NNMT在镍诱导的BEAS-2B细胞组蛋白H3甲基化中的作用研究
发布时间：2018-07-20 13:23
【摘要】：研究背景镍化合物在工业生产中的广泛应用不可避免地会造成职业人群的健康危害。流行病学研究表明,与在非镍暴露的场所中的工作人员相比,镍矿工人中因患肺癌的死亡率明显增高,镍的化合物已经被国际癌症研究中心(IARC)正式确立为一类致癌物。近来的研究表明,组蛋白甲基化参与了镍诱导肺癌的早期阶段。尼克酰胺-N-甲基转移酶(NNMT)与癌细胞的增殖、迁移等生物学特征密切相关,研究发现NNMT可以减少细胞内腺苷甲硫氨酸/腺苷同型半胱氨酸(SAM/SAH)比值导致组蛋白甲基化水平降低。而目前,尚未见文献报道镍暴露能通过影响NNMT的表达进而诱导组蛋白甲基化。本课题主要研究NNMT是否参与了镍引起的组蛋白甲基化,并初步探讨镍引NAD/NADH比值改变在镍导致的NNMT表达抑制以及H3K9me2水平升高中的作用,为阐明镍引起组蛋白甲基化的机制提供新的线索。研究内容(1)将支气管上皮细胞(BEAS-2B)暴露于不同浓度的氯化镍(Ni Cl2)72h以及在200μM Ni Cl2处理0h、12h、24h、48h和72h。采用CCK-8试剂盒检测氯化镍暴露后BEAS-2B细胞活性的变化。用Western blot检测氯化镍暴露后,组蛋白H3第9位赖氨酸单甲基化(H3K9me1)、双甲基化(H3K9me2)和三甲基化(H3K9me3)以及NNMT蛋白的表达水平,RT-PCR用于检测H3K9me2下游的肺癌抑癌基因MAP2K3和DKK1以及NNMT m RNA的表达水平。(2)采用细胞转染的方法使BEAS-2B细胞过表达NNMT,观察NNMT过表达后,经氯化镍处理后的细胞中H3K9me2及其下游抑癌基因MAP2K3和DKK1的表达变化,用HPLC检测细胞中SAM/SAH比值的变化。(3)用NADH氧化剂柠嗪酸(phenazine methosulfate,PMS)处理经氯化镍暴露后的细胞,检测细胞内NAD/NADH比值的变化,用Western blot和RT-PCR检测NNMT、H3K9me2及其下游抑癌基因MAP2K3和DKK1的表达变化。研究结果(1)氯化镍引起组蛋白H3第9位赖氨酸甲基化中H3K9me2水平增加,并下调其下游抑癌基因MAP2K3和DKK1的表达。(2)氯化镍抑制BEAS-2B细胞中NNMT的表达,NNMT可以通过降低细胞内SAM/SAH比值参与镍引起的H3K9me2升高以及MAP2K3和DKK1表达水平降低。(3)PMS可以抑制镍引起的H3K9me2升高以和抑癌基因MAP2K3和DKK1表达下调,但不能拮抗镍导致的BEAS-2B细胞中NNMT的表达抑制。研究结论根据本研究的结果,得出结论:镍导致的NNMT表达抑制参与了镍诱导的H3K9me2水平增加,NNMT可以通过改变细胞内SAM/SAH的比值调控镍引起的H3K9me2升高。氯化镍导致的NAD/NADH比值下降没有在镍抑制NNMT表达中起作用,但是NAD/NADH比值的改变参与了镍导致H3K9me2水平升高这一过程。本研究为解释镍引起组蛋白甲基化状态改变提供了新的假说,有助于进一步阐明镍致肺癌的表观遗传机制。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2133667
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
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