| Lung cancer has brought about major public health problems with the highest morbidity and mortality of all tumors in China and around the world.Thus,it is particularly important to carry out early prevention research on lung cancer.Numerous studies have shown that incident lung cancer is associated with individual’s genetic background.As a changeable factor,the role of environmental factors in tumorigenesis deserves further investigation.Traditional environmental risk factors,including smoking,benzopyrene,asbestos and heavy metals,are all important factors in promoting tumorigenesis.In contrast,as the essential micronutrients for the humans,essential metal deficiency leads to the occurrence of various diseases and pathological status,and even cancers.For example,increased incidences of pancreatic and liver cancer are associated with Fe deficiency,while supplementation of Se can reduce the risk of liver cancer.Associations of essential metals with tumorigenesis are usually explained by the biological process of oxidation and antioxidation,however,such associations cannot be completely explained by the biological mechanism of oxidation and antioxidation alone.In addition,as the most common tumor type,fewer studies have investigated the associations of essential metal exposure with incident lung cancer.Based on the finding above,we aimed to investigate the associations between essential metal exposure and the risk of lung cancer,and to explore the underlying biological mechanisms for these associations.First,a nested case-control study from Dongfeng-Tongji cohort including 440 new incident lung cancer cases and 1320 healthy controls(1:3 matched on age and gender)was used to investigate the associations of 11 plasma essential metals [Cobalt(Co),Copper(Cu),Fe,Manganese(Mn),Molybdenum(Mo),Rubidium(Rb),Se,Strontium(Sr),Tin(Sn),Vanadium(V),and Zn] with incident lung cancer.Then,a prospective cohort study of occupational coke-oven workers was used to investigate the relationship between baseline level of plasma Zn with 4 years’ telomere shortening,and plasma Zn was found associated with lung cancer in the nested case-controls study.To investigate the biological mechanism for the association of plasma Zn with incident lung cancer,we selected the genes whose expressions were associated with level of plasma Zn from gene expression files of 144 healthy subjects,and the associations of Zn level with selected gene expression were validated in paracancerous lung tissues from 87 surgical lung cancer cases.Further,the expression of the selected genes between lung cancer and normal tissues were compared both in our 87 surgical lung cancer cases and 106 lung cancer cases from The Cancer Genome Atlas(TCGA)database(https://cancergenome.nih.gov/).There are three main parts in this study as shown in the following.Part I.Associations of plasma essential metal elements with incident lung cancer: a nested case-control study Objectives: To investigate the associations of 11 plasma essential metals with incident lung cancer,and provide new clues for the early prevention of lung cancer.Methods: This study is based on Dongfeng-Tongji cohort study,which includes 27009 participants in 2008 and 14120 newly enrolled participants in 2013.A total of 440 incident lung cancer with plasma samples in the follow-up until December,2016,and 1320 healthy controls matched by age and gender were included in this study.The general demographic characteristics,lifestyles and dietary data were collected by a questionnaire from all subjects.Incident lung cancer cases were confirmed by reviewing on their medical records.Plasma levels of 11 essential metals were measured by quadrupole inductively coupled plasma-mass spectrometry(ICP-MS).To investigate the association of plasma metals with incident lung cancer,single and multiple metal models for essential metals were modeled by conditional logistic regression analysis,with adjustment for age,gender,BMI,education levels,smoking,pack-years smoked,alcohol drinking,regular physical activity status and family history of cancer.The restricted cubic splines linked to logistic regression model were also used to evaluate the linear associations between