| Lung cancer was the most common incident cancer and the leading cause of cancer death with an incidence of 1.8 million new cases and 1.59 million deaths in 2012according to the report from IARC（International Agency for Research on Cancer）.In China,there were approximately 0.733 million new lung cancer cases（509,300 males and 224,000 females）and 0.610 million lung cancer deaths（432,400 males and177,800 females）in 2015.Recently,owing to tobacco consumption,environmental pollution and aged tendency of population,lung cancer has become increasingly greatest threats to public health in our country.Therefore,it is important and urgent to strengthen the study of the etiology and pathogenesis for lung cancer and screen efficient,specific and sensitive biomarkers for the prevention.Epidemiological studies have shown that the development and progression of lung cancer is a complex result for the interplay of environmental,biological,genetic and epigenetic factors.Smoking is the environmental risk factor which most related to lung cancer risk.Even though evidences indicate that over 80%of lung cancer cases in males and over 50%in females can be attributed to tobacco consumption,but only less than20%of smokers finally developed into lung cancer,suggesting that there is an inter-individual variation in genetic susceptibility to lung cancer when exposing to enviromental risk factors.In recent years,genome-wide association study（GWAS）and Next-Generation Sequencing（NGS）contributed greatly to the oncological research.The GWAS study as a powerful tool to identify lung cancer susceptible genes with large sample size and multiple stage validation provided important clues to identify high risk people,guide individual prevention and treatment of lung cancer.GWAS has achieved great success in deciphering the genetic basis of susceptibility among complex diseases.Since the publication of the first lung cancer GWAS study in 2008,the researchers have discovered above 45 lung cancer susceptible loci,such as 1p36,2q23,3q28,5p15,5q31,5q32,6p21,6p22,6q24,10p14,10q25,12q23,13q12,13q13,15q25,17q24,18p11,19q13,20q13and 22q12.In 2007,29,266cases and 56,450 controls of European descent were genotyped on the Onco Array and combined with existing data for an aggregated genome-wide association study（GWAS）analysis of lung cancer.They identified 18 susceptibility loci achieving genome-wide significance,including 10 new loci:4 loci（1p31.1,6q27,8p21 and 15q21.1）for lung cancer and 6 loci（15q21,8p12,10q24,20q13.33,11q23.3 and 9p21.3）for lung adenocarcinoma.Their study partly contributed to the“missing heritability”defined as the fact that single genetic variations cannot account for much of the heritability of diseases,behaviors,and other phenotypes.However,the function of most of the lung cancer susceptibility loci that how to regulate the susceptibility gene expression remains unexplained.Previous studies have found that there is a diversity of mechanisms of gene expression regulation.Epigenetics is the study of heritable changes in gene activity and expression that do not involve changes in the DNA sequence.Epigenetics,mainly including histone modification,DNA methylation,RNA methylation and chromatin remodeling,play an important role in protein translational,transcriptional and post-transcriptional modification.The N6-methyl-adenosine（m6A）is the most prevalent m RNA modification in eukaryotes among about 100 types of RNA modifications identified and has diverse biological functions such as transcription splicing,nuclear RNA export,protein translation control,and cell fate determination.The m6A related disease or phenotype included obesity,embryonic development,gender decision,infertility and tumor.It has been shown recently to play essential roles in various cancers including leukemia,breast cancer and liver cancer.Modification of m6A played an important role in regulating gene expression.Evidence was emerging that m6A modification related regulatory proteins play critical roles in various cancers.In addition,the sequences around m6A sites were characterized as m6A motif（RRm6ACH,（[G/A/U][G>A]m6AC[U>A>C]））.The m6A motifs were highly conserved and were enriched in stop codons,3’UTR and m RNA coding region.However,the study on underlying regulatory mechanisms in lung cancers was limited.The association between m6A modifications related variants and the lung cancer susceptibility remained incompletely understood.Here,to support our hypothesis that genetic variants in the motifs may change the susceptibility of lung cancer,we systematically screened susceptibility loci in predicted m6A region by meta-analysis of multiple GWASs data.Then we validated that loci was related to m6A modification by using two public m6A database.After we analyzed fine-mapping and conducted functional annotation,we determined the function of