RÉSUMÉ
Objective:To establish a protein-protein interaction (PPI) network of 5 microRNA (miRNA) target genes differentially expressed in the plasma of coal-burning fluoride exposed population, and to screen genes and gene modules with important roles.Methods:Five miRNA (hsa-miRNA-3131, hsa-miRNA-4516, hsa-miRNA-6501-5p, hsa-miRNA-10b-5p, hsa-miRNA-4683) target genes differentially expressed in the plasma of coal-burning fluoride exposed population screened by our previous study were mapped to the STRING online database (https://string-db.org), and the PPI network was screened. The Cytoscape v3.6.0 software was used to visualize the PPI network, the topological attribute values degree and betweenness centrality were obtained by the NetworkAnalyzer plug-in, and the central node was filtered in the network (the node with the highest degree and the highest betweenness centrality). At the same time, the maximal clique centrality (MCC) analysis method in the CytoHubba plug-in was used to determine the important nodes in the PPI network. The cluster analysis was conducted by the MCODE plug-in, and the gene modules were screened in the PPI network. The protein names contained in the gene modules were submitted online to the KOBAS v3.0 database (http://kobas.cbi.pku.edu.cn/), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the gene modules selected by the MCODE plug-in.Results:The PPI network of target genes was consisted of 1 035 nodes and 4 346 edges. The degree (101) and betweenness centrality (0.010 723 89) of ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52) were the highest, which was the central node of the PPI network. According to MCC analysis, UBA52 was an important node in the PPI network. The top 5 gene modules were selected from the PPI network, and the highly enriched gene pathways in the KEGG pathway enrichment analysis of the 5 gene modules included ubiquitin mediated proteolysis, spliceosome, endocytosis, neuroactive ligand-receptor interaction and vesicular transport.Conclusion:The PPI network of 5 miRNA target genes differentially expressed in the plasma of people exposed to coal-burning pollution of fluoride is successfully established, and the UBA52 gene and the 5 main pathways of gene modules are selected.
RÉSUMÉ
Objective To establish an model of fluorosis with human primary osteoblasts in vitro and to detect the influences of different doses of sodium fluoride (NaF) on histone acetylation of CyclinD1,cyclindependent kinases 4 (CDK4) gene in human osteoblasts,then to explore the molecular mechanism of skeletal fluorosis from epigenetic perspective of the cell cycle regulation related genes.Methods Human primary osteoblasts from bone tissues of trauma surgery healthy people (car accident) were isolated by enzyme digestion and identified.The osteoblasts were treated with 0,125,250,500 and 1 000 μmol/L NaF for 72 h.The level of histone acetylation (H3K9,H3K14,H4K12,H4K16) in the transcription regulatory region (ChIP1 region) and in the coding region (ChIP2 region) of CyclinD1 and CDK4 genes were detected by quantitative chmmatin immuno-precipitation (Q-ChIP).Results ①After human osteoblasts were treated with 0,125,250,500 and 1 000 μmol/L NaF,respectively,the levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of CyclinD1 gene were 1.152 ± 0.104,1.174 ± 0.187,1.090 ± 0.176,1.170 ± 0.197 and 1.147 ± 0.097,respectively,the differences were not statistically significant (F =0.524,P ＞ 0.05);the average levels of histone acetylation of H3K14 were 1.495 ± 0.117,1.465 ± 0.069,1.470 ± 0.187,1.760 ± 1.089 and 1.341 ± 0.443,the differences were not statistically significant (F =0.841,P ＞ 0.05);the levels of histone acetylation of H4K12 were 1.239 ± 0.286,0.702 ± 0.063,0.765 ± 0.370,1.011 ± 0.321 and 1.319 ± 0.026,the differences were not statistically significant (F =2.329,P ＞ 0.05);the levels of histone acetylation of H4K16 were 1.452 ± 0.217,1.621 ± 0.165,1.462 ±0.090,1.510 ± 0.146 and 1.564 ± 0.154,the differences were not statistically significant (F =0.123,P ＞ 0.05).②The levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of CDK4 were 1.472 ± 0.163,1.580 ± 0.161,1.585 ± 0.132,1.451 ± 0.136 and 1.560 ± 0.039,the differences were not statistically significant (F =0.461,P ＞ 0.05);the levels of histone acetylation of H3K14 were 0.919 ± 0.149,0.900 ± 0.059,0.911 ±0.162,0.663 ± 0.049 and 0.841 ± 0.122,the differences were not statistically significant (F =0.974,P ＞ 0.05);the levels of histone acetylation of H4K12 were 0.456 ± 0.142,0.911 ± 0.126,0.969 ± 0.185,1.110 ± 0.146 and 0.931 ± 0.141,the differences were not statistically significant (F=5.459,P ＞ 0.05);the levels of histone acetylation of H4K16 were 1.315 ± 0.083,1.374 ± 0.153,1.423 ± 0.055,1.300 ± 0.132 and 1.385 ± 0.696,the differences were not statistically significant (F =1.663,P ＞ 0.05).③The differences of histone acetylation levels of H3K9,H3K14,H4K12 and H4K16 in ChIP2 coding region of CyclinD1 and CDK4 genes were not statistically significant between NaF treatment groups (F =0.392,0.823,0.999,0.397,0.705,0.049,1.065,0.196,P ＞ 0.05).Conclusion The histone acetylation of CyclinD1 and CDK4 may not be involved in the transcriptional regulation in human primary osteoblasts treated with fluoride.