metals and incident lung cancer.In addition,stratified analysis by lifestyles including smoking,drinking and regular physical activity status were also conducted for the association of plasma Zn with incident lung cancer.Further,combined effects of Zn and lifestyles on incident lung cancer were also conducted among males.In addition,linear regression was used to evaluated the associations between different kinds of food consumption per week and level of plasma Zn.Results: In single metal models,we found that each one-unit increase in ln-transformed plasma Zn was associated with 11% decreased risk of lung cancer(OR=0.89,95%CI=0.79-0.99)among all participants,and the protective effects of plasma Zn on lung cancer risk were mainly shown among males(OR=0.86,95%CI=0.74-0.99),but not among females(OR=1.00,95%CI=0.83-1.22).In the multiple-metal models with mutual correction of multiple metals,the protective effects of Zn on lung cancer remained unchanged among males,each one-unit increase in ln-transformed Zn was associated with a 29% decreased risk of lung cancer(OR=0.71,95%CI=0.55-0.93).In addition,linear association of Zn with lung cancer risk among males were also shown in the restricted cubic splines analysis(P=0.041).In the sensitivity analysis,after exclusion of the incident lung cancer cases within the first 6,12 or 24 months’ followup period,the protective effects of plasma Zn on lung cancer risk were essentially unchanged [OR(95%CI)=0.86(0.74,1.00),0.86(0.74,0.99)and 0.84(0.72,0.99),respectively].Further,all males were stratified into four subgroups(Q1-Q4)by the quartile levels of plasma Zn in controls.Compared with subjects within the lowest levels of Zn(Q1,970.10μg/L),33% and 36% decreased risk of lung cancer were found among participants with higher Zn in Q3(1275.48-3358.49μg/L)and Q4(>3358.49μg/L)groups [OR(95%CI)=0.67(0.45,0.99)and 0.64(0.43,0.95),respectively].A linear trend for the associations of Zn with lung cancer risk was also shown from Q1 to Q4 groups(Ptrend=0.014).Stratified analysis for the above associations were also conducted according to dichotomous lifestyles including smoking(ever,never),alcohol drinking status(ever,never)and regular physical activity(yes,no).The protective effects of higher levels of Zn(≥1275.48μg/L)on decreased incident lung cancer were mainly shown among nonsmokers [OR(95%CI)=0.70(0.39,1.28)],non-alcohol drinkers [OR(95%CI)=0.64(0.41,0.98)] and regular exercisers [OR(95%CI)=0.68(0.50,0.93)].In addition,the analysis for the combined effects of plasma Zn with lifestyles on incident lung cancer showed that non-smokers,non-alcohol drinkers and regular exercisers had combined effects with high plasma Zn(≥1275.48μg/L)on decreased risk of lung cancer [OR(95%CI)=0.71(0.40,1.27),0.58(0.37,0.88)and 0.59(0.32,0.86),respectively].In looking at the association of plasma Zn with the consumption of different kinds of foods,we found that higher nuts consumption(g/week)were positively associated with increased plasma level of Zn(Pearson r=0.086,P=0.014).However,such association was not found among other foods,including cereals,eggs,poultry,aquatic products,fresh vegetables,fresh fruits,milk or dairy products or beans consumptions.Conclusions: Increased plasma Zn was associated with decreased lung cancer risk,and such association was mainly shown among non-smokers,non-alcohol drinkers and regular exercisers.Weekly nut consumption is a possible food source of increased level of plasma Zn.Thus,Improving the level of plasma zinc may provide guidance for the early prevention of lung cancer.Currently,the mechanisms for the effects of plasma Zn on incident lung cancer remains unclear,and we will gradually carry out the research to explore the underlying mechanisms.Part II.Plasma zinc and telomere shortening: a prospective cohort study Objectives: The change of telomere length is one of the important biomarker for the early development of cancer.In the first part,we investigated the associations of 11 plasma essential metals with incident lung cancer,and found the protective effects of plasma Zn on incident lung cancer.Thus,in this part,we will focus on the effects of plasma Zn on telomere shortening,and provide early evidence