The Effects Of MiR-24,miR-28,and MiR-150 On The Associations Of Titanium With Chromosome Damage And Lung Cancer Development

Lung cancer is the most frequently occurring malignancy and the leading cause of mortality in cancer-related deaths in China.According to the reported nationwide cancer incidence and mortality in 2014,published by the National Cancer Registry of China(NCCRC),the incidence rate of lung cancer is 57.13 per 100,000 and the mortality rate is 45.80 per 100,000.The incidence and mortality rate of lung cancer are both at high levels and elevated than the level of 2013.The treatment of lung cancer is difficult and the 5-year survival rate of the patients is low.Among males,both the incidence rate and mortality rate of lung cancer rank first in all types of cancer.There were many environmental and genetic risk factors for lung cancer.For the occupational populations,coke-oven emissions(COEs),asbestos,chloromethyl ether and arsenic are risk factors for lung cancer.Lung cancer caused by coke oven emissions is one of the 11 statutory occupational cancers and is the most serious occupational disease among coke-oven workers.The coke oven emissions contain high levels of polycyclic aromatic hydrocarbons(PAHs),benzene series,and heavy metal particles.Exposure to these carcinogens can accelerate the oxidative stress-induced DNA and chromosome damage processes in coke-oven workers and eventually leads to occurrence of lung cancer.Chromosome damage is an important early effect event of lung cancer and is very important in the study of early prevention for lung cancer.Heavy metal exposure is a common risk factor that causes chromosome damage and cancer.Exposure to arsenic,cadmium,chromium,lead,mercury,and nickel may directly or indirectly induce the production of free radicals in the body,such as reactive oxygen species(ROS)and reactive nitrogen.The accumulation of free radicals further induces oxidative stress in cells and leads to chromosome damage.In addition,essential metals such as zinc,selenium and strontium can inhibit chromosome damage through anti-oxidative stress pathways.At present,the mechanisms underlying the effects of metals on the process of chromosome damage is not yet clear.MicroRNA(miRNA)is a short-chain,small-molecule RNA that can regulate the expression of specific genes through translational inhibition or degradation of target mRNA.Some studies have shown that metal exposure can indirectly regulate the expression patterns of related genes mediated by miRNAs and thus play a role in oxidative stress and DNA damage response(DDR).In our previous published works(Deng et.al,Environ Health Perspect.2014,122(7):719-25.),the plasma levels of five miRNAs,including miR-24,miR-27 a,miR-28,miR-142,and miR-150,have been found to be dose associated with micronucleus frequencies in lymphocytes,which indicate the chromosome damage levels.We hypothesized that these five candidate miRNAs could be involved in mediating the associations between metals exposure and chromosome damage,and may further affect the occurrence of lung cancer.This study was aimed to assess the associations between metals exposure and chromosome damage among coke-oven workers and to study the mediation effects of above 5 miRNAs(miR-24,miR-27 a,miR-28,miR-142,and miR-150);furthermore,we also tried to explore the effects of identified metal and miRNAs in lung cancergenesis and the the dose-response relationships between them.The study contains following two parts: Part I Associations of titanium with chromosome damage levels among coke-oven workers and the mediating roles of miRNAs Objectives: Chromosome damage is the driving force in the process of cell carcinogenesis,and it is also an early effect of tumorigenesis.The purpose of this section was to investigate the associations between the urinary concentrations of 23 metals and chromosome damage levels among coke-oven workers,and to further explore the mediation effects of five candidate miRNAs in these associations.Methods: In this study,365 healthy male coke-oven workers were included.The basic demographic information,lifestyles,history of diseases,and