its regulatory proteins by function experiments in cell line.The research process included:A total of 2,181,706 predicted m6A regions（RRACH,R:A/G,H:A/C/U）were selected in the study.After integrating with the SNPs from the 1000 Genomes Project（2015,phase3）,there were 249,465 SNPs left in above regions.Among them,12,025SNPs meeting the criteria described in the method after quality control and integration within all 4 GWAS databases（12,843 lung cancer cases and 12,639 controls）.We then removed 1,768 SNPs which showed apparent heterogeneity（i2>50%）.After Bonferroni-correction(Bonferroni P<4.16×10-6),4 SNPs（rs4246215（11q12.2）,rs10790248（11q23.3）,rs869638（11q23.3）and rs1805（11q23.3））were identified to be significantly associated with lung cancer.Linkage disequilibrium and conditional analyse showed that three SNPs（11q23.3）:rs10790248,rs869638 and rs1805 had high linkage disequilibrium with each other,which located in one independently linked signal.rs869638 could represent the independently linked signal.Rs4246215（11q12.2）showed low linkage disequilibrium with other 3 SNPs and located in other independently signal.Further,the subgroup analysis of Asian population versus European population showed that rs869638 in MPZL3(C>G:OR=1.10,95%CI=1.05–1.17,P=3.87×10-4)was more prominently associated with lung adenocarcinoma（LUAD）with no evidence for heterogeneity using the METAL fixed effects model.rs869638 could not reach genome-wide significance(P=1.98×10-1)for squamous cell carcinoma（LUSC）in NJMU and FLCCA date sets.Rs4246215 was prominently associated with LUAD(G>T:OR=0.9,95%CI=0.85–0.96,P=4.23×10-4)and LUSC(G>T:OR=0.86,95%CI=0.78–0.94,P=8.34×10-4).In addition,the rs869638(C>G:OR=1.09,95%CI=1.05-1.13,meta P=2.27×10-6),not rs4246215 was located next to an experiment-validated m6A site from Rmbase（RNA modification base）and m6Avar database reported by previous studies.Given that above results,we therefore considered that MPZL3 may be under the regulation of m6A mechanism by rs869638located on m6A regions.So the further study focused on rs869638.Then we conducted bioinformatics analysis and functional experiments in cell line.We firstly analyzed fine-mapping of rs869638 and refined an imputation region（500kb up and down stream of rs869638）using high-density genotyping in NJMU and FLCCA date sets.After correction(P=5×10–4),there were only one independent signal in this region.Secondly,In order to look for the potential causal SNPs traditionally,we conducted functional annotation for rs869638 and related SNPs（r2>0.8,in Asian or European population based on 1000 Genomes dataset）.As expected,all related SNPs were not annotated into promoter or enhancer regions of MPZL3.Secondly,we found that the risk G allele of rs869638 could decrease the m RNA expression of MPZL3 in GTEx V7 database(β=0.22,P=4.20×10–12).Confusingly,based on gene differential expression analysis of TCGA dataset,the expression of MPZL3 remarkably raised in adenocarcinoma tissues(P=8.28×10–3)compared with adjacent normal tissues,suggesting that m RNA expression of MPZL3 alone is unlikely to mediate this association.m6A-dependent regulatory mechanism may partly account for complex loci in 11q23.3 as the possible potential mechanisms.Lastly,CCK8 assays showed that knockdown of MPZL3 expression inhibited cell proliferation compared with the control cells.Similarly,the result of colony-formation assay revealed that clonogenic survival was decreased following knockdown of MPZL3.Then we knockdown three mainly“writers”（m6A methyltransferase,METTL3,METTL14 and WTAP）in A549 cells,interestingly,we found that knockdown of METTL14 could significantly induce the expression of MPZL3.Therefore,we think MPZL3 could be the susceptible gene for the association of the region,while m6A methylation might play a role.So,our results provided an alternative mechanism that the variant may modify the m6A sites nearby rs869638 and regulate the expression of MPZL3.In summary,we systematically evaluated the association between m6A related variants（SNPs in the region of predicted m6A region according to motif sequence）and lung cancer risk by meta-analysis of multiple GWASs data.In addition,the rs869638(C>G:OR=1.09,95%CI=1.05-1.13,meta P=2.27×10-6)in MPZL3 was located next to an experiment-validated m6A site from Rmbase and m6Avar database.Then we conducted bioinformatics analysis and functional experiments in cell line.We provide first evidence that susceptibility SNP of lung cancer（rs869638）at m6A motifs may alter the expression of MPZL3 by modifying the m6A methylation status.Further more,we present a role of this modification in regulating cancer susceptibility.The precious resources of GWAS and m6A sites will help advance understanding on the function of this epitranscriptomic modification in lung cancer. |
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