The Role Of ADRB2 Gene In The Development And Clinicopathological Staging Of Lung Cancer

Background:Lung cancer is the main cause of cancer-related deaths,accounting for about one-fifth of cancer deaths worldwide.Lung cancer is a malignant tumor originating from the bronchial mucosa or glands.However,its pathogenesis is not fully understood yet.Smoking and genetic predisposition are common causes of lung cancer.Cigarette smoke contains more than 60 kinds of carcinogens,among which nitrosamine 4-(methyl nitrosamine)-1(3-pyridyl)-1 butanone(NNK)is one of the main carcinogens in cigarette smoke.NNK can act as an agonist for β2-adrenergic receptor and cause the occurrence of lung cancer.The β2-adrenergic receptor is a member of the G protein-coupled receptor superfamily,which is encoded by ADRB2 gene.Activation of β2-adrenergic receptor by β-agonists,such as epinephrine,norepinephrine and NNK,is related to the proliferation,migration,apoptosis,metastasis,angiogenesis,drug resistance,and poor prognosis of lung cancer.Expression of ADRB2 gene in lung cancer is still controversial.X.Wu et al found that ADRB2 was highly expressed in lung adenocarcinoma(LUAC)through analyzing two datasets in GEO(Gene Expression Omnibus database)database(GSE2514 and GSE7670),while Z.Q.Tian et al found that expression of ADRB2 was downregulated in non-small cell lung cancer(NSCLC).Lijing Wang et al also found that ADRB2 was down-regulated in lung squamous cell carcinoma(LUSC)by microarray analysis in GEO database.Expression of ADRB2 was down-regulated in other malignancies such as prostate cancer and breast cancer.Furthermore,expression of ADRB2 was correlated with the clinicopathological characteristics of malignant tumors including breast cancer,gastric cancer,melanoma,colorectal cancer and oral squamous cell carcinoma.At present,the expression of ADRB2 gene in lung cancer(including lung adenocarcinoma,squamous cell carcinoma,small cell lung cancer,large cell lung cancer and pulmonary sarcomatoid carcinoma)and the relationship between the expression of ADRB2 gene and the clinical pathological staging of lung cancer are not clear.Two common SNPs(single nucleotide polymorphisms)in the ADRB2 gene are rs1042713 and rs 1042714,which are located in the protein coding region.Changes in DNA sequences lead to missense variants,specifically the replacement of a glycine(Gly)residue with an arginine(Arg)residue at position 16 and replacement of glutamate(Gln)with glutamine(Glu)at position 27.These two SNPs exhibited functional relevance in vitro and are correlated with β-agonist response heterogeneity.Studies have confirmed that polymorphisms of rs 1042713 and rs1042714 are associated with disease risk of breast cancer and pancreatic carcinoma.However,there is little evidence showing the correlation of ADRB2 gene with disease risk and clinicopathological features in lung cancer.As the important single nucleotide polymorphism sites of ADRB2 gene that regulate the expression and function of β2AR,it is necessary to further study the roles of their polymorphisms in the development of lung cancer.Purpose:The purpose of this study was to investigate the expression level of ADRB2 and the relationship between ADRB2 expression and clinicopathological characteristics of lung cancer(including lung adenocarcinoma,squamous cell carcinoma,small cell lung cancer,large cell lung cancer and pulmonary sarcomatoid carcinoma),as well as the association between genetic polymorphisms of rs1042713 and rs 1042714 and lung cancer risk,Methods:This thesis includes two parts:①Tumoral RNA-seq data and clinical information was downloaded from The Cancer Genome Atlas(TCGA)database.Normal tissue samples were obtained in the Genotype-Tissue Expression(GTEx)database.Statistical analyses were performed using R software.The differentially expression of ADRB2 mRNA between lung cancer and normal tissues was tested by Wilcox test or Kruskal-Wallis test.Oncomine database was used to verify the differential expression of ADRB2 gene between the lung cancer group and control group.Correlation analysis between ADRB2 expression and clinicopathological characteristics of lung cancer was estimated by chi-square test,Spearman correlation analysis,Mann-Whitney test and Kruskal-Wallis test.Bonferroni correction was used for multiple comparisons.②A case-control study was conducted with 119 lung cancer patients and 97 controls.Clinical records and demographic characteristics were obtained,and peripheral venous blood were collected from all participants.The genomic DNA was extracted from the peripheral blood samples of all subjects.Genotyping of rs1042713 and rs1042714 polymorphisms were carried out by the TaqMan probe.SPSS 26.0 was used for 1:1 propensity score matching between lung cancer group and control group.All statistical analyses were conducted with SPSS 26.0.Demographic differences between cases and controls were evaluated using Student’s t-test or non-parameter testing for continuous variables and Chi-square or Fisher’s exact test for categorical variables.Unconditional logistic regression analysis was used to analyze the associations between each variant and corresponding haplotype with lung cancer risk.Analysis of linkage disequilibrium between rs1042713 and rs 1042714 and the construction of haplotype model were conducted on SHEsis online software.P<0.05 indicates statistical significance.Results:1 Expression of ADRB2 gene in lung cancer and association between ADRB2 expression and clinicopathological characteristics of lung cancer Data from TCGA and Oncomine platform revealed that the expression of ADRB2 mRNA was significantly downregulated in lung cancer(including lung adenocarcinoma,squamous cell carcinoma,small cell lung cancer,large cell lung cancer and pulmonary sarcomatoid carcinoma).Subgroup analysis revealed that the expression of ADRB2 was lower in male patients than female patients(p<0.05),and the ADRB2 expression was lower in smokers compared with nonsmokers(p<0.05).Expression of ADRB2 in lung cancer patients with T3 staging was significantly lower than in patients with T1 staging(Bonferroni correction.p=0.002,α/6=0.008).ADRB2 expression in medium and advanced lung cancer(stage Ⅲ and Ⅳ)was also lower than in early stage lung cancer(stage Ⅰ and Ⅱ)(p=0.003).AJCC pTNM staging and pT staging were negatively correlated with ADRB2 expression(p<0.05).2 The role of ADRB2 gene polymorphisms in the development of lung cancer The AG genotype of rs1042713 was related to increased risk of lung cancer in smokers compared with GG genotype(OR=3.103,95%CI:1.162-8.289).As for rs1042714,the G allele was a risk factor for lung cancer(OR=2.297,95%CI:1.046-5.044).GC genotype of rs1042714 had a significantly higher risk of lung cancer(OR=2.777,95%CI:1.139-6.773),especially in smokers(OR=3.967,95%CI:1.051-14.970).Haplotype analysis revealed that the G-G haplotype was associated with a significant increased risk of lung cancer(OR=2.986,95%CI:1.122-7.947).Conclusion:ADRB2 expression is downregulated in lung cancer and the expression of ADRB2 is negatively correlated with pT staging and AJCC pTNM staging.Polymorphisms of rs1042713 and rs 1042714 are associated with lung cancer risk.AG genotype of rs1042713 is associated with increased risk of lung cancer in smokers.As for rs 1042714,G allele is the risk allele for lung cancer,and GC genotype has a significantly higher risk of lung cancer.In this study,we clarified the expression of ADRB2 gene in lung cancer and the association between the expression level of ADRB2 gene and the clinical pathological staging of lung cancer.Also,we analyzed the relationship between the polymorphisms of rs 1042713 and rs1042714 and the risk of lung cancer,which provides a new idea for further study the role of ADRB2 gene in regulating the development and progression of lung cancer.