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Objective To explore the association of H3K14 acetylation (ac) with arsenicosis induced by coal-burning and arsenic-induced genetic damage,which might help us to find an biomarker to monitor the arsenicosis and arsenic-induced toxicity.Methods Totally 151 arsenicosis subjects were recruited from Jiaole Village of Xingren County,Guizhou.According to "National Principle for Diagnosis of Arsenicosis" (WS/T 211-2001),the arsenicosis group was divided into 3 subgroups:mild poisoning (n =62),intermediate poisoning (n =50) and severe poisoning (n =39).The control group was comprised of 78 healthy villagers from Jiaole Village who were exhibited no signs of arseniasis.The hair,the urine and the peripheral blood samples were collected from the subjects.The contents of arsenic in the hair samples were analyzed with inductively coupled plasma mass spectrometry.Histones were extracted from human peripheral blood lymphocytes (PBLCs) using the method of acid extraction.The levels of H3K14ac was measured with a sandwich enzyme-linked immunosorbent assay (ELISA).The rate of micronucleus (MN) and chromosome aberration (CA) of peripheral blood lymphocytes were examined by genetic methods.The levels of urinary 8-hydroxy-2 deoxyguanosine (8-OHdG) in all the subjects were measured with the high performance liquid lhromatography-mass spectrometry (HPLC-MS).Results ①The association of arsenic-exposure with arsenicosis induced by coal-burning and H3K14ac:The levels of hair arsenic in arsenicosis group [0.27(0.15-0.39) μg/g] was significant higher than that in control group [0.15 (0.08-0.20) μg/g,F=10.736,P ＜ 0.01].The degree of arsenicosis was positive correlation with the hair-arsenic level (r =0.363,P ＜ 0.05).The levels of H3K14ac was also positive correlation with the hair-arsenic level (r =0.385,P ＜ 0.05).②The association of H3K14ac and arsenicosis induced by coal-burning:The levels of H3K14ac in arsenicosis group (4.07 ± 4.03) was 2.5-fold higher than that in control group (1.62-± 1.19,F =19.753,P ＜ 0.01).H3K14ac was a risk factor of arsenicosis,the risk of arsenicosis increased correspondingly with the levels of H3K14ac [OR (95％CI) =1.779 (1.323-2.392),P ＜ 0.01].③The correlation of H3K14ac and the degree of arsenicosis:Based on the degree of arsenicosis,we found a significant difference in the levels of H3K14ac among the four groups (F =7524,P ＜ 0.01).Compared with the non-poisoning group (1.62 ± 1.19),the levels of H3K14ac in mild poisoning,intermediate poisoning and severe poisoning subgroups (3.70 ± 3.20,4.95 ± 5.47,3.49 ± 2.62) were increased (all P ＜ 0.01),but there were no significant differences in the levels of H3K14ac between the mild poisoning,intermediate poisoning and severe poisoning subgroups (P ＞ 0.05).(④)The genetic damage of all subjects:The rate of MN (2.03 ± 1.55) and CA (12.44 ± 5.01) in arsenicosis group were significantly higher than those in control group (MN:1.17 ± 0.97,Wald =14.121;CA:6.29 ± 2.41,Wald =83.164,P ＜ 0.05).Urinary 8-OHdG was increased in arsenicosis group than that in control group [(3.80 ± 3.88),(2.33 ±1.34) μg/g Cr,F =6.116,P ＜ 0.05].⑤The association of H3K14ac with genetic damage:The results revealed that H3K14ac modification was positively correlated with the rate of CA (β =0.84,P ＜ 0.01).The level of H3K14ac was not significantly associated with the rate of MN and urinary 8-OHdG (MN:β =0.10,P ＞ 0.05;8-OHdG:β=0.05,P ＞ 0.05).Conclusions The increase of H3K14ac modification in human peripheral blood lymphocytes is a risk factor of arsenic poisoning.Additionally,the dysregulation of H3K14ac was significant association with arsenic-induced chromosomal aberrations.
