探討人類肝癌細胞HepG2經4-氨基聯苯處理過後miRNA-630對於同源重組修復相關蛋白MCM8的調控機制

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：54

、訪客IP：3.149.26.40

姓名

李佳芸(Chia-Yun Lee) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討人類肝癌細胞HepG2經4-氨基聯苯處理過後miRNA-630對於同源重組修復相關蛋白MCM8的調控機制
(Molecular mechanisms of microRNA-630 regulating MCM8 expression in 4-aminobiphenyl-treated HepG2 cells)

相關論文

★ 4-aminobiphenyl誘導HepG2細胞中的microRNAs表現並藉由microRNAs調控DNA修復機制	★ 研究Dicrotophos對HepG2細胞毒性之分子機制：CSA蛋白質在毒性扮演之角色
★ TNT經由ROS介導之內質網壓力及粒線體失衡誘導人類肝臟細胞凋亡	★ Pseudomonas sp. A46全基因組分析與重金屬復育基因工程菌開發
★ 4-Aminobiphenyl 調控 miR-630 抑制 RAD18 表現誘導 Hep3B 細胞產生氧化性 DNA 損傷	★ 三硝基甲苯之毒理機制及生物降解暨多氯乙烯汙染模場生物整治
★ 假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討	★ 開發新穎性包埋Dehalococcoides mccartyi及Clostridium butyricum之長效脫氯膠體
★ 探討DNA損傷反應與慢性暴露4-胺基聯苯產生之肝臟毒性	★ 以Lpp-OmpA工法建構新穎性基因工程菌強化鎘生物復育能力
★ 建構脫鹵球菌與固氮菌共培養系統促進氮源缺乏環境下的還原脫氯作用	★ 硒代胱氨酸通過誘導人肝細胞癌中的 DNA 損傷和抑制 DNA 修復途徑來增強順鉑敏感性
★ 轉錄體分析 Acetobacterium woodii 降解1,1,1-三氯乙烷機制並用以協助 Dehalococcoides進行還原脫氯	★ 以宏觀基因體分析新穎 Candidatus Dehalobacterium strain DLY 降解二氯甲烷機制
★ 研究雙特松對HepG2細胞之DNA修復的影響	★ 金屬硫蛋白在大腸桿菌的表達與金屬累積能力測試

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

4-氨基聯苯已被認定為人類致癌物質，且廣泛存在於日常生活中。先前的研究中證實，4-氨基聯苯會造成ROS的上升及誘導調控DNA修復蛋白的miRNA生成，但對於兩者在4-氨基聯苯對於人類肝癌細胞的毒理機制中扮演的角色仍所知甚少。研究中利用生物資訊軟體TargetScan預測，得知DNA雙股斷裂修復蛋白MCM8會受miRNA-630調控，再藉由觀察-H2AX的表現量偵測DNA雙股斷裂程度。經實驗證實miRNA-630會抑制MCM8的生成，並確實會導致同源重組修復效率下降，抑制DNA雙股斷裂修復。同時也在合併處理4-氨基聯苯與NAC(N-acetyl-1-cysteine)的細胞中，發現miR-630的生成及DNA損傷程度皆有降低，故得知ROS會誘導miRNA-630且參與DNA雙股斷裂修復的調控。
　　以上結果證實，經4-氨基聯苯處理後細胞會產生ROS，然後誘導miRNA-630表現，導致DNA修復蛋白MCM8表現量被抑制，最終造成的DNA損傷。

摘要(英)

4-ABP (4-Aminobiphenyl) is recognize as a human carcinogen, and commonly found in our daily life. Previous studies have shown that 4-ABP causes oxidative DNA damage and induces expression of some miRNAs involved in the regulation of DNA repair. However, the mechanism about the role of miRNAs involved in oxidative DNA damage in 4-ABP-treated hepatoma cells is unclear. By using TargetScan software, we found that miR-630 is a conserved target of MCM8, a DNA double-strand breaks repair protein. Therefore, we want to figure out the effects of miRNA on MCM8 expression. Our results proved that the elevated expression of miR-630 resulted in the decrease of MCM8 expression, causing the significant decrease of homologous recombination activity. Accordingly, the repair of DNA double-strand breaks (DSBs) was attenuated as evidenced from γ-H2AX analysis. In 4-ABP-treated cells, we found that the levels of miR-630 expression and DNA damage were attenuated by co-treatment with N-acetyl-1-cysteine (NAC), indicating that ROS-dependent miR-630 was involved in DSBs repair.
　　Collectively, 4-ABP induces ROS (Reactive oxygen species) generate, increases miRNA-630 expression, and then attenuates repair of DNA double-strand breaks by targeting MCM8.

