Study On Association And Mechanism Between Genetic Variants Of Cell Cycle Pathway Genes And The Risk Of Head And Neck Squamous Cell Carcinoma

Objective:Head and Neck Squamous Cell Carcinoma（HNSCC）has a concealed onset,most HNSCC cases are hard to be diagnosed at early stage and have poor prognosis.Cell cycle dysregulation is one of the hallmarks of cancer,and common genetic alterations in cell cycle pathway in HNSCC have been reported.However,there is a lack of comprehensive exploration of associations between genetic variants of cell cycle pathway and HNSCC,especially in the Chinese population.Therefore,this study aims to identify the genetic variants of cell cycle pathway genes associated with susceptibility of HNSCC in the Chinese population,and explore the molecular mechanism of genetic variants in HNSCC using bioinformatics and cell experiments,thus,providing new clues to the etiology of HNSCC.Methods:1.A two-stage matched case-control study was conducted in this study.In the discovery phase,248 pairs of 1:1 matched case-controls were recruited in Nanjing.In the replication phase,431 pairs of 1:1 matched case-controls were recruited in Wuhan.Epidemiological information and 5 m L peripheral venous blood of each participant were collected.The candidate genes and potential functional SNPs of cell cycle pathway were preliminary screened by consulting literatures and applying bioinformatics databases,and genotyping was performed using SNPscan^TM.Paired t test and Mc Nemar’s chi2 test were used to compare the differences in demographic characteristics and genotype distribution between cases and controls.Multivariate conditional logistic regression was used to analyze the association between genetic variants and the risk of HNSCC,as well as the interactions of gene-gene and gene-smoking on the risk of HNSCC,FDR correction was performed for multiple tests.Multivariate ordered logistic regression was used to analyze the association between genetic variants and tumor pathological differentiation,multivariate unconditional logistic regression was used to analyze the relationship between genetic variants and serum tumor markers.2.A variety of bioinformatics tools were utilized to annotate the function of genetic variants associated with HNSCC risk.RNAfold was adopted to evaluate the influence of SNP on the secondary structure of mRNA 3’-UTR.Then,Mir SNP,PolymiRTS and miRNASNP databases were used to predict the effects of SNP on miRNA-mRNA binding,which was further verified by dual luciferase reporter assay.Besides,TCGA and Kaplan-Meier Plotter databases were used for differential expression analysis and survival analysis.3.In vitro cell assays were performed to investigate the biological functions of miR-940.METTL3 and METTL1 were knocked down and the expression of mature miR-940 was measured to explore the effect of m⁶A and m⁷G on miR-940 expression.E2F2 overexpressed plasmids containing rs2075993 different alleles were co-transfected with miR-940,respectively,to further explore the molecular mechanism of rs2075993 in HNSCC.Result:1.Two-stage association study showed that CDKN1C rs452338 was significantly associated with the risk of HNSCC（dominant model:OR=0.61,95%CI=0.46-0.80;overdominant model:OR=1.57,95%CI=1.21-2.11;additive model:OR=0.63,95%CI=0.49-0.81）.Genetic variations of CDK4 rs2072052 increased the risk of HNSCC（additive model:OR=1.21,95%CI=1.02-1.42）.Genetic variations of E2F2rs3820028 increased the risk of HNSCC（dominant model:OR=1.88,95%CI=1.39-2.55;recessive model:OR=1.67,95%CI=1.20-2.34;additive model:OR=1.66,95%CI=1.34-2.05）.E2F2 rs2075993 was also found to be associated with the risk of HNSCC（dominant model:OR=1.83,95%CI=1.35-2.48;recessive model:OR=1.66,95%CI=1.17-2.35;additive model:OR=1.63,95%CI=1.31-2.03）.Combined effect analysis revealed that,compared to