The Roles Of NNMT In Nickel-induced Histone H3 Methylation In BEAS-2B Cells

BackgroundNickel compounds are widely used in mordern industry,as a result,they pose a health threat to occupational human.Epidemiological studies reported increased incidence of lung cancer among nickel mineral workers.Nickel compounds are classified as lung carcinogens in humans by the International Agency for Research on Cancer（IARC）.Histone methylation has been reported to exhibit significant role in nickel-induced carcinogenesis.Nicotinamide N-methyltransferase（NNMT）expression has been shown to correlate with the migration,invasion,proliferation and other characters of cancer cells.NNMT decreases histone methylation in several cancer cells by altering the cellular ratio of S-adenosylmethionine（SAM）to S-adenosylhomocysteine（SAH）.However,the role of NNMT in nickel-induced histone methylation remains unclear.Therefore,the purpose of our study is to investigate whether NNMT participates in nickel-induced histone methylation and to explore the mechanism in which nickel affect the expression of NNMT.This study illustrates a new molecular mechanism of nickel-induced histone methylation.Methods（1）BEAS-2B cells were exposed to different concentrations of nickel chloride（NiCl₂）for 72 h or 200 μM NiCl₂ for 0h,12 h,24h,48 h,72h.Histone H3 on lysine 9（H3K9）mono-,di-,and trimethylation and NNMT protein levels were measured by Western blot analysis.Expressions of NNMT mRNA and the H3k9me2-associated genes,mitogen-activated protein kinase 3（MAP2K3）and dickkopf1（DKK1）,were determined by RT-PCR analysis.（2）NNMT over-expression plasmid was transfected into cells exposed to nickel.H3k9me2 and H3k9me2-associated genes MAP2K3 and DKK1 were determined.The cellular SAM/SAH ratio was evaluated by HPLC.（3）The NADH oxidant phenazine methosulfate（PMS）was used to treat cells exposed to nickel.The cellular NAD/NADH ratio was determined.H3k9me2 and H3k9me2-associated genes MAP2K3 and DKK1 were assayed by Western blot and RT-PCR analysis.Results（1）Nickel increases H3K9 dimethylation in H3K9 methylation and down-regulates MAP2K3 and DKK1.（2）Nickel suppresses NNMT expression both in protein and mRNA levels.NNMT down-regulates H3K9me2 by altering the SAM/SAH ratio.（3）PMS reverses the nickel-induced increase in H3K9me2 and decrease in MAP2K3 and DKK1.PMS could not inhibited nickel-induced NNMT repression.ConclusionWe concluded that the repression of NNMT may underlie nickel-induced H3K9 dimethylation by altering the cellular SAM/SAH ratio.Alterations in NAD+/NADH were not involved in nickel-induced NNMT repression at both the mRNA and protein levels but participated in nickel-induced H3K9 dimethylation.Our study provides new insight into nickel-induced histone H3 methylation and contributes to further epigenetic mechanisms of nickel carcinogenesis.