Role Of ASPM In Toxicity Of Human Embryonic Lung Epithelial Cells Induced By Cadmium Chloride

1.Background and significanceCadmium?Cd?is known occupational hazardous chemicals and environmental contaminant,which is classified by the International Agency for Research on Cancer?IARC?as the first class of human carcinogens.Cadmium and its compounds can cause human systerm damage and related cancers such as prostate cancer and bladder cancer,head and neck cancer,pancreatic cancer,lung cancer,kidney cancer,etc.Prolonged exposure to cadmium can cause tumors,such as the skeletal system,the urinary system and the reproductive system.The liver and kidney are the main target organs.The molecular mechanism of cadmium-induced cancer is complex.It is related to DNA damage,gene expression changes,DNA methylation abnormalities and enhanced lipid peroxidation,apoptosis and other mechanisms.The ASPM?Abnormal spindle microtubule assembly?gene is located on chromosome 1 and is a gene encoding a protein of 3477 amino acids.The ASPM gene also plays an important role in the linkage of non-homologous ends of DNA double-strand break repair.Expression in tissues as well as in almost all human transformed cell lines.Studies have shown that ASPM mRNA expression is up-regulated in many tumors,such as medulloblastoma,glioma,hepatocellular carcinoma and pancreatic cancer,and prostate cancer.Among the molecular mechanisms by which ASPM causes tumors,studies have shown that it is associated with DNA damage.At present,ASPM gene has rarely been reported in the mechanism of lung injury caused by environmental chemicals.This sudy ombining with the role of.of ASPM gene in toxicity of cadmium-induced human bronchial epithelial cells.It is mainly from a cadmium-induced malignant transformed cell model and a subchronic cadmium-induced rat model.It provides a new idea for the possibility of ASPM gene as a new molecular marker for cadmium exposure and for the study of cadmium toxicity mechanism.2.Purposes2.1 Based on the 16HBE cell model of cadmium malignant transformation established in the previous group,the bioinformatics analysis of 16HBE cells showed that the expression profile of ASPM mRNA was differentially expressed.It was observed the expression of ASPM mRNA in tissues?lung,heart,liver and kidney?after cadmium exposure in the subchronic cadmium-induced rat model.Through further research,the data were analyzed to analyze the biological characteristics of the gene such as locus,morphology,size and conservation.2.2 It was investigated the differential expression of ASPM mRNA and protein in16HBE cells by treated with cadmium chloride?different time and concentration?.The expression of ASPM in cytotoxic acute injury induced by cadmium.To further investigate the expression changes of ASPM in acute injury induced by cadmium-induced 16HBE cytotoxicity.2.3 It is studied that the effect of low expression of ASPM on cell morphology,growth cycle,apoptosis and other general biological characteristicsby constructing ASPM gene stable low expression 16HBE cell line.2.4 It was observed that cell inhibition rate,apoptosis,DNA damage using cadmium chloride?different time,different concentration?to infect ASPM gene stable low expression cell line,and explored the role of ASPM in acute toxicity damage of cadmium-induced 16HBE cells.2.5 It was observed the release of cytochrome C using cadmium chloride?different time poins,and different concentrations?to affect ASPM gene stable low expression cell line,and explored the mechanism of ASPM in oxidative damage of cadmium-induced16HBE cells.3.Methods3.1 In the malignant transformation model constructed in the previous stage,qRT-PCR was used to detect the expression changes of ASPM in the process of cadmium-induced malignant transformation,and observe the expression pattern of ASPM.3.2 SPF grade SD rats were used in four dose groups:0.9%NaCl?control group?,0.306mg/kg?low dose group?,0.612 mg/kg?middle dose group?and 1.225 mg/kg?high dose group?.Intraperitoneal injection,continuous exposure for 14 weeks,5 times per week.ASPM expression in the main organs?lung,heart,liver and kidney tissues?in rats was detected by qRT-PCR technique.3.3 It was detected for the expression level of ASPM gene in different stages of cell line of cadmium chloride transformation model established in our group,and for different doses?0,10,20,30?mol/L?and different time?0,12,24,48h?the expression level of ASPM gene after acute exposure to cadmium chloride by qPCR experimental method.3.4 The short hairpin double-stranded RNA?shRNA?vector of ASPM was introduced into 16HBE cells using lentiviral vector-mediated cell transfection technique.It was named 16HBE-shASPM after screening with puromycin.It was verified for the expression of mRNA of ASPM gene-deficient cell line by qRT-PCR.It were observed for the general biological characteristics such as morphology,cell growth curve and cell cycle of the defective cells.3.5 This article data statistics processing application Microsoft Excel software entry,data analysis application software SPSS16.0.Mean±SD was used to describe the statistical results.One-way ANOVA was used to compare the mean of the three groups and three groups.The LSD method and the Dunnett T test were used for the comparison between the two groups.The test level was?=0.05.When the result was P<0.05,it is statistically significant.3.6 This article data statistics processing application Microsoft Excel software entry,data analysis application software SPSS16.0.Mean±SD was used to describe the statistical results.One-way ANOVA was used to compare the mean of the three groups and three groups.The LSD method or the Dunnett T test were used for the comparison between the two groups.The test level was?