關鍵字(中)

★ 4-氨基聯苯
★ miRNA-630
★ MCM8
★ DNA損傷
★ 同源重組

關鍵字(英)

★ 4-Aminobiphenyl
★ miR-630
★ MCM8
★ DNA Damage
★ Homologous Recombination

論文目次

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VII
表目錄 VIII
第一章、緒論 1
1-1 研究背景 1
1-1-1 4-胺基聯苯（4-aminobiphenyl；4-ABP） 1
1-1-2 4-胺基聯苯造成DNA損傷 1
1-1-3 4-胺基聯苯的致癌性 2
1-1-4 氧化壓力(Oxidative stress) 3
1-1-5 DNA損傷反應 (DNA damage response; DDR) 3
1-1-6 miRNA (microRNA; 微小RNA) 4
1-1-7 miRNA 的生成 4
1-1-8 miRNA對標靶基因mRNA的辨識與預測 6
1-1-9 環境毒素對於DNA修復蛋白之表觀遺傳調控 7
1-1-10 4-胺基聯苯所誘導的表觀遺傳學調控機制 7
1-2 研究目的 9
1-3 研究架構 9
第二章、材料與方法 10
2-1 實驗材料 10
2-1-1 藥品 10
2-1-2 抗體 11
2-1-3 套組試劑 11
2-1-4 溶液製備 11
2-2 實驗方法 13
2-2-1 細胞株來源及細胞培養 13
2-2-2 細胞計數與存活測試 14
2-2-3 化學物質處理 15
2-2-4 細胞存活率分析 15
2-2-5 DNA損傷測定-彗星試驗 15
2-2-6 西方免疫墨點法 16
2-2-7 HepG2 RNA萃取 20
2-2-8 mRNA 反轉錄成cDNA 21
2-2-9 DNA及RNA 電泳 22
2-2-10 即時定量聚合酶連鎖反應 22
2-2-11 質體抽取 24
2-2-12 質體DNA及小片段RNA轉染 25
2-2-13 免疫螢光染色 26
2-2-14 同源重組活性測試 26
第三章、結果 28
3-1 4-胺基聯苯誘導HepG2細胞造成細胞毒性 28
3-2 4-胺基聯苯誘導HepG2細胞DNA損傷 28
3-3 4-胺基聯苯對於miRNA-513a-5p 與miRNA-630的表現量的影響 28
3-4 4-胺基聯苯對於MCM8及MCM9之mRNA及蛋白質表現量的影響 29
3-5 miRNA-630會抑制MCM8表現量 29
3-6 ROS會誘導miRNA-630生成 30
3-7 MCM8表現量對於同源重組活性的影響 30
第四章、討論 32
4-1 4-胺基聯苯對於HepG2細胞造成的毒性 32
4-2 DNA損傷誘導miRNAs生成 33
4-3 miRNA-630對DNA修復蛋白MCM8的調控 34
4-4 MCM8對於同源重組活性的影響 35
第五章、結論 37
圖表 38
參考文獻 57

