerase chain reaction (polymerase chain reaction, PCR) to detect mRNA gene level. The extraction of exposure to The pyrolysis products of HPASMC in normoxic and hypoxic environment, through co immunoprecipitation experiments clearly KLF5 and hypoxia inducible factor -1 alpha (hypoxia inducible Factor-la, HIF-1 a) between whether there is correlation. Using small interfering RNA (siRNA small interfering RNA) expression of KLF5 alpha and HIF-1 HPASMC. In order to increase the level of KLF5, established the lentiviral expression vector of KLF5 gene and HPASMC. KLF5 gene infection construct short hairpin structure RNA (short hairpin RNA, shRNA) lentiviral vector, by intravenous injection of rats following KLF5 expression.
Results: the mean pulmonary artery pressure in hypoxia group rats increased significantly with distal pulmonary arterioles in membrane thickening and hypertrophy of right ventricle. Hypoxia induced pulmonary arteries of rats in the film PCNA, Ki67 positive cells, TUNEL positive cells decreased. To enhance the ability of proliferation and migration of HPASMC cells reduced apoptosis of hypoxia, silencing of KLF5 or HIF-1 alpha. These biological changes can be reversed. In smooth muscle cells, down-regulation of HIF-1 protein level of KLF5 silencing, overexpression of KLF5 can increase the levels of HIF-1, HIF-1 and alpha silence the expression of KLF5 was not significantly changed. The experiment showed that the KLF5 and HIF-1 alpha are direct binding immunoprecipitation, protein precipitation increased under hypoxia stimulation of cell cycle. Protein cyclin B1, D1 cyclin and apoptosis related proteins Bax, BC-2, survivin, caspase-3 and caspase-9 are involved in the regulation of.KLF5 KLF5-HIF-1 alpha axis on the growth of HPASMC silencing rat pulmonary artery in HIF- The expression of 1 alpha was significantly down, and the average pulmonary arterial pressure and pulmonary artery remodeling were significantly improved, and the results were in agreement with the results of cell experiments.
Conclusion: hypoxia induced proliferation of pulmonary artery smooth muscle cell migration increased, apoptosis, vascular remodeling formation. This study from the cellular level and in vivo validation of pulmonary artery smooth muscle cells KLF5-HIF-1 alpha axis exists in hypoxic rats, and hypoxic pulmonary vascular remodeling plays an important role in the.KLF5 is a potential target for the treatment of hypoxia pulmonary arterial hypertension.
The second part
Role of transcription factor KLF5 in the proliferation and apoptosis of non small cell lung cancer cells in anoxia
Purpose: the transcription factor KLF5 is widely expressed in tumor tissue, and is a prognostic factor in cancer. Then, the role of KLF5 in non small cell lung cancer has not been elucidated. Hypoxia plays an important role in the development of cancer by HIF-1 alpha. This study aimed to investigate the role of KLF5 in hypoxia induced lung cancer cell growth and the the potential association between KLF5 and HIF-1 alpha.
Methods: we exposed to normoxia (20% oxygen) or hypoxia (1% oxygen) environment A549 cell clone formation assay, CCK-8 test, CFSE labeling experiment, Annexin-V/PI assay, cell viability, colony forming ability, cell proliferation and apoptosis. The expression of "synthetic small interfering RNA to downregulation of KLF5 and HIF-1 alpha. MRNA and protein expression detected by PCR and Western blot.
Results: the study shows that hypoxia can enhance A549 cell viability, colony formation ability, promote the proliferation and inhibition of apoptosis. The expression of HIF-1 is upregulated by hypoxia and KLF5 alpha, is time dependent. The silencing of KLF5 and HIF-1 alpha through the down-regulation of cyclin B1, survivin, upregulation of Caspase-3 caused by lack of oxygen to reverse the increase in cell viability and apoptosis of experimental reduced. KLF5 and HIF-1 a direct link between the co immunoprecipitation and protein precipitation in the anoxic environment more. In addition, silencing of KLF5 can reduce the level of HIF-1 alpha, KLF5 expression was not affected by HIF-1 alpha, the KLF5 is the upstream regulatory factor HIF-1 alpha.
Conclusion: This study confirmed that hypoxia is a cancer promoting factor, and it plays a role in lung cancer cells by activating KLF5 to HIF-1 alpha to cyclin B1/survivin/caspase-3.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R544.1

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