=0.05.When the result was P<0.05,it is statistically significant.4.Results4.1 The expression of ASPM gene in malignant transformation and acute injury induced by subchronic cadmium in rats and cadmium-induced 16HBE cells.In the malignant transformation model established in the early stage,ASPM was up-regulated in the 14th and 15th generations of cadmium exposure.Compared with the the control group,14th and 35th increased by 2.92 times and 3.44 times,respectively.The difference was statistically significant?P<0.05?.In the subchronic cadmium-induced rat model,ASPM showed different fold increase in lung,heart,liver and kidney tissues.Compared with the control group,the difference was statistically significant?P<0.05?.16HBE cells were treated with different concentrations of CdCl₂ for 24h and16HBE cells were treated with 30?mol/LCdCl₂ for different time.The expression of ASPM mRNA increased with the increase of cadmium chloride dosage.Compared with the control group,the relative expression levels of ASPM mRNA in the 10,20,30?mol/LCdCl₂ treatment groups were 6.03±1.91,13.42±1.28,and 43.42±6.02,respectively.It was statistically significant?P<0.05?.As the time of exposure to cadmium chloride increased,the expression of ASPM mRNA gradually increased.Compared with the control group,the relative expression levels of ASPM mRNA were5.09±0.61,12.87±2.31,17.98±2.42,respectively,and the difference was statistically significant?P<0.05?.4.2 Construction of ASPM gene silencing stable cell lineUnder the same culture conditions,the low expression cell 16HBE-shASPM showed no significant change in cell morphology compared to 16HBE.Under the same culture conditions,the doubling time of 16HBE and 16HBE-shASPM was 22.09 h and 22.56h,respectively.At 96 h,the 16HBE-shASPM cell count was lower than 16HBE cells,the difference was statistically significant?P<0.05?.4.3 The effect of ASPM gene on DNA damageAfter treatment with H₂O₂,it was more serious for the DNA damage of 16HBE-shASPM cells compared with 16HBE.The difference was statistically significant?P<0.05?.After cells treated with different CdCl₂ dose for 24h,the DNA damage increased with the increase of CdCl₂ dose compared with the control group.The DNA damage index values of 16HBE-shASPM cells increased compared with 16HBE cells with the same treatment.The difference was statistically significant?P<0.05?.After cells treated with 30?mol/L CdCl₂ for different time,the DNA damage index values of16HBE cells increased with the prolongation of exposure time.The difference was statistically significant?P<0.05?.The four DNA damage indices of 16HBE-shASPM cells were higher than those of 16HBE cells under the same treatment,and the difference was statistically significant?P<0.05?.4.4 Effect of ASPM Gene on Cell Activity after Acute CdCl2 Exposure.The inhibition rate of 16HBE-shASPM cells was slightly higher than that of 16HBE cells when the concentration of CdCl₂ was 10 or 20?mol/L CdCl₂ at low concentration.The inhibition rate of 16HBE-shASPM cells was close to 100%when the concentration of CdCl₂ was 30 or 40?mol/L at high concentration.The inhibition rate of 16HBE-shASPM cells was higher than that of 16HBE cells at different time points with the prolongation of exposure time,and the difference was statistically significant?P<0.05?.4.5 The effect of ASPM gene on apoptosis of CdCl2-induced cell linesAfter treatment with different CdCl₂ dose,the early apoptotic rate of 16HBE-shASPM cells was higher than 16HBE that under the same treatment.The difference was statistically significant?P<0.05?.After treatment with 30?mol/L CdCl₂ for different time,the apoptotic rate of 16HBE-shASPM cells was higher than 16HBE that at the same time point.The difference was statistically significant?P<0.05?.4.6 Effect of ASPM gene on cytochrome C induced by CdCl2The release of Cyt-C from 16HBE cells and 16HBE-shASPM cells increased with increasing dose of CdCl₂.In the 30?mol/L CdCl₂ dose group,the release of 16HBE-shASPM cells was much higher than that of 16HBE cells,and the difference was statistically significant?P<0.05?.The release of 16HBE cells and 16HBE-shASPM cells showed an increasing trend with the prolongation of CdCl₂ exposure time.At 48h,the release of Cyt-C from 16HBE-shASPM cells was significantly higher than that of16HBE cells,and the difference was statistically significant?P<0.05?.4.7 Effect of ASPM gene on SOD and MDA after acute exposure to CdCl2Compared with 16HBE cells treated with CdCl₂,the dose and time of CdCl₂increased,SOD activity decreased,and MDA increased.Compared with 16HBE cells treated with the same CdCl₂ dose point and time point,The SOD content of ASPM-deficient cells was lower than that of 16HBE cells,while the MDA content was higher than that of 16HBE cells?P<0.05?.5.Conclusion5.1 The expression of ASPM gene in the tissues of cadmium-induced acute injury cells,malignant transformation of cells and subchronic poisoning rats increased with the increase of exposure time,and there was a significant dose-response relationship.It was suggested that ASPM gene was involved in the process of cadmium-induced acute and subchronic injury.5.2 It was successfully constructed the stable low expression cell line of ASPM gene.The cell morphology of the low expression cell line of ASPM was not different from that of the 16HBE cell line.The growth rate of 16HBE cells was faster than that of16HBE-shASPM cells at 96h.5.3 Compared with 16HBE cells treated with CdCl₂ at the same time and at the same CdCl₂ concentration at different time,the inhibition rate,DNA oxidative damage degree,apoptosis rate and cytochrome C release of ASPM cells with 16HBE-shASPM cells increased after cadmium treatment.It is suggested that ASPM gene will play an important role in the process of cadmium-induced acute cell injury.