| Background:Gastric cancer remains a high disease burden in China.According to the latest statistics,gastric cancer in China's male incidence of cancer ranked second,second only to lung cancer;in women in third place,inferior only to lung cancer and breast cancer.Although the five-year survival rate is relatively high of the early gastric cancer after surgery,however,early gastric cancer is often lack of typical clinical symptoms,and majority of patients are diagnosed into the late stage,and lost the best treatment period.Thus,the mortality rate in the population is still high,and ranks the second cause of death in cancer.And with the increasing population base and the intensification of aging,the situation is getting worse.Elucidating the risk factors associated with the occurrence of gastric cancer and actively taking tertiary preventive control measures has important public health significance.Gastric cancer is a tumor that is closely related to the environment.Helicobacter pylori infection,pickled foods,nitrates,polycyclic aromatic compounds,tobacco and alcohol exposure have been shown to be associated with the occurence of gastric cancer.In recent years,more and more studies have shown that the susceptibility to gastric cancer between individuals with different genetic backgrounds is also significantly different in the face of the same external environment,indicating that genetic factors are important exposure factors affecting the occurrence of gastric cancer.This hereditary difference in the population is more manifested as single nucleotide polymorphisms(SNP).Genome-wide association study(GWAS)is an efficient strategy for screening genetic susceptibility loci and confirms that 3q13.31(rs9841504)and 5 p13.1(rs13361707)regions are susceptible regions of gastric cancer in Chinese population.However,most of the GWAS-positive loci are located in areas without biological function,and the genetic association is still difficult to explain.In addition,GWAS often has high statistical requirements(P<10-7),thus,false negative appearance is inevitable.Thus,based on the candidate gene research strategy to explore the susceptibility of gastric cancer,especially the key carcinogenic/tumor suppressor gene susceptibility still plays an important role.Tumor cell stem characteristics are shown to have the potential for self-renewal and are also used to explain many tumor phenotypes.Although the concept of cell stem of the tumor is still controversial,the important role of the Wnt pathway in normal and tumor stem cell function is generally recognized.In contrast to precursor cells,stem cells have higher proliferative capacity and prolong life,so that there is more chance of accumulating inheritance and epigenetic variation,leading to tumorigenesis.In tissues that use Wnt signaling to perform self-renewal and repair,the tumor is produced when the signal is abnormally active;this has been fully demonstrated in colorectal cancer.Although the mechanism of Wnt signaling in the pathogenesis of gastric cancer is not as comprehensive and thorough in colorectal cancer,the abnormal activation of this signal is prevalent in gastric cancer and is associated with tumor progression and metastasis.Like other important signaling pathways,the Wnt signal is regulated by a series of antagonistic and agonist factors to maintain a dynamic equilibrium.Dickkopfs(DKKs)and secreted frizzled-related proteins(sFRPs)are important extracellular inhibitory factors in the Wnt signaling pathway,which bind to signal receptor(LRP)and ligand(Wnt),down-regulate the Wnt signaling by blocking the formation of ligand-receptor complexes.The genetic variation,especially the potential functional regional variation and the risk of tumor occurrence has been concerned by more and more researchers.DKKs and sFRPs gene genetic variants(SNPs)were found to be significantly associated with tumor risk in kidney cancer,bladder cancer,and breast cancer.Athough the relationship of DKKs and SFRPs with the occurrence of gastric cancer is demonstrated in the epigenetic analysis,whether individuals who carry the corresponding risk genotype are more likely to have gastric cancer have not been reported.This study was to investigate the possible relationship between DKKs gene and sFRPl gene functional region polymorphism and gastric cancer risk and the possible biological effects of related genetic variation by case-control study.Meanwile,we compared DKKs and sFRPl mRNA expression level between cancer and paired normal tissue by RNA-seq data from The Cancer Genome Altas,and explore the effect of mRNA expression on patients's prognosis.It could provide more theoretical basis for gastric cancer mechanism research.Subjects and methods:This study was designed by hospital-based case-control study.The patients were diagnosed with primary gastric cancer by histopathology.The patients were from the Jiangsu Provincial Hospital of Traditional Chinese Medicine.The control group was also from the Jiangsu Provincial Hospital of Traditional Chinese Medicine,which excluded cancer,severe organic disease and severe digestive diseases.Collection time was from January 2008 to July 2012.The DKK(1-4)and sFRPl genes were used as candidate genes,and the latent functional regions(5' untranslated,exon and the 3'untranslated region)were screened using NCBI(https://www.ncbi.nlm.nih.gov/projects/SNP/)with the lowest allele frequency>10%in the Han Chinese in Beijing(HCB).For sites located in the 3 'untranslated region and having significant differences in the case and control,the online SNP functional analysis forecast site SNPinfo(http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm)was used to predict the potential binding miRNA with Gibbs free energy value greater than 20.Recombinant plasmid pGL3-promoter-SFPRl rs1127379 A>G,internal reference plasmid pRL-SV40 were transfected into human gastric cancer cell line MGC803 with miRNA mimics or negative control miRNA to detect the comparison of firefly/sea kidney fluorescence,verifying that whether rs1127379 