| Objective:Genome-wide association studies(GWAS)were used to verify single nucleotide polymorphisms(single nucleotide polymorphisms,SNPs)rs884309 and rs1464938 in the Han population of Guangxi Zhuang Autonomous Region that may be associated with the onset of bladder transitional cell carcinoma,and explore at the same time Its correlation with pathological grade and clinical stage of bladder cancer.Methods:(1)Pathological tissues were collected from the Affiliated Hospital of Youjiang Medical College for Nationalities and the Northwest Affiliated Hospital,and excluded those who self-reported a family history of cancer(including a family history of non-melanoma skin cancer)and received chemotherapy or radiotherapy before surgery,of which 331 The patient was pathologically confirmed as a transitional cell carcinoma of the bladder.In addition,516 healthy volunteers who came to the Affiliated Hospital of Youjiang Medical College for Nationalities and the Northwest Affiliated Hospital of the Youjiang National Medical College and Northwest Affiliated Hospital did not suffer from tumors at the same time,and their age and gender were compared with the case group.Cases and controls The average age of the group was 68.5±9.2 years and 62.5±8.2 years.The ratio of male to female in the case and control group is about 4:1,and all subjects are non-relative Han people in Guangxi Zhuang Autonomous Region.4ml of venous blood was drawn and whole genome DNA was extracted,and the 3 SNPs were genotyped by the ABI 7500 Fast Real-Time PCR system using Taq Man assay(Applied Biosystems,Carlsbad,CA,USA).About 5% of samples were randomly selected for Sanger sequencing to verify its accuracy.According to the manufacturer’s manual,q PCR was performed using SYBR Green Master Mix(Qiagen),and FGF12 m RNA was quantified by GAPDH 2-ΔC standardization.Amplify the promoter region of FGF12,which contains rs1464938 A or rs1464938 G.After digestion with Nhe I and Hind III,insert the fragment into a p GL3-based vector(Promega,Madison,WI),and verify the position of the insert in the plasmid by Sanger sequencing and completeness.1μg p GL3-rs1464938 A or p GL3-rs1464938 G plasmid(Life Technologies)was transfected with Lipofectin 2000.The kidney grass luciferase vector p RL-SV40(50 ng)was used as an internal control(Promega)for co-transfection.The promoter activity of p GL3-rs1464938 A and p GL3-rs1464938 G was detected 48 h after transfection.(2)The student’s t test for continuous variables and the χ2 test for categorical variables were used to compare the differences in demographic characteristics between the cases and the control group.The goodness of fit χ2 test was used to evaluate the Hardy-Weinberg balance(HWE),genotype and allele frequency of each SNP.By using a logistic regression model adjusted for age and gender to calculate odds ratios(ORs)and 95%confidence intervals(CIs),the relationship between the three SNPs and the risk of TCC was estimated.In multiple comparisons,a Bonferroni correction was performed for the α level of0.0125(0.05/4).SHEsis online software(http://analysis.bio-x.cn/my Analysis.php)was used for haplotype analysis.The difference of luciferase activity in different plasmids was checked by t test.All tests used SPSS 11.0 software,and the test was defined as P <0.05 as the difference was statistically significant.Results:(1)Statistical analysis showed that the average age of the case group was significantly higher than that of the control group(P <0.001),indicating that age is a risk factor for bladder cancer.In terms of gender differences,there was no significant difference between the case group and the control group(P = 0.15).According to Hardy-Weinberg equilibrium law,the distribution frequencies of rs884309 and rs1464938 genotypes in the control group(P > 0.05)are well representative.(2)TCC patients rs884309 determine the adjusted ORs and 95% CIs to assess the association between rs884309 and TCC risk.These comparisons showed a slight increase in OR(GT vs GG: adjusted OR = 1.14,95% CI,0.82-1.57,P > 0.05;TT vs.GG: adjusted OR = 1.20,95% CI,0.77-1.87,P > 0.05;T vs.G:adjusted OR = 1.10,95% CI,0.89-1.36,P > 0.05).TCC patients rs710521 determined adjusted ORs and 95% CIs to assess the association between rs884309 and TCC risk.These comparisons showed a slight decrease in the visible OR(AG vs AA: adjusted OR=0.69,95%CI,0.43-1.10,P > 0.05;GGvs.AA: adjusted OR = 0.80,95% CI,0.33-1.93,P > 0.05;Gvs.A:Adjusted OR = 0.81,95% CI,0.56-1.16,P > 0.05).(3)The frequency of AA genotype and A allele of rs1464938 in TCC patients was significantly higher than that in the control group.Determine the adjusted ORs and 95% CIs to assess the association between rs1464938 and TCC risk.These comparisons showed a significant increase in OR(AA vs.GG: adjusted OR= 2.54,95% CI,1.49-4.35,P <0.001;AA vs.AG/GG: adjusted OR = 2.25,95% CI,1.36-3.71,P = 0.002;A vs.G: adjusted OR = 1.44,95% CI,1.15-1.80,P = 0.001).(4)Four common haplotypes were detected: rs884309Grs1464938 G,TG,GA,TA.The frequency of GA haplotypes in TCC patients was significantly higher than that in the control group.The correlation between haplotype and TCC risk was assessed by measuring ORs and 95% CIs.The comparison showed a significant increase in OR(GA vs GG: OR = 1.61,95% CI,1.23-2.11,P = 0.001).Conclusion:(1)SNP rs1464938 is significantly related to the risk of TCC in the Han population in Guangxi,but has no significant correlation with the classification and staging of TCC cases.(2)SNP rs884309 and rs710521 have no obvious correlation with the risk of TCC incidence,TCC case classification and staging in Guangxi Han population.(3)This study found that the genotype frequency of GA haplotypes in TCC patients was significantly higher than that in the control group,so GA haplotypes were a risk factor for TCC patients.(4)This study found that the rs1464938 AG/AA genotype showed higher levels of FGF12 m RNA by increasing the activity of the FGF12 promoter.This may enhance the cytokine effect of FGF12,thereby promoting tumor growth. |
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