for the results that were found in the nested case-control study of lung cancer.Methods: The occupational coke-oven worker cohort study including 1628 participants at baseline in 2010,and 1243 of them had been successfully followed-up in 2014.In this study,1243 coke-oven workers were enrolled in the analysis.Information on general characteristics,lifestyles and occupational history were collected at baseline,and peripheral venous blood was collected both in 2010 and 2014,respectively.Real time quantitative PCR and ICP-MS were used to measure levels of 4 years’ telomere shortening and plasma Zn,respectively.Generalized linear regression models were used to evaluated the independent and combined effects of Zn with lifestyles(smoking,drinking,and regularly physical activity status)on 4 years’ telomere shortening.Age,gender,BMI,lifestyles and exposure status of coke-oven emissions were adjusted in the above analysis.Results: Telomere length(in the year 2010 and 2014)and level of plasma Zn in 1196 and 744 coke-oven workers were successfully detected,respectively.Finally,a total of 724 subjects were included in the following analysis.Telomere length in peripheral venous lymphocytes from baseline of 2010 and the follow-up year of 2014 were both measured by PCR,and higher telomere length was found in coke-oven workers in 2010 than 2014(median=1.98 and 1.58,respectively,P<0.001).Decreased 4 years’ telomere shortening in peripheral venous lymphocyte with the increased level of plasma Zn in baseline were also showed [β(95%CI)=-0.086(-0.143,-0.030),P=0.003].Further,all subjects were divided into high and low plasma Zn groups according to the median level of plasma Zn(1250μg/L),and lower telomere shortening was found in subjects with high plasma Zn(≥1250μg/L)group than participants with lower plasma Zn(0.09 vs.0.18,P<0.001).In the stratified analysis by lifestyles,the protective effects of higher plasma Zn(≥1250μg/L)on 4 years’ telomere shortening were found both in smokers [β(95%CI)=-0.56(-0.97,-0.15)],non-smokers [β(95%CI)=-0.71(-1.33,-0.09)],alcohol drinkers [β(95%CI)=-0.77(-1.32,-0.23)],non-alcohol drinkers [β(95%CI)=-0.50(-0.94,-0.07)] and regular exercisers [β(95%CI)=-0.97(-1.42,-0.52)],but such associations were not found among non-regular exercisers [β(95%CI)=-0.31(-0.82,0.20)].In addition,the analysis for the combined effects of plasma Zn with lifestyles on telomere shortening showed that non-smokers,non-alcohol drinkers and regular exercisers had combined effects with high plasma Zn(≥1250μg/L)on decreased telomere shortening [β(95%CI)=-0.57(-1.16,0.01),-0.63(-1.17,-0.10)] and-0.73(-1.19,-0.27),respectively].Conclusions: High level of plasma Zn can protect against on telomere shortening in peripheral venous lymphocytes,and this effect was more pronounced among nonsmokers,non-drinkers,and regular exercisers.Since telomere shortening is an important biological event for the development of early lung cancer,our findings suggest that the protective effects of Zn on incident lung cancer may mainly through protecting against on telomeres damage.In addition,Zn has multiple biological functions,and the incident lung cancer was usually accompanied with changes of gene expression.Thus,we supposed that Zn may also play roles on regulating the expression of cancer-related genes.Part III.Associations of plasma zinc with gene expression and differences in the expression of plasma zinc-related genes between lung cancer and normal tissues Objectives: In the previous two parts of the prospective cohort study,we found that Zn had protective effects on telomere shortening and incident lung cancer.Although Zn takes part in a variety of biological processes in cells,the mechanisms for the protective effects of Zn needs further exploration.In this part,the associations of Zn with gene expressions were conducted both in plasma and lung tissues,and we also compared the gene expressions between lung cancer and normal tissues.This study may help to elucidate the mechanisms for the protective effects of Zn on incident lung cancer.Methods: 144 healthy subjects were selected from physical examination center of Dongfeng hospital in Shiyan.The general demographic information,lifestyles