The Role Of LTBP2 In The Damage Of 16HBE Cells Induced By Cadmium Chloride

1.BackgroundNumerous studies have found that cadmium can cause damage to the liver,kidneys,lungs,bones,brain and other organs and blood system through occupational exposure,smoking and eating contaminated food.Cadmium and its compounds are also associated with reproductive damage,cancer,diabetes and aging.So far,it is not clear for the molecular mechanism of cadmium carcinogenesis.There are many studies suggest that cadmium causes cancer through the following aspects:oxidative stress,inhibiting DNA damage repair,abnormal DNA methylation,inhibiting apoptosis,affecting cell cycle regulation,causing abnormal expression of a variety of genes,estrogen-like effect,promoting the growth of tμmor stem cells,and chronic inflammatory stimulation.LTBP2（Latent transforming growth factor-beta binding protein 2）is associated with the occurrence and development of tumor diseases such as thyroid cancer,liver cancer,multiple organ tumors and other cancer diseases.However,it has not been reported that its mechanisms interact with environmental chemical damage.The aim of this study was to detect the LTBP2 gene expression in human bronchial epithelial cells（16HBE）after acute injury induced by cadmium chloride,and to study the LTBP2 gene changes in LTBP2deficient cell model,and to explore the mechanism of LTBP2 gene in cadmium chloride induced injury toxicity.2 Objective2.1 Biological information of 16HBE cells was detected based on the previously established model of cadmium malignant transformation.The differential expression of LTBP2 gene was found in the 16HBE mRNA expression profile of different malignant transformation algebras It was further searched for the location,morphology and other biological characteristics of LTBP2.2.2 It was observed that the expression of LTBP2 mRNA in different tissues（heart,liver,kidney and lung）in the animal model which exposure subchronically to cadmium before to discuss the role of LTBP2 in animal model tissues induced by cadmium.and the role of LTBP2 in animal model tissues induced by cadmium was discussed.2.3 It was verified that differential expression of LTBP2 at different stages after malignant transformation in 16HBE cells induced by CdCl₂.After the establishment of a stable cell line with low expression of LTBP2,it was observed that the effects of low expression of LTBP2 on cell morphology,cell proliferation and other general biological characteristics,and studied.that the role of LTBP2 gene in cadmium malignant transformation toxicity.2.4 It wad detected that changes of LTBP2 mRNA expression in 16HBE cells exposed to CdCl₂ at different doses and times.LTBP2 low-expression cell lines were treated with CdCl₂ at different concentrations and times.It was to study that the effect of LTBP2 gene on acute toxic injury induced by cadmium with cell inhibition rate,apoptosis,DNA damage,oxidative stress,etc.3 methods3.1 The differential expression of LTBP2 gene was detected by qRT-PCR,and a stable cell line with low expression of LTBP2 gene was constructed by RNA interference technology and lentiviral transcription technology.It was detected that the changes in general biological characteristics.The effects of LTBP2 gene on cell inhibition rate,apoptosis,DNA damage,Cyt-C content,MDA content and SOD activity were detected by MTT,CCK8,flow cytometry,comet assay,Cyt-C and oxidative index kit.3.2 The experimental data were analyzed by SPSS 16.0 software.and the measurement data was represented by`x±s which subjects to normal distribution after normal test.Each sample was carried out for three parallel experiments and the experiment was repeated three times.Mean comparison among the three groups and above were used by One-way ANOVA（population variance homogeneity,further multiple comparisons by SNK-q test）or kruskal-wallis H test（population variance heterogeneity,further multiple comparisons by Dunnett’s T3 test）.T tests were used to compare the two groups of data.Linear regression with one variable was used to analyzed the relationship between the cadmium chloride dose and the corresponding indicators.It was 0.05 for the test level.4 result4.1 Expression of LTBP2 gene in cadmium-induced 16HBE malignant transformed cells:qRT-PCR was used to detect the relative expression of LTBP2 mRNA in cells.Compared with 16HBE control group,the LTBP2 expression was 2.04±0.36 and 5.93±0.60 respectively.It was statistically significant for the difference（P<0.05）.4.2 Expression of LTBP2 in subchronic infected rat tissues:In the rat model of subchronic cadmium exposure,it was increased for the expression of LTBP2 in lung,heart and liver tissues.The expression levels of LTBP2 in lung tissues were 1.98±0.15,1.40±0.26 and 1.32±0.10 at the low,medium and high dose groups respectively,which were higher than those in the normal group.The difference was statistically significant（P<0.05）.The expression levels of LTBP2 were 1.16±0.16,1.97±0.19,and 2.02±0.13,respectively,in the different heart tissue exposure groups.Compared with the normal group,it was increased for the expression levels of the medium and high dose groups.The difference was statistically significant（P<0.01）.The expression levels of LTBP2 was1.33±0.35,1.22±0.25,and 1.19±0.10 in liver