| Background:Lead is a nonessential element of the human body,its excessive exposure to human health hazards.Lead enters the body via the respiratory tract and digestive tract,and can be transported to various tissues and organs with the blood.Therefore,lead is toxic to the nervous system,circulatory system,urinary system,skeletal system,reproductive system and so on.About 90-95% of lead in human body is stored in bone tissue in the form of lead or lead protein complex,with the process of bone remodeling,bone lead will continue to release from the bone and cause sustained damage or even apoptosis to osteoblast.The purpose of this study was to explore the toxicity of lead and elucidate the molecular mechanisms of the disease,and toprovide a reliable basis for the comprehensive analysis of the hazards of lead exposure and the health of people exposed to lead.Methods:The effects of lead acetate on the biological behavior of osteoblasts were detected by CCK-8 assay,and flow cytometry.Realtime-PCR and Western blot were used to detect the expression of target gene mRNA and protein level.Gene expression microarray was used to detect the differentially expressed genes in osteoblasts after lead acetate treatment.Study on the effect of ATF4 on osteoblasts were performed by plasmid transfection and interference fragment.Mi RNA chip and LncRNA chip technology were used to detect lead exposed cell lines / lead poisoning groups with the common miRNA and LncRNA.Prediction of miRNA's target genes were screened by target gene prediction software.The regulational relationship between mi R-24-3p and FASL were carried out by luciferase reporter gene assay.RNA pull-down test was used to detect the ceRNA effect between KCNQ1OT1 and miR-24-3pStudy on the molecular mechanism of ATF4 regulation of target gene KCNQ1OT1 were performed by luciferase reporter gene assay,EMSA,Ch IP and gene interference.Results: 1.The treatment of lead in osteoblasts,could result in decreased proliferation of osteoblasts,genomic instability and apoptosis,the presence of which existed dose and time dependence.The expression of FASL gene in both protein level and m RNA level were significantly increased in the osteoblast after treated with lead acetate.After the interference fragment of FASL,could significantly restored the apoptosis of osteoblasts.After screening by microarray gene expression,we found there were a total of 1793 gene expression levels changed more than 2 times after lead exposure,of which 682 genes was up-regulated and 1111 genes decreased,of which 207 genes and transcription regulation function,185 genes and regulating the metabolism of RNA,101 programmed cell death and gene regulation,94 genes and RNA polymerase II promoter regulation,91 genes related with cell proliferation regulation,74 genes related with phosphorylation regulation,stress responses in 66 genes and cells,58 genes and mRNA 54 genes related to metabolism,and intracellular transport(transport of substances in various organelles between 37 genes),and cell cycle related genes,35 with vascular development.The ATF4 and CHOP appeared int he list of more than 6 times the genes.2.After treatment of lead acetate in U32 PB cells,PERK had no obvious change,the expression of p-PERK was significantly increased,p-eIF2 expression increased,e IF2 expression increased,and ATF4 expression was significantly increased.However,p-IRE1,IRE1 and ATF6 showed no significant change.ATF4 target gene CHOP and endoplasmic reticulum stress indicator molecules Bip and Grp94 expression were significantly increased.After exposure to lead,the interference fragment of ATF4 was found to decrease the expression of FASL and the apoptosis rate of osteoblasts.However,CHOP interference fragment did not influence the expression of FASL.3 the results of miRNA microarray and LncRNA microarray of both lead exposure cell lines and the bone marrow cells of lncRNA displayed that miR-24-3p and miR-29c-3p were down regulated and KCNQ1OT1 was up regulation.Software prediction analysis and 3 'UTR luciferase experiments confirmed that FASL is a downstream target gene regulated by miR-24-3p.Also and the expression of FASL gene was down regulated after the addition of miR-24-3p.In addition,KCNQ1OT1 and miR-24-3p in population verification was negatively related.By detected of KCNQ1OT1,there are two potential regions named chr11:2703855-2703875 and chr11:2717519-2717539 in KCNQ1OT1,which could combine with miR-24-3p,and the further luciferase test indentified the chr11:2717519-2717539 as the main binding region.4.According to the bioinformatics software,we found that there were 5 ATF4 binding sites in the upstream promoter region of KCNQ1OT1.In the truncation experiment,we constructed the KCNQ1OT1 TRUN 1-4 plasmids by truncating the 5 ATF4 sites,respectively.We found that luciferase activity of KCNQ1OT1 truncated TRUN3 decreased most significantly,which indicates that the AAATGACACCATT sequence in the KCNQ1OT1 promoter sequence were the main factor affect the expression of KCNQ1OT1.Through EMSA and Ch IP experiments,we verified the binding ability of ATF4 and KCNQ1OT1 in vivo and in vitro.After overexpression of ATF4,the expression of KCNQ1OT1,miR-24-3p and FASL was detected.The results suggested that the apoptotic pathway of ATF4-KCNQ1OT1-miR-24-3p-FASL exist in osteoblasts.Conclusion: 1 Lead exposure can induce the high expression of FASL in osteoblasts,and finally lead to the apoptosis of osteoblasts.2 In the lead-exposuring cells,the ATF4 signal transduction pathway in the endoplasmic reticulum stress signaling pathway was activated,but the expression of FASL was not affected by the ATF6 and XPB1 branches.3 Expression of CHOP,the downstream of ATF4,increased after lead exposure,but it did not regulate the expression of FASL.4.miR-24-3p and KCNQ1OT1 were negatively correlated as ceRNA after lead exposure,and miR-24-3p could regulate the expression of FASL.5.ATF4 can affect the expression of KCNQ1OT1 by binding to the upstream sequence of KCNQ1OT1.6.The effect of lead on osteoblast apoptosis is through the ATF4-KCNQ1OT1-miR-24-3p-FASL signaling pathway. |
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