Effects of inorganic arsenic-mediated up-regulation of As3MT on proliferation,apoptosis and AP-1 expression of 16HBE cells
Objective To explore the effect of inorganic arsenic(iAs)mediated arsenic(+3 oxidation state)methyltransferase[arsenic(+3 oxidation state)methyltransferase,As3MT]on the proliferation,apoptosis of 16HBE cells,activation of protein-1(AP-1)expression,so as to gain insight into the mechanism of action of inorganic arsenic in cell biology.Methods 16HBE cells were cultured in vitro and randomly divided into 2 groups:sodium arsenite(NaAsO2)with different concentrations(0,2,4,6 μmol/L)and the same concentration(0,6,6,6 μmol/L)monomethylarsenic acid(MMA),dimethylarsenic acid(DMA)and NaAsO2 were used as a group to infect 16HBE cells for 48 hours.The expression of As3MT protein in cells was detected by Western Blot.Silencing As3MT by small interfering RNA(siRNAs)transfection can produce a series of biological effects on the infected cells.CCK-8 kit was used to detect cell viability,JC-1 kit was used to detect early cell apoptosis and HO/PI double staining kit was used to detect late cell apoptosis and necrosis.The transcriptional activity of transcription factor AP-1 was detected by dual luciferase reporter gene.The protein expression levels of c-Jun(p-c-Jun)and c-Fos(p-c-Fos)were detected by Western Blot.Results It was found that the treatment of inorganic arsenic could significantly up-regulate the expression level of As3MT in 16HBE cells,and affect the proliferation and apoptosis of cells.Specifically,compared with the control group,the expression of As3MT in 16HBE cells after exposure increased with the increase of NaAsO2 concentration,showing a dose-effect relationship.At the same concentration,the ability to induce the expression of As3MT was NaAsO2＞DMA＞MMA.Cell transfection technology suggested that As3MT was successfully silenced.After silencing As3MT,the CCK-8 method showed that the cell viability decreased(NCmean=1.00,S1mean=0.94,S2mean=0.71),and the cell viability of the siAs3MTb1 group was significantly lower than that of the siCtrl group(P＜0.05),the cell viability of the siAs3MTb2 group was significantly lower than that of the siCtrl group(P＜0.05).JC-1 detection indicated that apoptosis increased(NCmean=1.80,S1mean=1.40,S2mean=1.18),and the 550 nm/485 nm ratio of siAs3MTb1 group was significantly lower than that of siCtrl group(P＜0.05),the 550 nm/485 nm ratio of siAs3MTb2 group was significantly lower than that of siCtrl group(P＜0.05).HO/PI double staining indicated increased apoptosis and necrosis,and the staining of cells in siAs3MT group was more obvious than that in siCtrl group.The results of dual luciferase reporter gene analysis showed that the activity of AP-1 increased(NCmean=48.83,S1mean=132.67,S2mean=163.83),and the transcriptional activity of AP-1 in the siAs3MTb1 group was significantly higher than that in the siCtrl group(P＜0.05),the transcriptional activity of AP-1 was significantly higher than that of siCtrl group(P＜0.05).Western Blot results detected that after silencing As3MT,the expression of c-Jun increased,the expression of p-c-Jun increased,the expression of c-Fos increased,and the expression of p-c-Fos decreased.Conclusions This study found that inorganic arsenic can significantly up-regulate the expression of As3MT,and affect the proliferation,apoptosis and AP-1 expression of 16HBE cells.Further experiments showed that the up regulation of As3MT mediated by inorganic arsenic was related to the AP-1 pathway.These results suggest that inorganic arsenic may affect cell proliferation,apoptosis and transcription factor expression by regulating the expression of As3MT.
ArsenicArsenic(+3 oxidation state)methyltransferaseActivation of protein-116HBEApoptosisCell proliferation
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