tissues respectively.There was no significant difference between the two groups（P>0.05）.Compared with the normal group,it was decreased for the expression in the low dose group and the medium dose group.The difference was statistically significant（P<0.01）.In poisoned rats,cadmium chloride exposure stimulated LTBP2 mRNA expression in lung,heart and liver tissues.especially in heart tissues.The expression of LTBP2 was the highest in the high dose group especially in the heart tissue.In lung tissues,LTBP2 decreased with the increase of the dose,and there was a dose-response relationship.4.3 Expression of LTBP2 gene in acute cadmium induced 16HBE injury:After16HBE cells treated with 10,20,30,40μmol/L CdCl₂ for 24 h,qRT-PCR was used to determined the relative expression of LTBP2 gene.Compared with control group,there was a significant difference（P<0.05）.The expression of LTBP2 mRNA gradually increased with the increase of CdCl₂ dose,showing a significant dose-effect relationship（P<0.01）.After 16HBE cells treated with 30μmol/L CdCl₂ for 12,24,and 48h respectively,the difference of LTBP2 expression was statistically significant compared to cells without CdCl₂（P<0.05）.The expression of LTBP2 mRNA gradually increased with the increase of the exposure time,and the change trend presented a significant time-effect relationship（P<0.01）.4.4 Effects of low LTBP2 expression on general biological traits:The low LTBP2expression cell lines（16HBE-shLTBP2 cells）were constructed successfully by Lentiviral vector mediated cell transfection.There was no significant effect on cell morphology and proliferation rate duing to LTBP2 low expression.4.5 Effects of low LTBP2 expression on cadmium induced cell growth inhibition:After treatment with different concentration CdCl₂ for 24 h,it was increased gradually for the cell inhibition rate of 16HBE cells,which showed a significant dose-effect relationship（P<0.01）.Compared with 16HBE,it was significantly reduced for the inhibition rate of16HBE-shLTBP2 treated with the same concentration of CdCl₂.The difference was statistically significant（P<0.01）.After treatment with different time for 24,48 and 72h with 30μmol/L CdCl_2,the inhibition rate of 16HBE-shLTBP2 cells was significantly lower than that of 16HBE cells treated at the same time.The difference was statistically significant（P<0.05）.4.6 Effect of low expression of LTBP2 on Cyt-C content:Cyt-C kit was used to detect the effect of acute exposure of 16HBE cells on apoptosis.After cells treated with30μmol/L CdCl₂ for different time（12\24\48\72 h）,the Cyt-C concentration of16HBE-shLTBP2 was significantly higher than that of 16HBE at 24,48,72 h（P<0.05）.After treatment with 10,20,30,40μmol/L CdCl₂ for 24h,the 16HBE-shLTBP2 Cyt-C concentration was significantly higher than 16HBE at 10,20,40μmol/L（P<0.05）.4.7 Effects of low expression of LTBP2 on oxidative damage of cells induced by cadmium:After treatment with different concentrations CdCl₂ for different time,the SOD level of 16HBE-shLTBP2 cells was significantly higher than that of 16HBE cells（P<0.01）,and the MDA content of 16HBE-shLTBP2 was significantly lower than that of 16HBE（P<0.05）.4.8 Effects of low expression of LTBP2 on cell apoptosis induced by cadmium:Flow cytometry was used to detected apoptosis rate of the LTBP2 series cell lines after treatment with CdCl₂ for different concentrations and different times.The apoptosis rate of16HBE-shLTBP2 cells was higher than that of normal 16HBE cells in the same treatment group at 24h.It was also higher than that of control cells in different treatment time with30μmol/L CdCl₂.4.9 Effects of low expression of LTBP2 on cadmium induced DNA damage in cells:For 16HBE-shLTBP2 group,damage values（TL,TD（%）,TM and OTM）increased with increase of cadmium dose when cells were treated with different CdCl₂ concentrations.Cmpared with the 16HBE cells in the same group,the damage values of the16HBE-shLTBP2 group were significantly higher than those of the 16HBE cells in the same group at each concentration point.It was statistical differences（P<0.05）.After exposure 12,24,48h with 30μmol/LCdCl₂,it was significantly different for the four injury indicators of 16HBE-shLTBP2 group compared with those in the 16HBE group（P<0.05）.At 72h,there was no statistically significant difference in the four injury indicators between the two groups（P>0.05）.5 conclusion5.1 The expression of LTBP2 gene increased with the increase of toxic dose or malignant transformation degree,which was in the process of cadmium-induced 16HBE malignant transformed cells,subchronic infected rat tissues,and CdCl₂ with different doses and times acute infection,indicating that LTBP2 gene is involved in the process of cadium-induced acute injury and malignant transformation.5.2 After the successful construction of LTBP2 low-expression cell lines,CdCl₂treatment showed that compared with 16HBE,16HBE-shLTBP2 had lower cell inhibition rate,lower MDA content,and higher SOD content,suggesting that LTBP2 may be involved in the process of acute cell injury by cadmium through oxidative stress.The apoptosis rate and DNA damage degree of 16HBE were both lower than that of16HBE-shLTBP2,suggesting that LTBP2 may inhibit apoptosis and participate in DNA damage in the process of cadium-induced acute cell injury.