site changes will affect the binding of miRNA.We based on the cancer genome database The Cancer Genome Altas(TCGA)(https://gdc-portal.nci.nih.gov/),extracted TCGA-STAD project RNA-seq data,and analyzed the the effect of DKKs and sFRPl on the occurrence and prognosis of gastric cancer combined with patient demographic information,clinical pathology.The t test and ?2 test were used to compare the demographic characteristics between the case group and the control group.The ?2 test was used to analyze whether the SNP was in Hardy-Weinberg equilibrium in the control population.The multivariate logistic regression model was used to calculate the age and sex adjusted OR and its 95%confidence interval.Bonferroni method was used to perform multiple comparison corrections in the group.The heterogeneity between subgroups was estimated by Q test based on x2 distribution.PHASE2.0 was used for hyplotype construction;Quanto software was used for statistical power calculation;TCGA data integration using Perl script.Data management and analysis was implemented by SPSS21.0(International Business Machines Corp.,IBM)and SAS 9.1.3(SAS Institute,Inc.,Cary,NC,USA)statistical software.Results:1?In this study,six SNPs were eligible in the DKKs gene:rs2241529(A>G),rs3733635(T>C),rs17037102(G>A),rs419764(C>T),rs3206824(G>A),and rs2073664(C>T).The six SNP loci were genotyped in the case group and the control group,and there was no significant difference between the two groups(two-sided ?2 test P>0.05).2.Two SNP loci rs1127379 and rs10088390 in 3' UTR were screened out in the sFRPl gene.(AA,AG,GG)of rs1127379 were 37.1%,46.1%and 16.8%in the control group;42.7%,46.3%and 11.0%in the case group;and distribution of the genotype between case and control group has significant differences(?2 = 7.652,P=0.022).After adjustment for age and sex,logistic regression analysis showed that the genotype of GG was significantly correlated with the reduced risk of gastric cancer(OR=0.53,95%CI:0.35-0.81)compared with AA genotype.Meanwhile,the recessive mode(GG vs AA/AG)also indicated the protective role of gastric cancer(OR = 0.55,95%CI:0.37-0.81).Based on our current sample size,statistical power was calculated by Quanto software,and we found the statistical power reached 91.66%to detect the OR=0.53,and 88.66%for OR=0.55.The correspondence parameters were G-allele frequency(0.4530)for rs1127379,the estimated gastric cancer prevalence(31.8/100000)in China,and recessive mode of inheritance.Thus,based on the high statistical power,we think the sample size was adequate for this analysis.3?We further evaluated the genetic effects of the sFRPl rs1127379 loci on gastric cancer cases according to the confirmed two histological types(intestinal and diffuse subtype),we found that the protective effect of the rs1127379 GG genotype remained significant in intestinal gastric cancer cases(adjusted OR=0.53,95%CI=0.34-0.84),but not for the diffuse type(P>0.05),while compared to the wild type AA genotype.When the gastric cancer cases were divided into gastric non-cardia and cardia cancer,according to the sites of tumor,we found that the protective effect was significant in the gastric non-cardia cases(adjusted OR=0.52,95%CI=0.33-0.82),but not for the gastric cardia cases(P>0.05).4?In the stratified analysis by age,gender,smoking and drinking status,the protective effect of the rs1127379 GG genotype remained significant in males(adjusted OR=0.46,95%CI=0.29-0.73),drinkers(adjusted OR=0.26,95%CI=0.08-0.88)and smokers(adjusted OR=0.21,95%CI=0.09-0.47)and both the youngers(adjusted OR=0.50,95%CI=0.26-0.94)and elders(adjusted OR=0.57,95%CI=0.35-0.94).In addition,we did not find any obvious effect for the rs10088390 on risk of GC in different strata.5?No significant association was detected between rs10088390 genotypes and risk of gastric cancer(adjusted OR=1.07,95%CI=0.81-1.43 for CG vs CC;adjusted OR=1.12,95%CI= 0.77-1.64 for GG vs CC,respectively).6?By using the PHASE 2.0 programs,we inferred four haplotypes on the observed genotype data(A rs1127379Crs10088390,A rs1127379Grs10088390,G rs1127379Grs10088390 and G rs1127379Crs10088390).A rs1127379Grs10088390 has shown the risk effect on the risk of GC as compared with the most common haplotype A rs1127379C rs10088390,OR was 2.54(1.76-3.67).7?miRNA-1182 was eligible for reseach based on the Gibbs free energy more than 20.Dual luciferase reporter gene assay showed that sFPRl rs1127379 A>G change could influence the regulation effect of miRNA-1182 on its target gene.8?According to transcriptome and clinical data of the cancergenome TCGA database,we found that the expression level of DKK2 mRNA in gastric cancer tissues was significantly higher than that in adjacent tissues(P=0.007).In contrast to DKK2,the expression level of DKK4 mRNA in gastric cancer tissues was significantly lower than that in adjacent tissues(P<0.0001).The expression of DKK1 and DKK3 mRNA was not significantly different between cancer tissues and adjacent tissues(P>0.05).However,the 5-year survival rate of DKK1 low expression group was significantly higher than that of high expression group(P=0.0086).Meanwhile,sFRPl mRNA was also lower in gastric cancer tissues than that in adjacent tissues(P<0.0001).however,the tumor-supression effect of sFRP1 was not supported by the seven paired tumor and adjacent tissues(P>0.05).Conclusions:1?The polymorphism of DKKs gene may not be associated with the risk of gastric cancer.2?sFRPl rs10088390(C>G)polymorphism has nothing to do with the incidence of gastric cancer.3?sFRPl rs1127379(A>G)was associated with intestinal carcinogenesis and noncardia gastric cancer risk,and A to G transition could influence the regulation of miRNA-1182 on its target gene.4?DKKs and sFRP1 play a certain biological role in the occurrence and development of gastric cancer.However,the potential tumor suppression effort of sFRP1was not confirmed in the seven-paired gastric cancer and adjacent tissues,indicating the race-heterogeneity of sFRP1. |
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