香烟PM2.5抑制RBM5诱导支气管上皮细胞恶性转化机制的研究
发布时间：2018-08-13 08:06
【摘要】：背景:近年来,空气污染与呼吸系统疾病的关系受到越来越多的关注。在过去的几年,中国各地区雾霾天气加重,导致空气质量的恶化,已经引起了全世界的关注。可吸入颗粒,又称PM2.5(particulate matter,颗粒直径小于2.5微米)能够携带多种有毒物质深入到达肺部,刺激腐蚀支气管上皮细胞和肺泡壁,从而损伤肺功能,甚至诱发肺癌。吸烟是迄今为止诱发肺癌的最危险的因素。与非吸烟人群相比,吸烟者罹患肺癌的风险高15-30倍,而90%肺癌患者也是由吸烟所致?香烟烟雾中含有5000余种化学物质,其中73种具有致癌性?尼古丁和亚硝胺4-(甲基)-1-(3-吡啶基)-1-丁酮(NNK)是香烟中的主要成分,能够刺激正常支气管上皮细胞及肺癌细胞的生长。燃烧的香烟烟雾中富含PM2.5,是室内PM2.5的罪魁祸首及主要来源,其致癌性更应受到重视。香烟PM2.5作为空气环境PM2.5的重要组成部分,目前对其诱发的支气管上皮细胞的恶性转化,特别是致肺癌发病机制的研究是国际上研究的热点。然而,对于香烟PM2.5诱发支气管上皮细胞恶性转化的精细机制知之甚少,本课题将在此方面进行初步的探索。RNA结合基序蛋白5(RBM5),也被称为LUCA-15或H37,是一种核RNA结合蛋白,广泛分布于哺乳动物组织中。RBM5最初作为一个候选肿瘤抑制基因被克隆,位于人类染色体3p21.3区域,该区域在人类多种癌症中表达缺失。前期研究发现在不同的肿瘤中RBM5表达都有降低,包括Ras转化的鼠胚胎成纤维细胞、乳腺癌、前列腺癌、人前庭神经鞘瘤、原发性肺癌等。大量研究表明,RBM5及其剪接变异体参与细胞凋亡、增殖和肿瘤的发生。也有报道显示,RBM5与吸烟所致肺癌的发生密切相关,但其具体调控机制目前尚无报道。Wnt信号通路是组织器官发育的关键调节因素,也是肺癌等许多癌症发生发展的重要通路。研究显示,香烟能够激活Wnt/β-catenin通路。在我们的前期工作中证实,暴露于香烟提取物的A549细胞,β-catenin的表达上调,而RBM5又参与Wnt/β-catenin通路的调控。因此,在香烟干预过程中,RBM5可能参与其中,并起到重要的调控作用。然而,在香烟PM2.5诱导支气管上皮细胞的过程中RBM5的表达情况尚不清楚,是否RBM5在香烟PM2.5诱发支气管上皮细胞恶性转化的过程中起到调控作用需进一步证实。本研究以人正常支气管上皮细胞为研究对象,探讨RBM5在香烟PM2.5暴露致支气管上皮细胞恶性转化过程中的调控作用及所涉及的具体分子机制,明确香烟PM2.5致恶性转化细胞中RBM5对Wnt/β-catenin信号通路的调节作用,为进一步明确吸烟相关肺部恶性肿瘤的分子发病机制提供科学依据,为空气环境污染诱发肺癌的防治提供新的思路。方法:1、香烟提取物(CSE)诱导支气管上皮细胞BEAS-2B恶性转化情况及RBM5的表达水平对数生长期的人正常支气管上皮细胞(BEAS-2B细胞),分别给予浓度为0μg/m L,25μg/m L,50μg/m L,100μg/m L的CSE及DMSO(CON),每天更换不同浓度新鲜含有CSE培养液,持续诱导8天后,恢复新鲜培养基继续培养2-3周。检测:(1)MTT法检测CSE诱导后各组转化细胞的生存率;(2)倒置显微镜观察转化细胞形态变化;(3)Transwell法及划痕实验评价转化细胞侵袭迁移能力;(4)real-time-PCR法及Western blot法检测转化细胞致癌基因m RNA和蛋白的表达水平;(5)建立CSE诱导的转化细胞裸鼠皮下移植瘤模型;(6)RT-PCR、real-time-PCR法及Western blot法检测CSE诱导的BEAS-2B转化细胞RBM5 m RNA和蛋白的表达水平。2、上调RBM5对CSE诱导的BEAS-2B恶性转化细胞的细胞周期、凋亡、侵袭和迁移及Wnt/β-catenin信号通路的影响(1)慢病毒转染LV-RBM5及LV-287,建立稳定表达RBM5的T3细胞(CSE诱导的恶性转化细胞),荧光显微镜观察转染效率;(2)MTT法及克隆形成实验检测转染后各组细胞的生存率及增殖能力;(3)Transwell法及划痕实验评价各组细胞侵袭迁移能力;(4)流式细胞术检测过表达RBM5后对转化细胞细胞周期的影响;(5)流式细胞术检测过表达RBM5后对转化细胞凋亡的影响;(6)免疫荧光标记法检测过表达RBM5后细胞内β-catenin的表达及分布;(7)荧光素酶报告基因检测过表达RBM5后细胞的转录因子TCF的活性;(8)real-time-PCR及Western blot法检测过表达RBM5后转化细胞的细胞周期、凋亡、侵袭和转移相关基因的表达;(9)Western blot检测过表达RBM5对T3细胞Wnt/β-catenin信号通路相关蛋白的表达。3、上调RBM5对CSE诱导的BEAS-2B恶性转化细胞的裸鼠皮下移植瘤细胞周期、凋亡、侵袭和迁移相关蛋白及Wnt/β-catenin信号通路相关蛋白表达的影响(1)建立RBM5过表达的T3细胞裸鼠皮下移植瘤模型,观察绘制肿瘤生长曲线;(2)Western blot检测过表达RBM5后肿瘤组织细胞周期、凋亡、增殖、侵袭和迁移以及Wnt/β-catenin信号通路相关蛋白表达情况。(3)免疫组化检测过表达RBM5后肿瘤组织部分细胞周期、凋亡、增殖、侵袭和迁移以及Wnt/β-catenin信号通路相关蛋白表达情况。结果:1、给予不同浓度的CSE持续诱导BEAS-2B细胞8天,恢复2周期间,与对照组相比,细胞表型发生改变,表现为增殖能力增强、倍增时间缩短、侵袭迁移能力增强;形态学变化显示,出现核质比异常,核固缩的现象,均以高剂量(100μg/m L CSE)组明显;各组转化细胞相关致癌基因(K-ras,C-myc,Cyclin A和D1)表达增加,高剂量(100μg/m L CSE)组转化细胞形成裸鼠皮下移植瘤,并标记该组细胞为“T3细胞”。揭示慢性暴露于CSE的正常支气管上皮细胞发生恶性转化,具有致瘤性。2、不同浓度CSE诱导的各组转化细胞,RBM5 m RNA及蛋白表达水平降低,呈剂量依赖性。提示RBM5参与CSE诱导的正常支气管上皮细胞发生恶性转化的过程。3、过表达RBM5能够明显抑制T3细胞(CSE诱导的恶性转化细胞)的增殖、克隆形成、侵袭迁移能力。转移相关基因HIF-1α、VEGF、MMP2和MMP9的表达明显下降;细胞周期阻滞在G1期,周期相关基因CDK4、CDK6、Cyclin D1和Cyclin A表达水平显著下调,而p53和p21表达水平明显上调;过表达RBM5能够促进T3细胞的凋亡,凋亡相关基因C-caspase 3、C-caspase 9和Bax表达水平显著上调,Bcl-2蛋白表达水平明显下调;在T3细胞中过表达RBM5能够抑制Wnt/β-catenin信号通路的激活。4、过表达RBM5能够抑制CSE诱导的恶性转化细胞皮下移植瘤的增长,移植瘤细胞发生细胞周期阻滞,凋亡增加,侵袭和转移相关蛋白表达下调,Wnt/β-catenin信号通路激活减少。进一步揭示RBM5在CSE诱导的正常支气管上皮细胞发生恶性转化过程具有调控作用。结论:我们证明了暴露于100μg/m L CSE的BEAS-2B细胞发生了恶性转化,具有裸鼠形成能力;在CSE转化的BEAS-2B细胞中RBM5的表达水平被抑制,而在发生恶性转化的细胞过表达RBM5能够明显抑制其增殖能力、诱导细胞周期阻滞在G1/S期、促进凋亡、抑制侵袭迁移及裸鼠成瘤能力,并且抑制Wnt/β-catenin信号通路的激活。因此,我们的发现可能为进一步明确吸烟相关肺部恶性肿瘤的分子发病机制提供科学依据,为明确RBM5抗肿瘤的精细机制提供新的见解,为空气环境污染诱发肺癌的防治提供新思路。