RÉSUMÉ
Objective To explore the association of H3K14 acetylation (ac) with arsenicosis induced by coal-burning and arsenic-induced genetic damage,which might help us to find an biomarker to monitor the arsenicosis and arsenic-induced toxicity.Methods Totally 151 arsenicosis subjects were recruited from Jiaole Village of Xingren County,Guizhou.According to "National Principle for Diagnosis of Arsenicosis" (WS/T 211-2001),the arsenicosis group was divided into 3 subgroups:mild poisoning (n =62),intermediate poisoning (n =50) and severe poisoning (n =39).The control group was comprised of 78 healthy villagers from Jiaole Village who were exhibited no signs of arseniasis.The hair,the urine and the peripheral blood samples were collected from the subjects.The contents of arsenic in the hair samples were analyzed with inductively coupled plasma mass spectrometry.Histones were extracted from human peripheral blood lymphocytes (PBLCs) using the method of acid extraction.The levels of H3K14ac was measured with a sandwich enzyme-linked immunosorbent assay (ELISA).The rate of micronucleus (MN) and chromosome aberration (CA) of peripheral blood lymphocytes were examined by genetic methods.The levels of urinary 8-hydroxy-2 deoxyguanosine (8-OHdG) in all the subjects were measured with the high performance liquid lhromatography-mass spectrometry (HPLC-MS).Results ①The association of arsenic-exposure with arsenicosis induced by coal-burning and H3K14ac:The levels of hair arsenic in arsenicosis group [0.27(0.15-0.39) μg/g] was significant higher than that in control group [0.15 (0.08-0.20) μg/g,F=10.736,P ＜ 0.01].The degree of arsenicosis was positive correlation with the hair-arsenic level (r =0.363,P ＜ 0.05).The levels of H3K14ac was also positive correlation with the hair-arsenic level (r =0.385,P ＜ 0.05).②The association of H3K14ac and arsenicosis induced by coal-burning:The levels of H3K14ac in arsenicosis group (4.07 ± 4.03) was 2.5-fold higher than that in control group (1.62-± 1.19,F =19.753,P ＜ 0.01).H3K14ac was a risk factor of arsenicosis,the risk of arsenicosis increased correspondingly with the levels of H3K14ac [OR (95％CI) =1.779 (1.323-2.392),P ＜ 0.01].③The correlation of H3K14ac and the degree of arsenicosis:Based on the degree of arsenicosis,we found a significant difference in the levels of H3K14ac among the four groups (F =7524,P ＜ 0.01).Compared with the non-poisoning group (1.62 ± 1.19),the levels of H3K14ac in mild poisoning,intermediate poisoning and severe poisoning subgroups (3.70 ± 3.20,4.95 ± 5.47,3.49 ± 2.62) were increased (all P ＜ 0.01),but there were no significant differences in the levels of H3K14ac between the mild poisoning,intermediate poisoning and severe poisoning subgroups (P ＞ 0.05).(④)The genetic damage of all subjects:The rate of MN (2.03 ± 1.55) and CA (12.44 ± 5.01) in arsenicosis group were significantly higher than those in control group (MN:1.17 ± 0.97,Wald =14.121;CA:6.29 ± 2.41,Wald =83.164,P ＜ 0.05).Urinary 8-OHdG was increased in arsenicosis group than that in control group [(3.80 ± 3.88),(2.33 ±1.34) μg/g Cr,F =6.116,P ＜ 0.05].⑤The association of H3K14ac with genetic damage:The results revealed that H3K14ac modification was positively correlated with the rate of CA (β =0.84,P ＜ 0.01).The level of H3K14ac was not significantly associated with the rate of MN and urinary 8-OHdG (MN:β =0.10,P ＞ 0.05;8-OHdG:β=0.05,P ＞ 0.05).Conclusions The increase of H3K14ac modification in human peripheral blood lymphocytes is a risk factor of arsenic poisoning.Additionally,the dysregulation of H3K14ac was significant association with arsenic-induced chromosomal aberrations.
