Functional Analysis And Association Study Of SNPs Within LncRNA-TUSC7 SNPs And Gastric Cancer Suseptibility

The incidence of gastric cancer?GC?among all cancers ranked fourth in male and fifth in female,which was high,especially in Korea,Japan and China.Although both of the morbidity and mortality of GC in China have declined in recent years,they still occupied top of the malignant tumors in digestive system.The increasing studies showed that long non-coding RNAs?lncRNAs?is related to the development,metastasis,prognosis,and drug resistance in GC.Among all the lncRNAs,tumor suppressor candidate gene 7?TUSC7?played an important role in the pogreesion of colorectal cancer,acute myeloid leukemia,glioma,gastric cancer and so on.However,the relationship between single nucleotide polymorphisms?SNPs?of TUSC7 and the susceptibility of gastric cancer and its underlying mechanism of TUSC7 have not been studied.ObjectivesThe aims of this study were to assess the association between three tagSNPs?rs12494960 rs2867837 rs1518338?of lncRNA-TUSC7 and GC susceptibility,then explored the preliminary function of lncRNA-TUSC7.Methods?1?Combined with a variety of database,the relevent literature of lncRNA about gastric cancer at home and abroad,the bioinformatics method for screening differentially expressed lncRNAs associated with gastric cancer,we selected the tagger SNPs?rs1518338,rs2867837 and rs12494960?by the software of haploview and predicted the function of these tagger SNPs by the website of http://bioinfo.life.hust.edu.cn/lncRNASNP/.?2?In this case-control study,according to the minimum allele frequency?0.01?and the research effect?0.9?,we use the software of PASS 8.0.14?http://www.ncss.com/pass.html?to calculate the minimum sample size.Finally,there were a total of 470 gastric cancer patients and 470 frequency matched cancer-free controls.rs2867837 and rs12494960 used the polymerase chain reaction-restriction fragment-length polymorphism?PCR-RFLP?methods to genotype and rs1518338used the created restriction fragment-length polymorphism?CRS-PFLP?to genotype.The Hardy-weinberg balance online tool was used to test the balance of the genotype results in the control group.The unconditional logistic regression model was used to analyze the association between different genotypes and the risk of gastric cancer by SPSS 21.0?SPSS Inc.,Shanghai,Co.,LTD,6761805c6989326cbf14,Shanghai,China?.The further analysis of the interaction between gene and environment used the software of MDR2.0.An online analysis software of SHEsis was used to analyze the haplotype of TUSC7.?3?qRT-PCR experiment was used to detect the expression level of lncRNA TUSC7 in the plasma of samples with different genotypes.Whether the genetic variationin the SNP loci affects the binding ablity of TUSC7 to its targeted miR-133a-3p was tested by the dual-luciferase reporter gene assay.Results?1?For rs12494960,AA?OR:1.16,95%CI:1.16,2.70?and CA+AA?O R:1.96,95%CI:1.47,2.59?genotypes were likely to increase the risk of gas tric cancer;for rs2867837,AG?OR:2.24,95%CI:1.12,4.47?and AG+GG?OR:1.51,95%CI:1.14,2.01?genotypes might increase the risk of gastric cancer.T he frequency of haplotype G_rs1518338A_rs2867837C_rs12494960?OR:0.505,95%CI:0.407,0.627?and C_rs1518338A_rs2867837A_rs12494960?OR:0.415,95%CI:0.253,0.679?were highe r in the control group than that in the case groups.Besides,the frequency of C_rs1518338A_rs2867837C_rs12494960?OR:2.290,95%CI:1.599,3.279?and G_rs1518338A_rs2867837A_rs12494960?OR:4.251,95%CI:2.841,6.361?were lower in the controls than that in cases.?2?The stratified analysis showed that based on the dominant model,respectively stratified by age?<50 and?50?,men or women,smoking?yes or no?,alcohol consumption?yes or no?and the family history of gastric cancer?yes or no?,after adjusting for other confounding factors,rs12494960A increased the risk of gastric cancer in all subgroups above.However,the rs1518338C was more likely to show the potentially risk effect gastric cancer in the positive family history than the nagetive family history?OR:3.71,95%CI:1.11,12.35?.?3?The interaction of gene-environment factor analysis showed that the smokers with rs12494960A may carry 2.34 times of GC risk than people without these two characteristics?OR:2.34,95%CI:1.79,3.13?;individuals carrying rs12494960A,rs2867837C,smoking,positive family history and alcohol consumption factors are likely to 3.57 times higher risk of gastric cancer than those free of these 5characteristics.?4?The dual-luciferase reporter gene assay showed that there was interaction between TUSC7 and miR-133a-3p,but the mutation of rs12494960C allele to A allele has not yet been found to change the activity between miR-133a-3p and TUSC7.?5?Results from the qRT-PCR showed that compared with rs12494960CC group?2.37±0.15?,the relative expression level of TUSC7 was significantly lower in rs12494960AC?1.70±0.61,n=23?and rs12494960AA?1.18±0.19,n=15?group?both P<0.05?.Conclusion?1?Both the genotypes of rs12494960 AC/AC+CC and the genotypes of rs2867837 AG/AG+GG may increase the risk of gastric cancer.?2?There existed binding site between TUSC7 and miR-133a-3p,but whether the variation of rs12494960 C to A leads to change the binding capacity remained to be further studied.?3?The plasmic expression level of TUSC7 was significantly decreased in the rs12494960 AA/CA genotype.