[Abstract]:BACKGROUND: In recent years, more and more attention has been paid to the relationship between air pollution and respiratory diseases. Over the past few years, fog and haze weather in various regions of China has aggravated the deterioration of air quality, which has attracted worldwide attention. Smoking is by far the most dangerous risk factor for lung cancer. Smokers are 15-30 times more likely to develop lung cancer than non-smokers, and 90% of lung cancer patients are also caused by smoking? More than 1000 chemicals, of which 73 are carcinogenic? Nicotine and nitrosamine 4 - (methyl) - 1 - (3-pyridyl) - 1-butanone (NNK) are the main components of cigarettes, which can stimulate the growth of normal bronchial epithelial cells and lung cancer cells. As an important part of PM2.5 in air environment, the malignant transformation of bronchial epithelial cells induced by cigarette PM2.5, especially the pathogenesis of lung cancer, is a hot topic in the world. However, little is known about the precise mechanism of malignant transformation of bronchial epithelial cells induced by cigarette PM2.5. RNA binding motif protein 5 (RBM5), also known as LUCA-15 or H37, is a nuclear RNA binding protein widely distributed in mammalian tissues. RBM5 was originally cloned as a candidate tumor suppressor gene in the human chromosome 3p21.3 region, which is absent in many human cancers. It has been found that RBM5 expression is decreased in various tumors, including Ras-transformed mouse embryonic fibroblasts, breast cancer, prostate cancer, human vestibular schwannoma, primary lung cancer and so on. Wnt signaling pathway is a key regulator of organ development and an important pathway for the development of many cancers such as lung cancer. Studies have shown that cigarettes can activate the Wnt / beta catenin pathway. The expression of beta-catenin is up-regulated, and RBM5 is involved in the regulation of Wnt/beta-catenin pathway. Therefore, RBM5 may be involved and play an important regulatory role in the process of cigarette intervention. The role of RBM5 in the regulation of malignant transformation of human normal bronchial epithelial cells was investigated in this study. The specific molecular mechanisms involved in the regulation of RBM5 in the malignant transformation of bronchial epithelial cells induced by exposure to cigarette PM2.5 were investigated. The regulation of beta-catenin signaling pathway provides a scientific basis for further clarifying the molecular pathogenesis of smoking-related pulmonary malignancies and providing new ideas for the prevention and treatment of lung cancer induced by air pollution. Long-term human normal bronchial epithelial cells (BEAS-2B cells) were treated with CSE and DMSO (CON) at concentrations of 0, 25, 50, 100 and 100 ug/ml respectively. After 8 days of continuous induction, the cells were cultured in fresh medium for 2-3 weeks. Detection: (1) MTT assay was used to detect the transformed cells in each group after induction of CSE. Survival rate; (2) inverted microscope observation of morphological changes of transformed cells; (3) Transwell method and scratch test to evaluate the invasion and migration of transformed cells; (4) real-time-PCR and Western blot to detect the expression of oncogene m RNA and protein in transformed cells; (5) establishment of CSE-induced subcutaneous transplantation tumor model of transformed cells in nude mice; (6) RT-PCR, real-blot The expression levels of RBM5 m RNA and protein in