參考文獻

[1] Vineis P. Epidemiology of Cancer from Exposure to Arylamines. Environmental Health Perspectives. 1994;102:7-10.
[2] Luceri F, Pieraccini G, Moneti G, Dolara P. Primary aromatic amines from side-stream cigarette smoke are common contaminants of indoor air. Toxicology and industrial health. 1993;9:405-13.
[3] Tokiwa H, Nakagawa R, Horikawa K. Mutagenic/carcinogenic agents in indoor pollutants; the dinitropyrenes generated by kerosene heaters and fuel gas and liquefied petroleum gas burners. Mutat Res. 1985;157:39-47.
[4] Manabe S, Izumikawa S, Asakuno K, Wada O, Kanai Y. Detection of carcinogenic amino-alpha-carbolines and amino-gamma-carbolines in diesel-exhaust particles. Environmental pollution (Barking, Essex : 1987). 1991;70:255-65.
[5] Chiang TA, Pei-Fen W, Ying LS, Wang LF, Ko YC. Mutagenicity and aromatic amine content of fumes from heated cooking oils produced in Taiwan. Food Chem Toxicol. 1999;37:125-34.
[6] Garrigos MC, Reche F, Marin ML, Jimenez A. Determination of aromatic amines formed from azo colorants in toy products. Journal of chromatography A. 2002;976:309-17.
[7] Yoon JI, Kim SI, Tommasi S, Besaratinia A. Organ specificity of the bladder carcinogen 4-aminobiphenyl in inducing DNA damage and mutation in mice. Cancer prevention research (Philadelphia, Pa). 2012;5:299-308.
[8] Talaska G, Schamer M, Skipper P, Tannenbaum S, Caporaso N, Unruh L, et al. Detection of carcinogen-DNA adducts in exfoliated urothelial cells of cigarette smokers: association with smoking, hemoglobin adducts, and urinary mutagenicity. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 1991;1:61-6.
[9] International Agency for Research on Cancer (2004) IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans,
Tobacco smoking, . In: Cancer. IAfRo, editor. International Agency for Research on Cancer: Lyon, France.2004.
[10] Stolz A. Basic and applied aspects in the microbial degradation of azo dyes. Applied microbiology and biotechnology. 2001;56:69-80.
[11] (US). CfDCaPUNCfCDPaHPUOoSaH. How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. . Available from: https://www.ncbi.nlm.nih.gov/books/NBK53017/: Atlanta (GA): Centers for Disease Control and Prevention (US). 2010.
[12] Ricicki EM, Soglia JR, Teitel C, Kane R, Kadlubar F, Vouros P. Detection and Quantification of N-(Deoxyguanosin-8-yl)-4-aminobiphenyl Adducts in Human Pancreas Tissue Using Capillary Liquid Chromatography-Microelectrospray Mass Spectrometry. Chemical Research in Toxicology. 2005;18:692-9.
[13] Murata M, Kawanishi S. Mechanisms of oxidative DNA damage induced by carcinogenic arylamines. Frontiers in bioscience (Landmark edition). 2011;16:1132-43.
[14] Lee HW, Wang HT, Weng MW, Hu Y, Chen WS, Chou D, et al. Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells. Oncotarget. 2014;5:3526-40.
[15] Cohen SM, Boobis AR, Meek ME, Preston RJ, McGregor DB. 4-Aminobiphenyl and DNA reactivity: case study within the context of the 2006 IPCS Human Relevance Framework for Analysis of a cancer mode of action for humans. Critical reviews in toxicology. 2006;36:803-19.
[16] Ramirez T, Strigun A, Verlohner A, Huener HA, Peter E, Herold M, et al. Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells. Arch Toxicol. 2017.
[17] Rodriguez-Antona C, Ingelman-Sundberg M. Cytochrome P450 pharmacogenetics and cancer. Oncogene. 2006;25:1679.
[18] Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Molecular and cellular biochemistry. 2004;266:37-56.
[19] Siti HN, Kamisah Y, Kamsiah J. The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular disease (a review). Vascular pharmacology. 2015;71:40-56.
[20] Tangvarasittichai S. Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus. World journal of diabetes. 2015;6:456-80.