occupation history of each worker were collected by using a standardized questionnaire.Inductively coupled plasma mass spectrometry(ICP-MS)was used to determine the urinary concentrations of 23 metals,including Al,As,Ba,Cd,Co,Cr,Cu,Fe,Mn,Mo,Ni,Pb,Rb,Sb,Se,Sn,Sr,Ti,Tl,U,V,W,and Zn;Gas chromatography-mass spectrometry(GC-MS)and enzyme-linked immunosorbent assay(ELISA)were used to determine the concentrations of 12 metabolites of urinary PAHs and plasma BPDE-albumin adducts,respectively,which can reflect the levels of internal PAHs exposure.The MN frequencies in peripheral blood lymphocytes were detected by using cytokinesis-block micronucleus assay,to reflect the degree of chromosome damage in the body.In addition,we also selected five candidate miRNAs(miR-24,miR-27 a,miR-28,miR142,and miR-150),and measured the expression levels of these miRNAs in plasma by using the real-time quantitative PCR method.The multivariate Poission regression models were used to analyze the dose-response relationships between 23 urinary metals and MN frequencies in peripheral blood lymphocytes.We also analyzed the associations between the significantly MN frequency-associated metal and the relative expression levels of the five miRNAs,by conducting the generalized linear regression models.The mediation effects of these miRNAs on the associations of metal exposure with MN frequencies were further evaluated by the causal mediation analysis.Results: With the increasing levels of urinary Pb,Ti and Zn,the MN frequencies of peripheral blood lymphocytes decreased significantly(P<0.05).With one unit increase in logarithmic transformed Pb,Ti,and Zn concentrations,the MN frequencies decreased by 10%,12%,and 10%,respectively,and the FR(95% CIs)were 0.90(0.82,1.00),0.88(0.82,0.94),and 0.90(0.82,0.99),respectively.The dose-response relationship between urinary Ti and reduced MN frequencies was still significant after FDR correction [FR(95% CI)= 0.88(0.82,0.94),P=0.009 after FDR correction].While the FDR adjusted P values for Pb and Zn were 0.208 and 0.192,respectively.As a result,the associations between urinary Ti and the expression levels of five plasma miRNA were further analyzed.The results showed that,for a doubling in urinary concentration of Ti,the plasma levels of miR-24,miR-27 a,miR-28,miR-142,and miR-150 decreased by 38.17%,24.07%,26.68%,17.10%,and 14.72%,respectively [PD(95% CIs)were:-38.17(-51.06,-21.88),-24.07(-35.99,-9.93),-26.68(-39.50,-11.13),-17.10(-31.28,0.02),-14.72(-23.77,-4.60),P values were: <0.001,0.002,0.002,0.050,and 0.007].Mediation analysis showed that miR-24,miR-28,and miR-150 mediated 25%(8%,70%),20%(6%,52%),12%(2%,37%)of the associations between Ti and MN frequencies respectively(P values were 0.002,0.004,and 0.016,respectively).However,the mediation effects of miR-27 a and miR-142 only reached the marginal significant level,with proportion mediated: 11%(0%,39%)and 8.6%(0%,31%),respectively(both P=0.054).Conclusions: There was a significant dose-response relationship between elevated Ti and reduced levels of chromosome damage among coke-oven workers.The urinary Ti was also associated with reduced plasma levels of miR-24,miR-28 and miR-150,which contribute to part of the associations of Ti with chromosome damage.This result was probably the first to identify the protective effect of Ti on chromosome damage,and this effect may be mediated by miR-24,miR-28,and miR-150,but the biological mechanisms need further explorations.In addition,since coke-oven workers are at high risk of lung cancer and chromosome damage is an important early biological event of lung cancer,the correlations between Ti exposure and the expression levels of above three miRNAs need to be tested in lung tissues and the biological effects of above miRNAs in oncogenesis of lung cancer should be investigated in further studies.Part II Levels of titanium and miR-24,miR-28,miR-150 expressions between lung cancerous and normal tissues and their assoiations Objectives: To study the dose-response relationship of Ti with miR-24,miR-28,and miR-150 on the basis of part I and explore the effects of them on lung cancergenesis.Methods: A total of 43 male lung cancer patients,who underwent lung cancer resection at the Department of Thoracic Surgery,Tongji Hospital Affiliated to Huazhong University of Science and Technology,were recruited in this study.The demographic characteristics,history of diseases,and surgical records were collected.The concentrations of Ti in the tumorous and normal lung tissues were detected by using ICP-MS,and the the expression levels of miR-24,miR28,and miR-150 relative to internal reference RNU6(U6)in the tumorous and normal lung tissues were measured by using the real-time quantitative PCR(RT-qPCR)methods.Paired t-test was used to compare the differences of Ti and miRNA expression levels between tumor and normal lung tissues.The linear regression models were used to evaluate the associations between Ti and the expression levels of these miRNAs in normal lung tissues.We further compared the expression levels of above three miRNAs between tumor and normal lung tissues of lung squamous cell carcinoma(SCC)and lung adenocarcinoma(AC),respectively.The miRNA expression data from the public database of The Cancer Genome Atlas(TCGA,http://cancergenome.nih.gov),including 500 cases of lung adenocarcinoma and 472 cases of lung squamous cell carcinoma were also investigated to validate our results.Results: Ti levels in lung tumorous tissues were lower than normal lung tissues(cancer vs.normal: 0.05 vs.0.20 μg/g),but the difference was not statistically significant(P=0.118).After classifying the patients into SCC and AC,it was found that the level of Ti(Ln-transformed)was significantly lower in lung SCC than the corresponding normal lung tissues(cancer vs.normal:-0.22 vs.0.22 μg/g,P=0.010).In AC patients,there was no such significant differences.Among the whole cancer patients,a doubling in the concentration of Ti in normal lung tissue resulted in 53.4%,47.2%,and 39.6% decrease in the expression of miR-24,miR-28,and miR-150(All P<0.05).For SCC patients,a doubling in the concentration of Ti in normal lung tissue was associated with 64.4%,63.4%,and 51.4% reduced expression of miR-24,miR-28,and miR-150(All P<0.05).In AC patients,there was no such significant correlation.The expression of miR-24 in cancerous tissues was significantly higher than in normal tissues for lung SCC patients(cancer vs.normal:-1.46 vs.-2.47,P=0.033),however,there was no such significant difference found in AC or total patients.The expression level of miR-28 in lung cancer tissues was significantly higher than in normal lung tissues(cancer vs.normal:-4.88 vs.-4.78,P=0.048).For lung SCC patients,the expression level of miR-28 was significantly higher in cancer tissues than in normal tissues(cancer vs.normal:-4.05 vs.-4.81,P=0.033),whereas for AC patients,there was no such significant difference.The expression of miR-150 in all lung cancer tissues was significantly lower than that in normal lung tissues(cancer vs.normal:-1.72 vs.-0.86,P<0.001),and this difference was mainly shown among SCC patients(cancer vs.normal:-1.71 vs.-0.85,P<0.007),but not seen among AC patients.Data from TCGA database showed that,in SCC cases,the expression levels of miR-24 and miR-28 in cancer tissues were significantly higher than those in normal lung tissues [cancer vs.normal: 11.33 vs.10.98(miR-24)and 6.97 vs.6.66(miR-28)],both P< 0.001.However,the expression level of miR-150 expression was significantly lower in tumor than in normal lung tissues(cancer vs.normal: 9.50 vs.10.33)(P<0.001 for all above).These results were consistent with the results from our study population.In addition,the TCGA data also showed increased expression of miR-28 in AC tissues(cancer vs.normal: 6.56 vs.5.91,P<0.001).But there was no significant difference in the expression levels of miR-24 and miR-150 between AC and normal lung tissues.Conclusions: These results confirmed the dose-response relationships between Ti and miR-24,miR-28,and miR-150 expression at the tissue level,suggesting that Ti may inhibit both miR-24 and miR-28 levels and further involve in the development of lung cancer.However,the effect of miR-150 in lung cancer development was still unclear.