BEAS-2B transformed cells induced by CSE were detected by time-PCR and Western blot. 2. The effects of RBM5 on cell cycle, apoptosis, invasion and migration of BEAS-2B malignant transformed cells induced by CSE were up-regulated. (1) Lentiviral transfection of LV-RBM5 and LV-287 to establish T3 cells stably expressing RBM5 (C The transfection efficiency of SE-induced malignant transformed cells was observed by fluorescence microscopy; (2) MTT assay and clone formation assay were used to detect the survival rate and proliferation ability of the transfected cells; (3) Transwell assay and scratch assay were used to evaluate the invasive and migratory ability of the cells in each group; (4) Flow cytometry was used to detect the effect of overexpression of RBM5 on the cell cycle of transformed cells; (5) Flow cytometry was used to detect the effect of overexpression of RBM5 on apoptosis of transformed cells; (6) Immunofluorescence labeling was used to detect the expression and distribution of beta catenin in cells after overexpression of RBM5; (7) Luciferase reporter gene was used to detect the activity of transcription factor TCF after overexpression of RBM5; (8) Real-time-PCR and Western blot were used to detect the transformation of transformed cells after overexpression of RBM5. Expression of cell cycle, apoptosis, invasion and metastasis related genes; (9) Overexpression of RBM5 on Wnt/beta-catenin signaling pathway related proteins in T3 cells was detected by Western blot, and RBM5 up-regulated cell cycle, apoptosis, invasion and metastasis related proteins and Wnt/beta-catenin related proteins in subcutaneous transplantation of BEAS-2B malignant transformed cells induced by CSE in nude mice. Effects of signal pathway-related protein expression (1) Establishment of a subcutaneous transplantation tumor model of T3 cells overexpressing RBM5, observation and drawing of tumor growth curve; (2) Western blot detection of tumor cell cycle, apoptosis, proliferation, invasion and migration, and Wnt/beta-catenin signaling pathway-related protein expression after overexpressing RBM5. (3) Immunohistochemical examination. The expression of some cell cycle, apoptosis, proliferation, invasion and migration, and the expression of Wnt/beta-catenin signaling pathway-related proteins in tumor tissues were measured after overexpression of RBM5. Results: 1. BEAS-2B cells were continuously induced by CSE at different concentrations for 8 days and restored for 2 weeks. Morphological changes showed that abnormal nuclear-cytoplasmic ratio and nuclear pyknosis were observed in the high dose group (100ug/m L CSE); the expression of K-ras, C-myc, Cyclin A and D1 was increased in each group, and the transformed cells in the high dose group (100ug/m L CSE) formed subcutaneous transplanted tumors in nude mice and labeled the same group. The results showed that the normal bronchial epithelial cells exposed to CSE had malignant transformation, which was oncogenic. 