[21] Karaaslan C, Suzen S. Antioxidant properties of melatonin and its potential action in diseases. Current topics in medicinal chemistry. 2015;15:894-903.
[22] Cervelli T, Borghini A, Galli A, Andreassi MG. DNA damage and repair in atherosclerosis: current insights and future perspectives. International Journal of Molecular Sciences. 2012;13:16929-44.
[23] Sirbu BM, Cortez D. DNA Damage Response: Three Levels of DNA Repair Regulation. Cold Spring Harbor perspectives in biology.5.
[24] .
[25] Qin Q, Xie H, Wise SS, Browning CL, Thompson KN, Holmes AL, et al. Homologous recombination repair signaling in chemical carcinogenesis: prolonged particulate hexavalent chromium exposure suppresses the Rad51 response in human lung cells. Toxicological sciences : an official journal of the Society of Toxicology. 2014;142:117-25.
[26] Park J, Long DT, Lee KY, Abbas T, Shibata E, Negishi M, et al. The MCM8-MCM9 complex promotes RAD51 recruitment at DNA damage sites to facilitate homologous recombination. Mol Cell Biol. 2013;33:1632-44.
[27] Lee KY, Im JS, Shibata E, Park J, Handa N, Kowalczykowski SC, et al. MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex. Nat Commun. 2015;6:7744.
[28] He M, Zhou W, Li C, Guo M. MicroRNAs, DNA Damage Response, and Cancer Treatment. International Journal of Molecular Sciences. 2016;17.
[29] Wang Y, Taniguchi T. MicroRNAs and DNA damage response: implications for cancer therapy. Cell Cycle. 2013;12:32-42.
[30] Cha HJ, Kim O, Lee GT, Lee KS, Lee JH, Park I, et al. Identification of ultraviolet B radiation-induced microRNAs in normal human dermal papilla cells. Molecular Medicine Reports. 2014;10:1663-70.
[31] Ruvkun G, Giusto J. The Caenorhabditis elegans heterochronic gene lin-14 encodes a nuclear protein that forms a temporal developmental switch. Nature. 1989;338:313-9.
[32] Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;75:843-54.
[33] Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993;75:855-62.
[34] Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, et al. MicroRNA genes are transcribed by RNA polymerase II. The EMBO journal. 2004;23:4051-60.
[35] Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425:415-9.
[36] Kim VN. MicroRNA biogenesis: coordinated cropping and dicing. Nature reviews Molecular cell biology. 2005;6:376-85.
[37] Pisarello MJ, Loarca L, Ivanics T, Morton L, LaRusso N. MicroRNAs in the Cholangiopathies: Pathogenesis, Diagnosis, and Treatment. Journal of clinical medicine. 2015;4:1688-712.
[38] Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome research. 2009;19:92-105.
[39] Saito T, Saetrom P. MicroRNAs--targeting and target prediction. New biotechnology. 2010;27:243-9.
[40] Lal A, Pan Y, Navarro F, Dykxhoorn DM, Moreau L, Meire E, et al. miR-24-mediated downregulation of H2AX suppresses DNA repair in terminally differentiated blood cells. Nature structural & molecular biology. 2009;16:492-8.
[41] Cao Y, Yu SL, Wang Y, Guo GY, Ding Q, An RH. MicroRNA-dependent regulation of PTEN after arsenic trioxide treatment in bladder cancer cell line T24. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2011;32:179-88.
[42] Williams AE, Perry MM, Moschos SA, Larner-Svensson HM, Lindsay MA. Role of miRNA-146a in the regulation of the innate immune response and cancer. Biochemical Society transactions. 2008;36:1211-5.
[43] Chandra S, Khatoon R, Pandey A, Saini S, Vimal D, Singh P, et al. Dme-miR-314-3p modulation in Cr(VI) exposed Drosophila affects DNA damage repair by targeting mus309. Journal of hazardous materials. 2016;304:360-9.
[44] Hou L, Zhang X, Wang D, Baccarelli A. Environmental chemical exposures and human epigenetics. International journal of epidemiology. 2012;41:79-105.
