| Objectives:1.Analyzed the morphology and concentration of three arsenic species in the occupational arsenic exposed workers.The expression of MEG3 mRNA in peripheral blood was examed by qRT-PCR.Explore the associations between concentrations of three arsenic species and MEG3 gene expression.2.Detect the expression level of MEG3 under the different arsenic concentrations in A549 cells.Detection of MEG3 gene expression level induced by three arsenic species in A549 cells.Explore the effects of the expression of MEG3 mRNA induced by S-adenosy-L-methionine(SAM)and reducing agent glutathione(GSH)combination with i As.3.Knockdown the expression of MEG3 in A549 cells and observe the effects of cell apoptosis.Detect the cell viability of the MEG3 knockdown groups induced by sodium arsenite,and exam the downstream genes of MEG3 regulated apoptosis.Furthermore,we explored the the mechanism of how MEG3 regulates arsenic-induced apoptosis,and provide relevant references for the prevention and treatment of diseases related to arsenic exposure.Method:1.Occupational arsenic exposure group and control group were extracted from the arsenic smelting plant and local residents in Yunnan Province,and the cluster sampling method was utilized.Collect the biological materials from two groups,such as urine samples and peripheral blood samples.2.The concentration of three urinary arsenic species(iAs,MMA and DMA)were detected in Yunnan CDC.and calculate the methylation indexes(PMI and SMI)and the percentages of urinary arsenic species(TiAs%,iAs%,MMA%and DMA%).3.After RN A extraction and reverse transcription.The genes expression levels of in arsenic exposed workers and A549 cells were detected by qRT-PCR.Explored the arsenic-induced apoptosis through the MEG3 downstream genes,such as API5,CASP7,CCND3 and APAF1.Analyzed the relationship between arsenic exposure and MEG3 gene expression,and explored the effect of cell apoptosis induced by MEG3,4.MEG3 was successfully knocked down by transfection with MEG3 siRNA.Apoptosis and necrosis level detected through double staining by Hoechst 33342 and PI.6.The data were compared between the two groups by t-test.The data of multiple groups were compared by analysis of variance and LSD.Pearson correlation coefficient was utilized to explore associations between concentrations of three arsenic species and gene expression.Result:1.There were no significant differences in age,sex,smoking and drinking between two groups(P>0.05).The concentrations of three urinary arsenic species,TiAs and MMA%were significantly higher in the exposure group(P<0.05).The PMI and SMI in the exposure group were lower than those in the control group(P<0.05).2.The expression level of MEG3 was positively correlated with and three urinary arsenic species(iAs,R2=0.12,P<0.1、MMA,R2=0.14,P<0.01、DMA,R2=0.12,P<0.01),MMA%(r=0.291)and TiAs(r=0.353)(P<0.05).The median values(percentile P25,percentile P75)of the four groups,(high,low.PMI groups and high,low SMI groups)were 0.92(0.89,0.95),0.81(0.74,0.84),0.88(0.81,0.91),and 0.72(0.69,0.76).The expression level of MEG3 was lower in high PMI and high SMI group(P<0.05).3.The expression of MEG3 induced by MMA and DMA was lower than that induced by iAs(P<0.05).SAM in combination with iAs decreased the expression of MEG3(P<0.05).However,GSH in combination with iAs did not change the expression of MEG3.4.The cell viability was significantly increased after MEG3-siRNA transfection in A549 cells(P<0.05).A549 cells were incubated with iAs the apoptosis increased after MEG3 knockdown.Knockdown of MEG3 inhibited the mRNA expression levels of anti-apoptotic genes BCL2A1 and API5.Moreover,increased expression of Caspase-7,cyclin D3 and APAF-1 mRNA(P<0.05).In addition,the expression of the PMAIP1,FAS and BCL2 have no statistical significance(P>0.05).Conclusion:1.The concentrations of three arsenic species in workers increased significantly.The level of TiAs was significantly higher than the latest urinary total arsenic limit 35μg/ml which established by the American government industrial hygienists conference.The results indicated that the potential occupational hazards of arsenic exposure workers.2.The expression of lncRNA MEG3 was regulated by arsenic exposure.There was a positive correlation between urinary arsenic species and MEG3 mRNA expression in occupational arsenic exposed group.3.Our experiments confirmed that knockdown the expression of MEG3 increased the cell viability in A549 cells and arsenic-induced apoptosis.This results indicated that the ability of MEG3 to inhibit cancer decreased.MEG3 regulated apoptosis via down-regulate API5 while up-regulate CASP7,CCND3 and APAF1.It is further proved that arsenic-induced apoptosis increased by regulating these genes after knockdown of MEG3.In addition,compared with previous experiments,our studies reported that API5 is regulated by MEG3 for the first time.Moreover,our work provides experimental evidence and possible mechanisms for further studies on the effects of arsenic on human health. |
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