【摘要】：背景:近年來,空氣污染與呼吸系統(tǒng)疾病的關系受到越來越多的關注。在過去的幾年,中國各地區(qū)霧霾天氣加重,導致空氣質量的惡化,已經(jīng)引起了全世界的關注。可吸入顆粒,又稱PM2.5(particulate matter,顆粒直徑小于2.5微米)能夠攜帶多種有毒物質深入到達肺部,刺激腐蝕支氣管上皮細胞和肺泡壁,從而損傷肺功能,甚至誘發(fā)肺癌。吸煙是迄今為止誘發(fā)肺癌的最危險的因素。與非吸煙人群相比,吸煙者罹患肺癌的風險高15-30倍,而90%肺癌患者也是由吸煙所致?香煙煙霧中含有5000余種化學物質,其中73種具有致癌性?尼古丁和亞硝胺4-(甲基)-1-(3-吡啶基)-1-丁酮(NNK)是香煙中的主要成分,能夠刺激正常支氣管上皮細胞及肺癌細胞的生長。燃燒的香煙煙霧中富含PM2.5,是室內PM2.5的罪魁禍首及主要來源,其致癌性更應受到重視。香煙PM2.5作為空氣環(huán)境PM2.5的重要組成部分,目前對其誘發(fā)的支氣管上皮細胞的惡性轉化,特別是致肺癌發(fā)病機制的研究是國際上研究的熱點。然而,對于香煙PM2.5誘發(fā)支氣管上皮細胞惡性轉化的精細機制知之甚少,本課題將在此方面進行初步的探索。RNA結合基序蛋白5(RBM5),也被稱為LUCA-15或H37,是一種核RNA結合蛋白,廣泛分布于哺乳動物組織中。RBM5最初作為一個候選腫瘤抑制基因被克隆,位于人類染色體3p21.3區(qū)域,該區(qū)域在人類多種癌癥中表達缺失。前期研究發(fā)現(xiàn)在不同的腫瘤中RBM5表達都有降低,包括Ras轉化的鼠胚胎成纖維細胞、乳腺癌、前列腺癌、人前庭神經(jīng)鞘瘤、原發(fā)性肺癌等。大量研究表明,RBM5及其剪接變異體參與細胞凋亡、增殖和腫瘤的發(fā)生。也有報道顯示,RBM5與吸煙所致肺癌的發(fā)生密切相關,但其具體調控機制目前尚無報道。Wnt信號通路是組織器官發(fā)育的關鍵調節(jié)因素,也是肺癌等許多癌癥發(fā)生發(fā)展的重要通路。研究顯示,香煙能夠激活Wnt/β-catenin通路。在我們的前期工作中證實,暴露于香煙提取物的A549細胞,β-catenin的表達上調,而RBM5又參與Wnt/β-catenin通路的調控。因此,在香煙干預過程中,RBM5可能參與其中,并起到重要的調控作用。然而,在香煙PM2.5誘導支氣管上皮細胞的過程中RBM5的表達情況尚不清楚,是否RBM5在香煙PM2.5誘發(fā)支氣管上皮細胞惡性轉化的過程中起到調控作用需進一步證實。本研究以人正常支氣管上皮細胞為研究對象,探討RBM5在香煙PM2.5暴露致支氣管上皮細胞惡性轉化過程中的調控作用及所涉及的具體分子機制,明確香煙PM2.5致惡性轉化細胞中RBM5對Wnt/β-catenin信號通路的調節(jié)作用,為進一步明確吸煙相關肺部惡性腫瘤的分子發(fā)病機制提供科學依據(jù),為空氣環(huán)境污染誘發(fā)肺癌的防治提供新的思路。方法:1、香煙提取物(CSE)誘導支氣管上皮細胞BEAS-2B惡性轉化情況及RBM5的表達水平對數(shù)生長期的人正常支氣管上皮細胞(BEAS-2B細胞),分別給予濃度為0μg/m L,25μg/m L,50μg/m L,100μg/m L的CSE及DMSO(CON),每天更換不同濃度新鮮含有CSE培養(yǎng)液,持續(xù)誘導8天后,恢復新鮮培養(yǎng)基繼續(xù)培養(yǎng)2-3周。檢測:(1)MTT法檢測CSE誘導后各組轉化細胞的生存率;(2)倒置顯微鏡觀察轉化細胞形態(tài)變化;(3)Transwell法及劃痕實驗評價轉化細胞侵襲遷移能力;(4)real-time-PCR法及Western blot法檢測轉化細胞致癌基因m RNA和蛋白的表達水平;(5)建立CSE誘導的轉化細胞裸鼠皮下移植瘤模型;(6)RT-PCR、real-time-PCR法及Western blot法檢測CSE誘導的BEAS-2B轉化細胞RBM5 m RNA和蛋白的表達水平。