The Selection And Functional Characterization Of Long Non-coding RNAs And Functional SNPs Associated With Gastric Cancer Susceptibility Based On Bioinformation

The incidence and mortality of gastric cancer has been both ranking second in the number of the cases and deaths of various cancers across China,with both the early diagnosis rate and 5-year survival rate in advanced gastric cancer less than 20%.Furthermore,the incidence of gastric cancer tends to be increasing and the age of onset tends to be younger.Long non-coding RNAs(lncRNAs)are a class of RNA molecules with more than 200 bases that function as RNAs with little or no protein-coding capacity.Generally,lncRNAs regulate gene expression at three levels: epigenetics,transcriptional regulation and post-transcriptional regulation,widely involving in the physiological and pathological processes.Accumulating studies have demonstrated that lncRNAs may play an active role in tumorigenesis,metastasis,prognosis and drug resistance of gastric cancer.LncRNAs are expected to be the biomarkers for diagnosis and prognosis of gastric cancer.But the underlying mechanisms remain to be clarified.ObjectiveTo screen the gastric cancer–associated lncRNAs and their functional single nucleotide polymorphisms(SNPs)based on bioinformation and validate the relationship between the candidate SNPs and gastric cancer susceptibility,then extend to investigate the potential function of the lncRNAs and their SNPs.Methods1.Screening by bioinformation toolsSearch in the GEO database and download the microarray data of gastric cancer(GSE50710,GSE53137,GSE58828)based on Chinese population.We managed to integrate several databases(Ensembl,refseq,lncRNAdb.v2.0,GENCODE.v24,NONCODE2016,LNCIpedia.v3.1,CCDS.v18),remove the redundancy and re-annotate the high-throughput microarrays by local BLASTN algorithm.Eventually,we selected the differentially expressed lncRNAs between gastric cancers and the normal by bioinformation methods and predicted the potential functions of the candidate lncRNAs.Those lncRNAs with SNPs having possible binding sites with miRNAs would be validated in large population.2.Validation of the association in the large populationBased on the case-control design,we determined the number of the sample through PASS 8.0.14(http://www.ncss.com/pass.html).Around 470 gastric cancers finally diagnosed by histopathological examination and 470 healthy controls from communities individual-matching with cases by age(±3 years)and sex by SAS 9.1 were involved in the study.Polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP)was used to determine the genotypes of SNPs in lncRNAs genes.The statistical analyses were conducted by SAS 9.1(SAS Institute Inc.,Cary,North Carolina,USA).Hardy-Weinberg equilibrium in the control group was tested by Hardy-Weinberg online tool.MDR2.0 was used to analyze the gene-environment interaction.3.Primary research into the biological fuctionQuantitative reverse transcription-polymerase chain reaction(qRT-PCR)was adopted to measure the relative expression of lnc RNAs in the plasma of 41 samples with different mutation of SNPs.The luciferase assays were used to determine whether the mutation of the SNPs could have impact on the binding between the lncRNAs and the predicted corresponding miRNAs.Results1.We managed to construct the protein-coding database including 120,018 reads and long non-coding RNA database involving 206,510 reads.189 differentially expressed lncRNAs were found between gastric cancers and the normal and 4 lncRNAs with SNPs having possible binding sites with miRNAs were selected to get validated in large population,including lnc-EVX1-3:3(rs1859168),lnc-MACC1-1:7(rs3815254),lnc-AMFR-1:1(rs4784659)and lnc-ZNF33B-2:1(rs579501).2.No statistical differences were found in the distribution of age,gender and drinking between case and control groups(P>0.05).The percentages of the smoker and individuals with family history of tumors in the case group were significantly higher than those in the control group(P<0.05).When adjusted for age,gender,smoking,alcohol drinking and family history of tumor,the unconditional multiple logistic regression based on the dominant(OR=0.64,95%CI: 0.47-0.86)and recessive genetic model(OR=1.77,95%CI: 1.34-2.35)consistently showed rs1859168 A was significantly associated with lower risk of gastric cancer.The dominant(OR=0.42,95%CI: 0.31-0.57)and additive(OR=0.52,95%CI: 0.40-0.67)genetic model also revealed that rs4784659 T could lower the risk of gastric cancer.Similarly,the dominant(OR=0.72,95%CI: 0.52-0.98)and additive(OR=0.73,95%CI: 0.56-0.97)genetic model also showed that the individuals with rs579501 C had lower risk of gastric cancer.Whereas no statistical association between rs3815254(C>T)and gastric cancer were consistently found in the dominant,codominant,recessive and additive genetic models(all P>0.05).3.After stratified analyses based on dominant model by age(<50 vs.≥50),gender(men vs.women),smoking(yes vs.no),alcohol drinking(yes vs.no),family history of cancer(yes vs.no),and adjusted for other factors,rs1859168 A and rs579501 G were not associated with decreasing risk of gastric cancer in parts of the subgroups.Notably,rs4784659 T consistently confered the potential to reduce gastric cancer risk in all subgroups above.The potential protective effect from gastric cancer in non-smokers(OR=0.23,95%CI: 0.14,0.38)was more significant than that in smokers(OR=0.65,95%CI: 0.43,0.98)(P<0.001).4.The optimal gene-environment interactive model revealed that the smokers with family history of tumor and rs4784659 CC and rs1859168 CC/AC and rs579501 AA have 5.03 times higher risk of gastric cancer over the individuals without combinations above(OR: 5.03;95%CI: 3.81-6.65).5.Luciferase reporter assays demonstrated that the luciferase activity of the construct with the risk rs579501 A allele decreased when transfected with miR-5002-5p mimics,but increased when transfected with miR-5002-5p inhibitor.6.As predicted by bioinformation methods,rs4784659 G>A mutation gained a binding site for hsa-miR-6780a-3p and lnc-AMFR-1:1,which might reduce the expression and effect of lnc-AMFR-1:1 and increase the expression of genes targeted by hsa-miR-6780a-3p.7.As shown by qRT-PCR,the expression of lnc-AMFR-1:1in the plasma of individuals with rs4784659 AG(0.52±0.33,n=14)and rs4784659 AA(0.21±0.14,n=9)were both lower(both P<0.0167)than those with rs4784659 GG(0.87±0.34,n=18).Conclusion1.rs4784659 T、rs579501 C and rs1859168 A may potentially reduce the susceptibility of gastric cancer.2.The functional rs579501 A mutation proved to promote the binding between hsa-miR-5002-5p and lnc-ZNF33B-2:1,and further inhibite the expression of lnc-ZNF33B-2:1.Whether the functional rs4784659 G>A mutation influenced the binding between hsa-miR-6780a-3p and lnc-AMFR-1:1 remained to be further clarified through biological experiments.3.The study provided potential biomarkers for the early diagnosis and therapy of gastric cancer and gave important clues for further studying the function and mechanism of lncRNAs in the tumorgenesis of gastric cancer.