| Part one: Screening and verification of differentially expressed genes in serum of occupational mercury workersBackground and objective: Mercury is one of the typical toxic heavy metals and is commonly distributed in nature.Metallic mercury is widely present in scientific instruments,electrical equipment,dental amalgam,felt manufacturing,disinfectants,caustic soda production,and disk batteries.In the process of producing related products,mercury workers will be more likely to inhale mercury vapor,because mercury vapor has good fat solubility will cause some damage to the human body.Long noncoding RNA(lnc RNA)has the characteristics of low expression,although the conserved type of the sequence is low,but it has a high tissue specificity,and plays an important biological role in the development of a variety of diseases.In this study,the purpose of this experiment was to screen for lnc RNAs with differential expression of high concentrations of mercury exposure and low concentrations of mercury exposure by lnc RNA chip experiments and to verify their value as biomarkers.Methods: The research objects of this experiment are 291 employees participating in occupational health examinations in a mercury thermometer manufacturing plant in Jiangsu Province,and the physical examination data of all employees is relatively complete and they have signed an informed consent form.According to the epidemiological method,10 people with high and low concentrations of mercury exposure were selected as experimental subjects in this study.The RNA of 10 people in each group was extracted and mixed into an RNA pool for lnc RNA expression profiling chip experiments.Several lnc RNAs were selected according to the difference multiple,and the population expansion verification of the candidate lnc RNAs was carried out by quantitative reverse transcription polymerase chain reaction(q RT-PCR),and the data of the two groups were analyzed by independent sample t-test to finally determine the differential expression of lnc RNAs related to mercury exposure.Results: The data showed that there were 1022 differentially expressed lnc RNAs in the two groups,compared with the low concentration mercury exposure group,compared with the low concentration mercury exposure group,there were 743 upregulated lncRNAs and 279 lnc RNAs expressed down-regulation in the high concentration mercury exposure group.Five lncRNAs were selected based on the difference multiple(FC≥2 or ≤0.5),including LOC146880,ZFAS1,LINC00265,GAS5,and FER1L4.Subsequent enlarged verification showed that the GAS5 expression in the high-exposure group was significantly increased,about 13.95 times that of the low-concentration mercury exposure group,and the expression difference between the two groups was statistically significant(P=0.001).At the same time,the correlation analysis showed that the relative expression of GAS5 was positively correlated with the urine mercury value(r=0.195,P=0.041).The ANALYSIS OF ROC curve showed that the AUC was 0.716(0.603-0.830),which was statistically significant compared with AUC=0.5(P<0.001).Conclusion: Mercury exposure in occupational populations is associated with LncRNA GAS5 expression and there is a dose-response relationship.GAS5 can be used as a potential biomarker for mercury exposure,and its mechanism of action still needs to be further explored.Part two: Differential expression and function of GAS5 in Mercury-infected HK-2 cellsBackground and Objective: Growth Arrest-specific Transcripts 5(GAS5)belong to one of the GAS family and play an important role in multiple biological processes,both positive and negative,during cell growth and development.In addition to participating in a variety of signaling pathways to regulate disease development,GAS5 can also be used as a sensitizer for apoptosis,promoting apoptosis under certain conditions.Based on the first part of the study,this study further explores the functional performance of lnc RNA GAS5 in mercurycontaminated HK-2 cells and whether it is involved in the mechanism of apoptosis.Methods: The survival rate of cells after mercury chloride infection was calculated by MTT cell activity assay experiment,and the mercury concentration at 70% survival rate was used as the highest infection concentration,and the final infection concentration was determined(0 μM,10 μM,30 μM).A mercury-contaminated HK-2 cell model was constructed,and the expression of lnc RNA GAS5 was determined by q RT-PCR technology,and the expression of COX2 protein,caspase 3 protein and caspase 8 protein was determined by Western Blot technology.A cell model of GAS5 overexpression was constructed by constructing an overexpressed plasmid model of GAS5,and then overexpressing GAS5 in HK-2 cells(human renal tubular epithelial cells)using transfection techniques,and the key gene and protein expression levels of downstream signaling pathways were detected using q RT-PCR technology and Western Blot technology.Results: In the HK-2 cell model infected with mercury chloride,the expression of GAS5 in the 30 μM and 10 μM mercury infected cell groups was significantly increased,which was 1.21 times and 1.22 times that of the 0 μM group,respectively,and the difference was statistically significant(P=0.002,P=0.019),while the relative expression of GAS5 in the 30 μM group was about 1.48 times that of the 0 μM group,and the difference was statistically significant(P=0.003).Compared with the 0 μM and 10 μM poisoning groups,the expression of COX2 protein in cells with a poisoning concentration of 30 μM was significantly increased,which was about 1.45 times and 1.42 times that of the 0 μM infected group and the 10 μM infected group,and the differences were statistically different(P<0.001).The expression level of caspase 3 protein in the high concentration and low concentration infected cell groups was increased,and the 30 μM infected cell group was 1.72 and 2.08 times higher than that of the 10 μM infected group and the 0 μM infected group,respectively,and the difference was statistically significant(P <0.001),the 10 μM poisoned cell group was 1.21 times that of the 0 μM poisoned cell group,and the difference was statistically significant(P=0.003).At the same time,the expression of caspase 8 protein was significantly increased in the 30 μM infected group,and the 30 μM infected cell group was 2.12 and 2.32 times that of the 10 μM infected group and the 0 μM infected group,respectively,and the difference was statistically significant(P<0.001).In the HK-2 cell model overexpressing GAS5,the relative expression level of mi R-26b-5p decreased by about 80% of the control group,and the difference was statistically significant(P=0.008).At the same time,the expression level of COX2 protein was increased,which was about 1.77 times that of the control group,and the difference was statistically different(P<0.001).The expression of caspase 3 and caspase 8 proteins was significantly increased,about 1.56 and 1.62 times that of the control group,respectively,and the differences were statistically significant(P<0.001).Conclusion: In in vitro renal cell models,mercury exposure upregulated lncRNA GAS5 expression and COX2 expression upregulated,resulting in increased apoptosis.After overexpression of GAS5,mi R-26b-5p expression was downregulated,and phenotypic changes similar to mercury exposure occurred,namely COX2 and upregulation of apoptosis protein expression.Thus,GAS5 may play a role in apoptosis caused by mercury exposure by activating the mi R-26b-5p-COX2-caspase 3/8 pathway,which can partially explain the mechanism of kidney damage caused by mercury exposure. |
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