RÉSUMÉ
<p><b>OBJECTIVE</b>To investigate the changes of genetic damage in patients with arsenism caused by coal-burning in 9 years. To analyze the relationship between the changes of genetic damage and disease progression and provide a basis for condition monitoring.</p><p><b>METHODS</b>Of 206 arsenism patients from the area with endemic arsenism in Guizhou province were tracking surveyed in February 1998 and divided into 4 groups, including suspicious, mild, moderate and severe poisoning group. Another 67 healthy residents from a neighbour township 12 km away where arsenic was not prevalent were surveyed. Over a 9-year follow-up, 131 arsenism patients and 45 controls with the complete biochemical indexes among them were selected as subjects in December 2006. Arsenic (As) concentration of urine and hair were detected by silver diethyldithiocarbamate spectrophotometry (Ag-DDC). Micronucleis (MN) and chromosome aberrations (CA) were analyzed by conventional methods. DNA single-strand breaks of peripheral blood were measured by single cell gel electrophoresis (SCGE), and the tail lengths of comet were used to measure DNA damage.</p><p><b>RESULTS</b>Among the control, suspicious, mild, moderate and severe arsenic poisoning group, the As contents of urine and hair were respectively (34.16 ± 10.25), (52.35 ± 22.41), (62.26 ± 31.13), (71.43 ± 49.92), (78.45 ± 50.64) µg/L and (1.37 ± 0.56), (3.69 ± 1.78), (4.88 ± 3.49), (5.21 ± 3.10), (6.25 ± 4.04) µg/g in 2006, which were lower than that 9 years before (urine as contents were (36.07 ± 20.70), (73.65 ± 41.33) , (90.92 ± 82.14) , (126.55 ± 107.31) and (139.44 ± 90.90) µg/L, and hair As contents were (1.41 ± 1.18), (4.85 ± 4.20), (5.72 ± 4.07) , (6.43 ± 4.32) and (7.19 ± 4.68) µg/g, respectively, F value was 10.63, 7.72, 14.66, 11.00 respectively, all P values were < 0.05). Except for suspicious poisoning group, the differences of urine As contents in the other groups all showed significance (P < 0.05). The incidences of MN were (0.238 ± 0.130) %, (0.268 ± 0.192) %, (0.283 ± 0.157) % and (0.391 ± 0.233)%; the incidences of CA were (14.36 ± 5.44) %, (18.09 ± 6.49) %, (19.38 ± 5.63)% and (19.83 ± 5.84) %; the tail lengths of comet were (29.88 ± 13.81) , (29.84 ± 12.80) , (34.50 ± 9.88) and (41.58 ± 12.98) µm respectively in 2006 for all poisoning groups; which were higher than that 9 years before(the incidences of MN were (0.163 ± 0.051) %, (0.186 ± 0.117) %, (0.196 ± 0.104) % and (0.273 ± 0.142) %; the incidences of CA were (13.18 ± 5.17)%, (14.48 ± 6.61)%, (15.67 ± 8.49) % and (16.90 ± 8.38) %; the tail lengths of comet were (15.07 ± 12.93) , (19.57 ± 8.80) , (27.03 ± 10.77) and (34.71 ± 14.95) µm) , except for the incidences of MN and CA in suspicious poisoning group and of MN in mild poisoning group , the differences of the three indexes in the other groups were significant (P < 0.05) . The state of illness of arsenic poisoning patients aggravated 9 years later. With the increase of urine and hair As contents and the development of arsenism, the incidences of MN, CA and the tail lengths of comet of all poisoning groups increased. There were positive correlations among them (r values were respectively 0.212, 0.316, 0.232, 0.263, 0.321, 0.654 and 0.760) (P < 0.05).</p><p><b>CONCLUSION</b>The exacerbation of genetic damage was related to constantly high arsenic loads. The accumulation of genetic damage and its irreversibility might be one of the important reasons of the development of arsenism and cancer.</p>