2. The expression of RBM5 m RNA and protein in the transformed cells induced by different concentrations of CSE decreased in a dose-dependent manner, suggesting that RBM5 was involved in the malignant transformation of normal bronchial epithelial cells induced by CSE. Overexpression of RBM5 significantly inhibited the proliferation, cloning, invasion and migration of T3 cells (malignant transformation cells induced by CSE). The expression of metastasis-related genes HIF-1a, VEGF, MMP2 and MMP9 decreased significantly. Cell cycle arrest in G1 phase resulted in a significant decrease in the expression levels of cycle-related genes CDK4, CDK6, Cyclin D 1 and Cyclin A, while the expression of p53 and p21 water decreased significantly. Overexpression of RBM5 could promote apoptosis of T3 cells, up-regulate the expression of apoptosis-related genes C-caspase 3, C-caspase 9 and Bax, and down-regulate the expression of Bcl-2 protein. Overexpression of RBM5 in T3 cells could inhibit the activation of Wnt/beta-catenin signaling pathway. Overexpression of RBM5 could inhibit the CSE-induced malignant transformation cells. The growth of subcutaneous transplanted tumor, cell cycle arrest, apoptosis increase, expression of invasion and metastasis-related proteins down-regulated, and activation of Wnt/beta-catenin signaling pathway decreased. The expression of RBM5 in BEAS-2B cells transformed by MLCSE was inhibited, while the proliferation of the cells transformed by MLCSE was significantly inhibited by over-expression of RBM5, which could induce cell cycle arrest in G1/S phase, promote apoptosis, inhibit invasion, migration and tumor formation in nude mice. Therefore, our findings may provide a scientific basis for further clarifying the molecular pathogenesis of smoking-related lung malignancies, and provide new insights for clarifying the fine mechanism of RBM5 anti-tumor, and provide new ideas for the prevention and treatment of lung cancer induced by air pollution.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
，
本文编号：2180383
[Abstract]:BACKGROUND: In recent years, more and more attention has been paid to the relationship between air pollution and respiratory diseases. Over the past few years, fog and haze weather in various regions of China has aggravated the deterioration of air quality, which has attracted worldwide attention. Smoking is by far the most dangerous risk factor for lung cancer. Smokers are 15-30 times more likely to develop lung cancer than non-smokers, and 90% of lung cancer patients are also caused by smoking? More than 1000 chemicals, of which 73 are carcinogenic? Nicotine and nitrosamine 4 - (methyl) - 1 - (3-pyridyl) - 1-butanone (NNK) are the main components of cigarettes, which can stimulate the growth of normal bronchial epithelial cells and lung cancer cells. As an important part of PM2.5 in air environment, the malignant transformation of bronchial epithelial cells induced by cigarette PM2.5, especially the pathogenesis of lung cancer, is a hot topic in the world. However, little is known about the precise mechanism of malignant transformation of bronchial epithelial cells induced by cigarette PM2.5. RNA binding motif protein 5 (RBM5), also known as LUCA-15 or H37, is a nuclear RNA binding protein widely distributed in mammalian tissues. RBM5 was originally cloned as a candidate tumor suppressor gene in the human chromosome 3p21.3 region, which is absent in many human cancers. It has been found that RBM5 expression is decreased in various tumors, including Ras-transformed mouse embryonic fibroblasts, breast cancer, prostate cancer, human vestibular schwannoma, primary lung cancer and so on. Wnt signaling pathway is a key regulator of organ development and an important pathway for the development of many cancers such as lung cancer. Studies have shown