[45] Zhang L, Feng G, Zhang X, Ding Y, Wang X. microRNA630 promotes cell proliferation and inhibits apoptosis in the HCT116 human colorectal cancer cell line. Molecular Medicine Reports. 2017;16:4843-8.
[46] Chappell G, Pogribny IP, Guyton KZ, Rusyn I. Epigenetic alterations induced by genotoxic occupational and environmental human chemical carcinogens: A systematic literature review. Mutation research Reviews in mutation research. 2016;768:27-45.
[47] Huan LC, Wu JC, Chiou BH, Chen CH, Ma N, Chang CY, et al. MicroRNA regulation of DNA repair gene expression in 4-aminobiphenyl-treated HepG2 cells. Toxicology. 2014;322:69-77.
[48] Wang ZY, Zhang W, Yang JJ, Song DK, Wei JX. Expression of miRNA-630 in bladder urothelial carcinoma and its clinical significance. Journal of Huazhong University of Science and Technology Medical sciences = Hua zhong ke ji da xue xue bao Yi xue Ying De wen ban = Huazhong keji daxue xuebao Yixue Yingdewen ban. 2016;36:705-9.
[49] Wang SC, Chung JG, Chen CH, Chen SC. 2- and 4-Aminobiphenyls induce oxidative DNA damage in human hepatoma (Hep G2) cells via different mechanisms. Mutat Res. 2006;593:9-21.
[50] Nauwelaers G, Bessette EE, Gu D, Tang Y, Rageul J, Fessard V, et al. DNA adduct formation of 4-aminobiphenyl and heterocyclic aromatic amines in human hepatocytes. Chem Res Toxicol. 2011;24:913-25.
[51] He J, Jiang BH. Interplay between Reactive oxygen Species and MicroRNAs in Cancer. Current pharmacology reports. 2016;2:82-90.
[52] Lutzmann M, Grey C, Traver S, Ganier O, Maya-Mendoza A, Ranisavljevic N, et al. MCM8- and MCM9-deficient mice reveal gametogenesis defects and genome instability due to impaired homologous recombination. Mol Cell. 2012;47:523-34.
[53] Ramirez T, Strigun A, Verlohner A, Huener HA, Peter E, Herold M, et al. Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells. Arch Toxicol. 2018;92:893-906.
[54] Weeden CE, Asselin-Labat ML. Mechanisms of DNA damage repair in adult stem cells and implications for cancer formation. Biochimica et biophysica acta. 2018;1864:89-101.
[55] Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine actions. Cellular and molecular life sciences : CMLS. 2003;60:6-20.
[56] Deng Q, Huang S, Zhang X, Zhang W, Feng J, Wang T, et al. Plasma microRNA Expression and Micronuclei Frequency in Workers Exposed to Polycyclic Aromatic Hydrocarbons. Environ Health Perspect. 2014;122:719-25.
[57] Chu D, Zheng J, Li J, Li Y, Zhang J, Zhao Q, et al. MicroRNA-630 is a prognostic marker for patients with colorectal cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2014;35:9787-92.
[58] Corcoran C, Rani S, Breslin S, Gogarty M, Ghobrial IM, Crown J, et al. miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in HER2 over-expressing breast cancer. Molecular Cancer. 2014;13:71.
[59] Wu L, Chen Z, Zhang J, Xing Y. Effect of miR-513a-5p on etoposide-stimulating B7-H1 expression in retinoblastoma cells. Journal of Huazhong University of Science and Technology Medical sciences = Hua zhong ke ji da xue xue bao Yi xue Ying De wen ban = Huazhong keji daxue xuebao Yixue Yingdewen ban. 2012;32:601-6.
[60] Nan D, Qing Y, Cun Y, Zhong Z, Li C, Zhang S, et al. miR-513a-5p regulates radiosensitivity of osteosarcoma by targeting human apurinic/apyrimidinic endonuclease2016.
[61] Nishimura K, Ishiai M, Horikawa K, Fukagawa T, Takata M, Takisawa H, et al. Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks. Mol Cell. 2012;47:511-22.
[62] Katyal S, Lee Y, Nitiss KC, Downing SM, Li Y, Shimada M, et al. Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes. Nature neuroscience. 2014;17:813-21.
[63] Truong LN, Li Y, Sun E, Ang K, Hwang PY, Wu X. Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication. The Journal of biological chemistry. 2014;289:28910-23.

指導教授

陳師慶(Ssu-Ching Chen)

審核日期

2018-6-28

推文