2、上調RBM5對CSE誘導的BEAS-2B惡性轉化細胞的細胞周期、凋亡、侵襲和遷移及Wnt/β-catenin信號通路的影響(1)慢病毒轉染LV-RBM5及LV-287,建立穩(wěn)定表達RBM5的T3細胞(CSE誘導的惡性轉化細胞),熒光顯微鏡觀察轉染效率;(2)MTT法及克隆形成實驗檢測轉染后各組細胞的生存率及增殖能力;(3)Transwell法及劃痕實驗評價各組細胞侵襲遷移能力;(4)流式細胞術檢測過表達RBM5后對轉化細胞細胞周期的影響;(5)流式細胞術檢測過表達RBM5后對轉化細胞凋亡的影響;(6)免疫熒光標記法檢測過表達RBM5后細胞內β-catenin的表達及分布;(7)熒光素酶報告基因檢測過表達RBM5后細胞的轉錄因子TCF的活性;(8)real-time-PCR及Western blot法檢測過表達RBM5后轉化細胞的細胞周期、凋亡、侵襲和轉移相關基因的表達;(9)Western blot檢測過表達RBM5對T3細胞Wnt/β-catenin信號通路相關蛋白的表達。3、上調RBM5對CSE誘導的BEAS-2B惡性轉化細胞的裸鼠皮下移植瘤細胞周期、凋亡、侵襲和遷移相關蛋白及Wnt/β-catenin信號通路相關蛋白表達的影響(1)建立RBM5過表達的T3細胞裸鼠皮下移植瘤模型,觀察繪制腫瘤生長曲線;(2)Western blot檢測過表達RBM5后腫瘤組織細胞周期、凋亡、增殖、侵襲和遷移以及Wnt/β-catenin信號通路相關蛋白表達情況。(3)免疫組化檢測過表達RBM5后腫瘤組織部分細胞周期、凋亡、增殖、侵襲和遷移以及Wnt/β-catenin信號通路相關蛋白表達情況。結果:1、給予不同濃度的CSE持續(xù)誘導BEAS-2B細胞8天,恢復2周期間,與對照組相比,細胞表型發(fā)生改變,表現(xiàn)為增殖能力增強、倍增時間縮短、侵襲遷移能力增強;形態(tài)學變化顯示,出現(xiàn)核質比異常,核固縮的現(xiàn)象,均以高劑量(100μg/m L CSE)組明顯;各組轉化細胞相關致癌基因(K-ras,C-myc,Cyclin A和D1)表達增加,高劑量(100μg/m L CSE)組轉化細胞形成裸鼠皮下移植瘤,并標記該組細胞為“T3細胞”。揭示慢性暴露于CSE的正常支氣管上皮細胞發(fā)生惡性轉化,具有致瘤性。2、不同濃度CSE誘導的各組轉化細胞,RBM5 m RNA及蛋白表達水平降低,呈劑量依賴性。提示RBM5參與CSE誘導的正常支氣管上皮細胞發(fā)生惡性轉化的過程。3、過表達RBM5能夠明顯抑制T3細胞(CSE誘導的惡性轉化細胞)的增殖、克隆形成、侵襲遷移能力。轉移相關基因HIF-1α、VEGF、MMP2和MMP9的表達明顯下降;細胞周期阻滯在G1期,周期相關基因CDK4、CDK6、Cyclin D1和Cyclin A表達水平顯著下調,而p53和p21表達水平明顯上調;過表達RBM5能夠促進T3細胞的凋亡,凋亡相關基因C-caspase 3、C-caspase 9和Bax表達水平顯著上調,Bcl-2蛋白表達水平明顯下調;在T3細胞中過表達RBM5能夠抑制Wnt/β-catenin信號通路的激活。4、過表達RBM5能夠抑制CSE誘導的惡性轉化細胞皮下移植瘤的增長,移植瘤細胞發(fā)生細胞周期阻滯,凋亡增加,侵襲和轉移相關蛋白表達下調,Wnt/β-catenin信號通路激活減少。進一步揭示RBM5在CSE誘導的正常支氣管上皮細胞發(fā)生惡性轉化過程具有調控作用。結論:我們證明了暴露于100μg/m L CSE的BEAS-2B細胞發(fā)生了惡性轉化,具有裸鼠形成能力;在CSE轉化的BEAS-2B細胞中RBM5的表達水平被抑制,而在發(fā)生惡性轉化的細胞過表達RBM5能夠明顯抑制其增殖能力、誘導細胞周期阻滯在G1/S期、促進凋亡、抑制侵襲遷移及裸鼠成瘤能力,并且抑制Wnt/β-catenin信號通路的激活。因此,我們的發(fā)現(xiàn)可能為進一步明確吸煙相關肺部惡性腫瘤的分子發(fā)病機制提供科學依據(jù),為明確RBM5抗腫瘤的精細機制提供新的見解,為空氣環(huán)境污染誘發(fā)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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2
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本文編號：2180383