| Part One: Screening and Verification of DifferentiallyExpressed Genes in Serum of Workers Exposed to Mercury.Backgrounds and Objectives: Mercury is chemically stable and can be persistent in the environment.It can form a biogeochemical cycle through the atmosphere,water,soil,and plant microorganisms when entering environment.It is widely used in metal smelting,instrument manufacturing,polyvinyl chloride catalysts and other industries.In the process of production and usage,workers tend to inhale air containing mercury vapor.On account of its lipid solubility,more than 70% of the inhaled mercury vapor can be absorbed by the human body.Studies have confirmed that mercury has varying degrees of toxicity to different organs of the human body.Mercury exposure can induce symptoms such as confusion,reduced vision,deafness,ataxia,and dysarthria.High levels of inorganic or organic mercury exposure will lead to cell apoptosis,embryonic or fetal death,and serious consequences such as cerebral palsy,deafness,and visual impairment.The purpose of this study is to screen and verify the differentially expressed genes in the serum of occupational mercury workers.Methods: The study population consists of 291 employees of a thermometer manufacturer in Jiangsu Province in 2016.We tested the mercury concentration in the working environment,and conducted questionnaire surveys and occupational health examinations for all employees.According to the basic information collected,40 subjects in the high-concentration mercury exposure group and 40 subjects in the lowconcentration mercury exposure group(control group)were matched,and total RNA was extracted from each blood sample.Mixed RNA pools were made by 10 samples of each groups for gene expression profiling chip experiment.The experimental results were selected according to the screening criteria to find differentially expressed genes,and imported into the Metascape website for GO and KEGG enrichment analysis,and the differentially expressed genes with the highest enrichment scores were selected as candidate genes for this experiment.A q RT-PCR technology was performed to carry out an expanded verification experiment on the remaining 60 people,the relative expression of the candidate gene is-△Ct =(Ct(candidate gene)-Ct(GAPDH))×(-1)and expression 2^(-△Ct),and we performed paired t-test on the expression of the two groups of candidate genes by SPSS 22.0,a scatter plot of the relative expression of each gene was made by Graphpad Prism8.0 software,and make a correlation analysis between the relative expression of candidate genes and urine mercury value to finally confirme the differentially expressed genes of mercury exposure.Results: The gene expression microarray results showed that compared with the control group,269 differentially expressed genes were detected in the highconcentration mercury exposure group.Among them,203 genes were up-regulated in the high-concentration mercury exposure group,and 66 genes were down-regulated in the high-concentration mercury exposure group.After the gene expression chip experiment results were analyzed by GO and KEGG enrichment,the PTEN pathway,PTEN,RNF2 genes in PI3K/AKT pathway;WNT pathway,SOX6 gene in PTEN pathway;PTEN pathway,KDM1 A gene in p53-mediated intrinsic apoptosis signaling pathway;SOX8 gene which is related to cell development and differentiation regulation,as candidate genes for subsequent population expansion verification.The population expansion verification experiment found that the PTEN gene expression in the highconcentration mercury exposure group was down-regulated compared with the control group(P=0.004),and the PTEN expression in the high-concentration mercury exposure group was 21.86% of that in the control group,the difference was statistically significant.Correlation analysis showed that the relative expression of PTEN(r=-0.36,P=0.005)and RNF2(r=-0.37,P=0.005)genes were both negatively correlated with urine mercury values.Conclusion: Mercury exposure lead to a down-regulate the expression of PTEN,and there is a negative correlation between the relative expression of PTEN gene and urine mercury.Part Two: The Differential Expression and FunctionalMechanism of PTEN in 293 T Cells Exposed to Mercury.Backgrounds and Objectives: PTEN is a protein phosphatase and lipid phosphatase active gene,and plays a vital role in the regulation of cell growth and survival signal pathways.PTEN can negatively regulate the PI3K/AKT signal pathway,and can also regulate focal adhesion,cell migration,proliferation,apoptosis,angiogenesis,DNA damage response,and chromosome stability through its potential protein phosphatase activity and/or protein-protein interaction.Based on the first part of the study,we explored the differential expression and functional mechanism of PTEN in mercuryexposed 293 T cells in this part of study.Methods: The CCK8 experiment calculates the survival rate of cells after mercury exposure,and takes the mercury concentration of 80% survival rate as the highest exposure concentration,and determines the exposure concentration as(25μM,10μM,0μM).A mercury-infected 293 T cell model was constructed.In this model,q RT-PCR technology and Western Blot technology were further used to detect the expression of PTEN gene and protein,AKT protein,and PI3 K protein.Using si RNA transfection technology to silence the expression of PTEN in 293 T cells,construct a cell model with low PTEN expression,and perform q RT-PCR technology and Western Blot technology to explore the effect of low expression PTEN on downstream signal pathways.Enzymelinked immunosorbent assay(ELISA)was made to detect the level of interleukin 6(IL-6)in the culture medium of infected cells and cells with low PTEN expression.Results: In the mercury-exposed 293 T cell model,compared with the control group,the expression of PTEN in the 25μM and 10μM mercury-exposed cell groups decreased,which was 87.55% and 84.90% of the expression in the control group;the expression of PTEN protein decreased,which was 40.31% and 52.09% of the expression in the control group,the difference was statistically significant(P<0.05).The expression level of PI3 K protein in the 25μM and 10μM mercury-exposed cell groups increased,1.73 times and 1.49 times higher than the control group;the expression level of AKT protein increased,2.02 times and 1.99 times higher than the control group,the difference was statistically significant(P <0.001).The level of IL-6 in the culture medium of the 25μM and 10μM mercury-exposed cell groups increased,which was3.69 times and 2.87 times higher than the control group,and the difference was statistically significant.In the PTEN low-expressing cell model,the expression of PI3 K and AKT protein was 1.36 times and 1.55 times higher than the control group,and the difference was statistically significant(P<0.001).The expression of IL-6 in PTEN lowexpressing cell culture medium increased,which was 3.66 times higher than the control group,and the difference was statistically significant(P<0.001).Conclusion: Mercury exposure can down-regulate the expression of PTEN,activate the PI3K/AKT regulatory pathway in 293 T cells,and increase the expression of the inflammatory factor IL-6 in the kidney. |
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