【摘要】：研究背景鎳化合物在工業(yè)生產(chǎn)中的廣泛應用不可避免地會造成職業(yè)人群的健康危害。流行病學研究表明,與在非鎳暴露的場所中的工作人員相比,鎳礦工人中因患肺癌的死亡率明顯增高,鎳的化合物已經(jīng)被國際癌癥研究中心(IARC)正式確立為一類致癌物。近來的研究表明,組蛋白甲基化參與了鎳誘導肺癌的早期階段。尼克酰胺-N-甲基轉(zhuǎn)移酶(NNMT)與癌細胞的增殖、遷移等生物學特征密切相關,研究發(fā)現(xiàn)NNMT可以減少細胞內(nèi)腺苷甲硫氨酸/腺苷同型半胱氨酸(SAM/SAH)比值導致組蛋白甲基化水平降低。而目前,尚未見文獻報道鎳暴露能通過影響NNMT的表達進而誘導組蛋白甲基化。本課題主要研究NNMT是否參與了鎳引起的組蛋白甲基化,并初步探討鎳引NAD/NADH比值改變在鎳導致的NNMT表達抑制以及H3K9me2水平升高中的作用,為闡明鎳引起組蛋白甲基化的機制提供新的線索。研究內(nèi)容(1)將支氣管上皮細胞(BEAS-2B)暴露于不同濃度的氯化鎳(Ni Cl2)72h以及在200μM Ni Cl2處理0h、12h、24h、48h和72h。采用CCK-8試劑盒檢測氯化鎳暴露后BEAS-2B細胞活性的變化。用Western blot檢測氯化鎳暴露后,組蛋白H3第9位賴氨酸單甲基化(H3K9me1)、雙甲基化(H3K9me2)和三甲基化(H3K9me3)以及NNMT蛋白的表達水平,RT-PCR用于檢測H3K9me2下游的肺癌抑癌基因MAP2K3和DKK1以及NNMT m RNA的表達水平。(2)采用細胞轉(zhuǎn)染的方法使BEAS-2B細胞過表達NNMT,觀察NNMT過表達后,經(jīng)氯化鎳處理后的細胞中H3K9me2及其下游抑癌基因MAP2K3和DKK1的表達變化,用HPLC檢測細胞中SAM/SAH比值的變化。(3)用NADH氧化劑檸嗪酸(phenazine methosulfate,PMS)處理經(jīng)氯化鎳暴露后的細胞,檢測細胞內(nèi)NAD/NADH比值的變化,用Western blot和RT-PCR檢測NNMT、H3K9me2及其下游抑癌基因MAP2K3和DKK1的表達變化。研究結果(1)氯化鎳引起組蛋白H3第9位賴氨酸甲基化中H3K9me2水平增加,并下調(diào)其下游抑癌基因MAP2K3和DKK1的表達。(2)氯化鎳抑制BEAS-2B細胞中NNMT的表達,NNMT可以通過降低細胞內(nèi)SAM/SAH比值參與鎳引起的H3K9me2升高以及MAP2K3和DKK1表達水平降低。(3)PMS可以抑制鎳引起的H3K9me2升高以和抑癌基因MAP2K3和DKK1表達下調(diào),但不能拮抗鎳導致的BEAS-2B細胞中NNMT的表達抑制。研究結論根據(jù)本研究的結果,得出結論:鎳導致的NNMT表達抑制參與了鎳誘導的H3K9me2水平增加,NNMT可以通過改變細胞內(nèi)SAM/SAH的比值調(diào)控鎳引起的H3K9me2升高。氯化鎳導致的NAD/NADH比值下降沒有在鎳抑制NNMT表達中起作用,但是NAD/NADH比值的改變參與了鎳導致H3K9me2水平升高這一過程。本研究為解釋鎳引起組蛋白甲基化狀態(tài)改變提供了新的假說,有助于進一步闡明鎳致肺癌的表觀遺傳機制。
[Abstract]:Background the widespread application of nickel compounds in industrial production will inevitably cause health hazards to the occupational population. Epidemiological studies show that the mortality rate of lung cancer in nickel miners is significantly higher than that in workers exposed to non-nickel, and nickel compounds have been officially established as carcinogens by the International Center for Cancer Research (IARC). Recent studies have shown that histone methylation is involved in the early stage of nickel-induced lung cancer. NNMT is closely related to the proliferation and migration of cancer cells. It has been found that NNMT can reduce the ratio of adenosine methionine to adenosine homocysteine (SAM / SAH) and decrease the level of histone methylation. However, it has not been reported that nickel exposure can induce histone methylation by affecting the expression of NNMT. The purpose of this study was to investigate whether NNMT was involved in the histone methylation induced by nickel, and to explore the role of NNMT induced by nickel in the inhibition of NNMT expression and the increase of H3K9me2 level. It provides a new clue for elucidating the mechanism of histone methylation induced by nickel. Contents (1) bronchial epithelial cells (BEAS-2B) were exposed to different concentrations of nickel chloride (Ni Cl 2) for 72 h and 200 渭 M Ni Cl 2 for 0 h, 12 h, 24 h, 48 h and 72 h. CCK-8 kit was used to detect the activity of BEAS- 2B cells after exposure to nickel chloride. After exposure to nickel chloride, nickel chloride was detected by Western blot. The expression of Lysine Monome1 (H3K9me1), H3K9me2 (H3K9me2) and NMT protein (H3K9me3), and the expression of NMT protein in histone H3. RT-PCR was used to detect the expression levels of MAP2K3, DKK1 and NMT mRNA in the lung cancer suppressor gene MAP2K3 and DKK1 downstream of H3K9me2. The staining method made BEAS-2B cells overexpression NNMT.After observing the overexpression of NNMT, The expression of H3K9me2 and its downstream tumor suppressor genes MAP2K3 and DKK1 were detected by HPLC. (3) the cells exposed to nickel chloride were treated with nadh oxidant phenazine methosulfate PMS. The expression of NMTH3K9me2 and its downstream tumor suppressor genes MAP2K3 and DKK1 were detected by Western blot and RT-PCR. Results (1) the level of H3K9me2 in histone H3 methylation induced by nickel chloride was increased. (2) Nickel chloride inhibited the expression of NNMT in BEAS-2B cells. (3) PMS could inhibit the expression of NNMT in BEAS-2B cells by decreasing the ratio of SAM / SAH in cells to increase H3K9me2 induced by nickel and decrease the expression of MAP2K3 and DKK1 in BEAS-2B cells. (3) PMS could inhibit the expression of NNMT in BEAS-2B cells. H3K9me2 increased and the expression of MAP2K3 and DKK1 were down-regulated. However, NNMT expression in BEAS-2B cells induced by nickel could not be antagonized. Conclusion according to the results of this study, it is concluded that the inhibition of NNMT expression induced by nickel is involved in the increase of H3K9me2 level induced by nickel. NNMT can regulate the increase of H3K9me2 induced by nickel by changing the ratio of intracellular SAM / SAH. The decrease of NAD / nadh ratio induced by nickel chloride did not play a role in the inhibition of NNMT expression by nickel, but the change of NAD / nadh ratio was involved in the increase of H3K9me2 level induced by nickel. This study provides a new hypothesis to explain the change of histone methylation induced by nickel, which is helpful to clarify the epigenetic mechanism of nickel induced lung cancer.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

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