| Objective: 1.To explore the association between the rs10224902,rs197367,rs3735400 and rs3815484 polymorphisms of the actin-binding protein(ANLN)gene and the gene-environment interaction with bladder cancer susceptibility;2.To explore the diagnostic and prognostic value of ANLN in bladder cancer and its relationship with immune infiltration and drug sensitivity by bioinformatics methods.Methods: 1.A case control study with 1:1matching was used to collect patients with newly diagnosed bladder cancer from January2018 to December 2022 at the Department of Urology of the People’s Hospital of Inner Mongolia Autonomous Region and the Affiliated Hospital of Inner Mongolia Medical University as the case group,and a healthy population with physical examinations at the two tertiary hospitals during the same period as the control group,and the two groups were matched according to the same sex and age ± 5 years.Basic information about the study subjects was collected through questionnaires and pathological findings and blood specimens were collected from the study subjects.The ANLN gene rs10224902,rs197367,rs3735400,rs3815484 locus polymorphisms were detected using the multiplex high-temperature ligase detection reaction(im LDR)technique.Multivariate conditional logistic regression analysis was used to calculate the Odds Ratios(ORs)and corresponding 95% confidence intervals(CIs)for the association of diet and lifestyle habits with the risk of bladder cancer;the association between the five genetic models and bladder cancer was analyzed using SNPstats online software;the generalized multifactor dimensionality reduction(GMDR)method was used for the analysis of gene-environment interactions.2.Gene expression data and clinical data for bladder cancer were downloaded from The Cancer Genome Atlas(TCGA)database.The relationship between ANLN expression and clinicopathological features was assessed using the Wilcoxon rank sum test.Receiver Operating Characteristic(ROC)curves were used to assess the diagnostic value of ANLN expression in bladder cancer.Kaplan-Meier and Cox regression analyses were used to investigate the effect of ANLN expression on overall survival.Gene Set Enrichment Analysis(GSEA)was used to identify signaling pathways associated with ANLN in bladder cancer.The relationship between ANLN expression and immune infiltration was analyzed using the Tumor Immune Estimation Resource(TIMER)database and the R "reshape2" package.The relationship between ANLN and drug sensitivity was calculated using the Cell Miner database.ANLN protein expression in serum was measured by enzyme-linked immunosorbent assay(ELISA).Results:(1)Univariate analysis showed that compared to the control group,the case group had statistically significant differences in pesticide exposure,factory work,education level,smoking,labor intensity,urine holding habits,fuel for heating and cooking,salty diet,fruit intake,meat and vegetable diet,type of cooking oil,and type of drinking water(P < 0.05).(2)Multivariate conditional logistic regression results showed that high education was a protective factor for bladder cancer(OR=0.581)and that a history of pesticide exposure,history of factory work,smoking,a diet based on meat,and a diet based on animal oils were risk factors for bladder cancer(OR 2.323,2.639,2.246,1.762,2.488 respectively).(3)The differences in the distribution of genotype frequencies at the rs10224902 and rs3735400 loci of the ANLN gene were statistically significant in the two populations(P < 0.05).rs10224902 loci recessive model was statistically significant(OR = 0.35)and the optimal genetic model;rs3735400 loci co-dominant and recessive models were statistically significant(OR = 0.29,0.28 respectively)and recessive model was the optimal genetic model.(4)The differences in allele frequency and genotype frequency at the rs197367 locus of the ANLN gene were statistically significant in T-staging(P < 0.05)and the differences in genotype frequency at the rs3815484 locus were statistically significant in pathological grading(P < 0.05).(5)The GMDR results showed a potential interaction between literacy,factory work history,smoking and dietary meat and vegetables in the risk of bladder cancer.(6)The results based on the TCGA database showed that ANLN m RNA expression was significantly upregulated in bladder cancer tissues,and the difference in expression was statistically significant in the clinical stage and histological grading of bladder cancer(P < 0.05);ROC analysis showed that ANLN has a high diagnostic value in bladder cancer.(7)Kaplan-Meier survival curve analysis showed that high ANLN expression was significantly associated with poor prognosis;multivariate Cox regression analysis confirmed that high ANLN expression was an independent risk factor for overall survival in bladder cancer patients.(8)GSEA analysis showed that ANLN was associated with multiple signaling pathways;immunologically,ANLN was closely associated with immune infiltration and immune checkpoint molecules in bladder cancer;and ANLN m RNA expression was negatively correlated with the sensitivity of most antitumor drugs.(9)ANLN protein expression in the serum of bladder cancer patients was significantly higher than that of the control group(P < 0.05).Conclusion:(1)Pesticide exposure,factory work,smoking,eating habits based on meat and cooking oil based on animal oil were risk factors for bladder cancer.CC genotype at rs10224902,GG genotype at rs3735400 and education level were protective factors for bladder cancer.(2)The genotype frequency of rs197367 and rs3815484 had statistically significant differences in T staging and pathological grading,and the allele frequency of rs197367 had statistically significant differences in T staging.(3)There is an interaction between literacy,history of factory work,smoking and dietary meat and vegetables in the development of bladder cancer.(4)ANLN m RNA expression was upregulated in bladder cancer tissues and had high diagnostic value;and high expression was associated with poorer overall survival in bladder cancer patients and was an independent risk factor for overall survival in bladder cancer patients.(5)ANLN regulates multiple signaling pathways in bladder cancer and is closely associated with immune infiltration and immune checkpoint molecules.(6)ANLN m RNA expression was negatively correlated with the sensitivity of most antitumor drugs.(7)ANLN protein expression in the serum of bladder cancer patients was higher than that of the control population. |
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