and medical history,as well as peripheral venous blood were also collected from all subjects.Level of plasma Zn were measured by ICP-MS and gene expression profile of 47323 gene locus were measured by HumanHT-12 v4 Expression BeadChip.Linear regression models were used to evaluated the association between level of plasma Zn and gene expression.We selected the gene whose expression was associated with level of plasma Zn according to the following criterias:(1)the genes in peripheral blood lymphocytes whose expression were associated with level of plasma Zn(P<0.05)and had been reported on the Zn related signals pathways;or(2)the topmost 40 genes(ranked according to the P value)whose expression were associated level of plasma Zn,and their annotation of gene function are associated with tumorigenesis according to NCBI databases.In addition,87 surgical lung cancer cases from Tongji hospital,Wuhan were also enrolled in this study.Their lung cancer and normal tissues were collected and stored in liquid nitrogen.Levels of Zn and gene expression in tissues were evaluated by ICP-MS and Real-time fluorescence quantitative PCR assay respectively,and the linear associations of Zn with the selected gene expressions were also evaluated in normal lung tissues.The differences of Zn and gene expressions between lung cancer and normal tissues in 87 lung cancer cases were tested by paired Wilcoxon rank test.Further,a public database of The Cancer Genome Atlas(TCGA)with gene expression of 106 paired of lung cancer and normal tissues was also used to evaluate the differentially expressed Zn related genes in the above.Results: Based on the above selection criterias,expression of 13 genes in peripheral blood lymphocytes,including gene with anxioxidant function(SOD1),genes with DNA repair function(APEX1 and TP53BP1),genes taking part in the process of cancer promotion(WDR33,SNX33,MAGED2,ARMC10,LAPTM4 B,TRIT1,HUWE1,ZNF813,and ZNF717),and gene with function of cancer metastasis(MMP25)were found to be linearly associated with level of plasma Zn.Increased level of plasma Zn was associated with increased expression of TP53BP1 [β(95%CI)=1.19(0.25,2.12),P=0.001],HUWE1 [β(95%CI)=1.49(0.57,2.40),P=0.002)] and MMP25 [β(95%CI)=0.96(0.02,1.91),P=0.045).In contrast,decreased expression of other 10 genes were founded in subjects with increased level of plasma Zn(all β<-0.97,P <0.044).Further,we conducted a validation study for the above associations in normal lung tissues among 87 surgical lung cancer cases,and we validated that increased level of tissue Zn was associated with increased expression level of TP53BP1 in normal lung tissues [β(95%CI)=0.509(-0.033,1.051),P=0.066],however,no other consistent results were found between the finding and validation study.In addition,lower level of Zn was found in lung cancer tissues than normal tissues(25.19 vs.35.70μg/g,P<0.001),while 9(SOD1,APEX1,TP53BP1,WDR33,MAGED2,LAPTM4 B,TRIT1,HUWE1 and ZNF813)of the 13 genes were highly expressed(P<0.05)in lung cancer tissues than normal tissues both in our study of 87 surgical lung cancer cases.We also compared the expression of 13 genes in lung cancer and normal tissues of 106 lung cancer cases from TCGA database,and found that expression of SOD1,APEX1,TP53BP1,WDR33,ARMC10,LAPTM4 B,TRIT1,HUWE1 and ZNF813 were highly expressed in lung cancer tissues than normal lung tissues.In total,8 genes(SOD1,APEX1,TP53BP1,WDR33,LAPTM4 B,TRIT1,HUWE1 and ZNF813)were highly expressed both in our 87 lung cancer cases and TCGA database.Conclusions:Plasma Zn was linearly associated with expression of 8 genes,including gene with anxioxidant function(SOD1),genes with DNA repair function(APEX1 and TP53BP1),and genes taking part in the process of cancer promotion(WDR33,LAPTM4 B,TRIT1,HUWE1 and ZNF813),in peripheral venous lymphocytes,and they were highly expressed in lung cancer tissues than normal lung tissues.This suggests that plasma Zn may play against on the oncogenesis and development of lung cancer by regulating the expression of these genes,which may help to elucidate the underlying mechanisms for the protective effects of Zn on incident lung cancer. |
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