that cigarettes can activate the Wnt / beta catenin pathway. The expression of beta-catenin is up-regulated, and RBM5 is involved in the regulation of Wnt/beta-catenin pathway. Therefore, RBM5 may be involved and play an important regulatory role in the process of cigarette intervention. The role of RBM5 in the regulation of malignant transformation of human normal bronchial epithelial cells was investigated in this study. The specific molecular mechanisms involved in the regulation of RBM5 in the malignant transformation of bronchial epithelial cells induced by exposure to cigarette PM2.5 were investigated. The regulation of beta-catenin signaling pathway provides a scientific basis for further clarifying the molecular pathogenesis of smoking-related pulmonary malignancies and providing new ideas for the prevention and treatment of lung cancer induced by air pollution. Long-term human normal bronchial epithelial cells (BEAS-2B cells) were treated with CSE and DMSO (CON) at concentrations of 0, 25, 50, 100 and 100 ug/ml respectively. After 8 days of continuous induction, the cells were cultured in fresh medium for 2-3 weeks. Detection: (1) MTT assay was used to detect the transformed cells in each group after induction of CSE. Survival rate; (2) inverted microscope observation of morphological changes of transformed cells; (3) Transwell method and scratch test to evaluate the invasion and migration of transformed cells; (4) real-time-PCR and Western blot to detect the expression of oncogene m RNA and protein in transformed cells; (5) establishment of CSE-induced subcutaneous transplantation tumor model of transformed cells in nude mice; (6) RT-PCR, real-blot The expression levels of RBM5 m RNA and protein in BEAS-2B transformed cells induced by CSE were detected by time-PCR and Western blot. 2. The effects of RBM5 on cell cycle, apoptosis, invasion and migration of BEAS-2B malignant transformed cells induced by CSE were up-regulated. (1) Lentiviral transfection of LV-RBM5 and LV-287 to establish T3 cells stably expressing RBM5 (C The transfection efficiency of SE-induced malignant transformed cells was observed by fluorescence microscopy; (2) MTT assay and clone formation assay were used to detect the survival rate and proliferation ability of the transfected cells; (3) Transwell assay and scratch assay were used to evaluate the invasive and migratory ability of the cells in each group; (4) Flow cytometry was used to detect the effect of overexpression of RBM5 on the cell cycle of transformed cells; (5) Flow cytometry was used to detect the effect of overexpression of RBM5 on apoptosis of transformed cells; (6) Immunofluorescence labeling was used to detect the expression and distribution of beta catenin in cells after overexpression of RBM5; (7) Luciferase reporter gene was used to detect the activity of transcription factor TCF after overexpression of RBM5; (8) Real-time-PCR and Western blot were used to detect the transformation of transformed cells after overexpression of RBM5. Expression of cell cycle, apoptosis, invasion and metastasis related genes; (9) Overexpression of RBM5 on Wnt/beta-catenin signaling pathway related proteins in T3 cells was detected by Western blot, and RBM5 up-regulated cell cycle, apoptosis, invasion and metastasis related proteins and Wnt/beta-catenin related proteins in subcutaneous transplantation of BEAS-2B malignant