Association And Functional Analysis Of SNPs In Long Non-coding RNA NEAT1 With Gastric Cancer Suseptibility

Gastric cancer（GC）is still an important cancer that endangers human health worldwide.Its morbidity and mortality ranked fifth and third among the global malignant tumors in 2018,respectively.Among them,the age-standardized incidence of GC in Eastern Asia was the highest.Although the incidence and mortality of GC in China have declined in recent years,the actual number of cases and deaths of GC are still increasing due to the aging of the population.GC is a complex disease which is caused by various risk factors including environmental and genetic factors.In recent years,many studies have demonstrated that the abnormal expression of long noncoding RNA（lncRNA）may play an important role in the progress of GC carcinogenesis.The relationship between single nucleotide polymorphisms（SNPs）of lncRNA and genetic susceptibility of GC has attracted an increasing attention.Numerous studies have revealed that lncRNA NEAT1 showed abnormal expression in many tumors including GC,thus participating in the tumorigenesis,metastasis and prognosis of tumors.However,the relationship between SNPs of NEAT1 and the susceptibility of GC has not been studied.ObjectiveThe aims of our study were to investigate the relationships between four tagging SNPs（rs550894,rs3825071,rs580933,and rs7943779）of NEAT1 and GC susceptibility,and to conduct the possible mechanism for the significant SNPs in tumorigenesis of GC.Methods（1）Screening SNPs of NEAT1 by bioinformation tools With the combination of a variety of databases,including NCBI and Ensembl,the tagging SNPs of NEAT1 in CHB population were selected by Haploview 4.2 software.Then the selected tagging SNPs were preliminarily predicted by the website of lncRNASNP2 and RNAfold,and potential functional SNPs were screened out.（2）Genotyping experiment A frequency-matched case-control design was used,with the control and case groups matched by gender and age（±2 years）.The sample size was calculated by PASS 15.0 software according to the minimum allele frequency（MAF）and study efficacy.Finally,484 GC cases and 484 healthy controls were included.The polymerase chain reaction-restriction fragment-length polymorphism（PCR-RFLP）method was used to genotype for rs550894,rs3825071,and rs7943779;the created restriction site-PCR-PFLP（CRS-PCR-PFLP）method was used to genotype for rs580933.（3）Quantitative reverse transcription-polymerase chain reaction（qRT-PCR）The relative expression level of NEAT1 in the plasma of different genotypes of rs3825071 was detected by qRT-PCR experiment.（4）Statistical analysisThe data were analyzed by SPSS 21.0 software.The t test and Chi-square（χ²）test were used to evaluate the difference of basic characteristics between the cases and the controls.The Hardy-Weinberg equilibrium of genotype in control group was tested by the goodness-of-fitχ² test.The unconditional logistic regression model was performed to detect the relationship of SNPs with GC susceptibility.The online SHEsis was applied to test NEAT1 haplotype analysis and the software of MDR 2.0was used to evaluate the potential gene-environment factors interactions.Results（1）NEAT1 rs3825071 and rs7943779 were associated with the genetic susceptibility of gastric cancer.For rs3825071,the risk allele was A;AG（OR:1.74,95%CI:1.28-2.37）and AG+AA（OR:1.72,95%CI:1.27-2.32）genotypes had a higher risk of gastric cancer compared with the GG genotype.The risk allele of rs7943779 was A,AG（OR:1.63,95%CI:1.19-2.23）and AG+AA（OR:1.63,95%CI:1.19-2.22）genotypes were associated with higher risk of gastric cancer compared with the GG genotype.（2）Based on the dominant model,the results of stratified analysis showed that rs3825071 A and rs7943779 A allele had a higher risk of gastric cancer in all or part of the subgroups.However,rs550894 T allele had a higher risk of gastric cancer only in the non-smokers and non-drinkers.（3）The haplotype Grs7943779Grs580933Grs550894Grs3825071 showed the highest frequency in the case group（51.82%）and the control group（57.16%）,which was associated with lower risk of gastric cancer（OR:0.83,95%CI:0.69-0.99）.The haplotype Ars7943779Grs580933Grs550894Ars3825071 increased the risk of gastric cancer（OR:1.56,95%CI:1.15-2.11）.（4）With the increase of the number of mutated alleles in NEAT1,the risk of gastric cancer increased(P_trend=0.009).（5）The results of gene-environment interaction showed that the second-order interaction model including alcohol consumption and rs3825071 was the optimal model.The risk of gastric cancer in the population with drinking history and rs3825071 A allele was 2.10 times higher than that in the population without the two factors（OR:2.10;95%CI:1.63-2.72）.（6）The results of qRT-PCR showed that the relative expression level of NEAT1was higher in rs3825071 AG（1.42±0.69）and AA（1.70±0.65）genotype compared with GG（1.06±0.53）genotype.Conclusions（1）Both the genotypes of rs3825071 AG/AG+AA and the genotypes of rs7943779 AG/AG+AA could raise the risk of gastric cancer.（2）The haplotype Grs7943779Grs580933Grs550894Grs3825071 may have the lower risk of gastric cancer.The haplotype Ars7943779Grs580933Grs550894Ars3825071 may be related to increasing risk of gastric cancer.（3）The gene-environment interactions of drinking and rs3825071 could increase the risk of gastric cancer.（4）rs3825071 genotype was related to the relative expression level of NEAT1 in plasma.Carriers with rs3825071 risk allele A（AG+AA genotypes）has the higher expression level of NEAT1 compared with GG genotype.