transformed cells induced by CSE in nude mice. Effects of signal pathway-related protein expression (1) Establishment of a subcutaneous transplantation tumor model of T3 cells overexpressing RBM5, observation and drawing of tumor growth curve; (2) Western blot detection of tumor cell cycle, apoptosis, proliferation, invasion and migration, and Wnt/beta-catenin signaling pathway-related protein expression after overexpressing RBM5. (3) Immunohistochemical examination. The expression of some cell cycle, apoptosis, proliferation, invasion and migration, and the expression of Wnt/beta-catenin signaling pathway-related proteins in tumor tissues were measured after overexpression of RBM5. Results: 1. BEAS-2B cells were continuously induced by CSE at different concentrations for 8 days and restored for 2 weeks. Morphological changes showed that abnormal nuclear-cytoplasmic ratio and nuclear pyknosis were observed in the high dose group (100ug/m L CSE); the expression of K-ras, C-myc, Cyclin A and D1 was increased in each group, and the transformed cells in the high dose group (100ug/m L CSE) formed subcutaneous transplanted tumors in nude mice and labeled the same group. The results showed that the normal bronchial epithelial cells exposed to CSE had malignant transformation, which was oncogenic. 2. The expression of RBM5 m RNA and protein in the transformed cells induced by different concentrations of CSE decreased in a dose-dependent manner, suggesting that RBM5 was involved in the malignant transformation of normal bronchial epithelial cells induced by CSE. Overexpression of RBM5 significantly inhibited the proliferation, cloning, invasion and migration of T3 cells (malignant transformation cells induced by CSE). The expression of metastasis-related genes HIF-1a, VEGF, MMP2 and MMP9 decreased significantly. Cell cycle arrest in G1 phase resulted in a significant decrease in the expression levels of cycle-related genes CDK4, CDK6, Cyclin D 1 and Cyclin A, while the expression of p53 and p21 water decreased significantly. Overexpression of RBM5 could promote apoptosis of T3 cells, up-regulate the expression of apoptosis-related genes C-caspase 3, C-caspase 9 and Bax, and down-regulate the expression of Bcl-2 protein. Overexpression of RBM5 in T3 cells could inhibit the activation of Wnt/beta-catenin signaling pathway. Overexpression of RBM5 could inhibit the CSE-induced malignant transformation cells. The growth of subcutaneous transplanted tumor, cell cycle arrest, apoptosis increase, expression of invasion and metastasis-related proteins down-regulated, and activation of Wnt/beta-catenin signaling pathway decreased. The expression of RBM5 in BEAS-2B cells transformed by MLCSE was inhibited, while the proliferation of the cells transformed by MLCSE was significantly inhibited by over-expression of RBM5, which could induce cell cycle arrest in G1/S phase, promote apoptosis, inhibit invasion, migration and tumor formation in nude mice. Therefore, our findings may provide a scientific basis for further clarifying the molecular pathogenesis of smoking-related lung malignancies, and provide new insights for clarifying the fine mechanism of RBM5 anti-tumor, and provide new ideas for the prevention and treatment of lung cancer induced by air pollution.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
，
本文编号：2180383
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