individuals without risk genotypes of associated SNPs,individuals carrying one risk genotype had a 0.94-fold increased risk of HNSCC（OR=1.94,95%CI=1.17-3.22）,and individuals with two risk genotypes had a 1.27-fold increased risk of HNSCC（OR=2.27,95%CI=1.32-3.90）,and individuals with 3-4 risk genotypes had a 2.87-fold increased risk of HNSCC（OR=3.87,95%CI=2.32-6.45）.As the number of risk genotypes increased,the risk of HNSCC displayed an increasing trend(P_trend<0.001).The analysis of gene-gene interaction showed that there were multiplicative interactions between rs452338 and rs2072052,rs2072052 and rs3820028,rs2072052 and rs2075993,and analysis of gene-smoking interaction showed that there was positive additive interaction between rs452338 and smoking.Moreover,genetic variations of rs3820028 and rs2075993 were associated with pathological differentiation of laryngeal squamous cell carcinoma（rs3820028 AG vs.AA:OR=3.12,95%CI=1.22-7.97;overdominant model:OR=0.36,95%CI=0.16-0.80）（rs2075993 GA vs.GG:OR=3.22,95%CI=1.27-8.21;overdominant model:OR=0.32,95%CI=0.14-0.73）.Genetic variations of rs452338 was associated with aberrant level of ferritin（GT vs.GG:OR=4.66,95%CI=1.09-20.00）,and rs2072052 was associated with aberrant level of neuron-specific enolase（overdominant model:OR=0.40,95%CI=0.16-0.98）.2.Function annotation revealed that E2F2 rs2075993 might be the most likely functional SNP and was predicted to be associated with miRNA binding.Rs2075993T>C increased the minimum free energy of E2F2 3’-UTR secondary structure,and through database prediction and dual luciferase reporter assay,rs2075993 T>C was found to disrupt the binding of miR-940 to E2F2.Furthermore,miR-940 was up-regulated in HNSCC tumor tissues（P=2.9e-8）,and higher expression of miR-940 was correlated with poor overall survival（HR=1.39,95%CI=1.02-1.90）,while higher expression of E2F2 was correlated with favorable overall survival（HR=0.64,95%CI=0.48-0.85）.3.The overexpression of miR-940 promoted the proliferation,migration and invasion,and inhibited the senescence and autophagy of tumor cells.After knockdown of the expression of METTL3,the expression of mature miR-940 was decreased,suggesting that m⁶A might affect the expression of miR-940.In addition,miR-940regulated the expression of E2F2,however,this regulation was affected by rs2075993,rs2075993 T>C disrupted the translation inhibition of E2F2 by miR-940.Moreover,compared with E2F2 rs2075993 C allele,when E2F2 rs2075993 T allele was co-transfected with miR-940,cell proliferation,migration and invasion were increased while cell senescence was decreased,whereas no effect on autophagy was observed.Conclusions:CDKN1C rs452338,CDK4 rs2072052,E2F2 rs3820028 and E2F2 rs2075993were associated with the risk of HNSCC,and there was a combined effect among these four SNPs.Genetic variants rs452338,rs3820028 and rs2075993 were interactive with rs2072052 to modify HNSCC susceptibility,and genetic variant rs452338 was synergistic with smoking to influence HNSCC susceptibility.Besides,genetic variants rs3820028 and rs2075993 were associated with pathological differentiation of laryngeal squamous cell carcinoma,rs452338 and rs2072052 were associated with aberrant levels of ferritin and neuron-specific enolase,respectively.In terms of mechanism,rs2075993T>C reduced the stability of E2F2 3’-UTR secondary structure and destroyed the binding of miR-940 to E2F2,rs2075993 T>C further interfered the translation inhibition of E2F2 by miR-940,which changed the interaction between miR-940 and E2F2,thus affecting the proliferation,migration,invasion and cell senescence of tumor cells.Moreover,miR-940 could be as a potential biomarker of HNSCC.Mi R-940 was up-regulated in HNSCC and was associated with poor prognosis of HNSCC,and its overexpression promoted the proliferation,migration and invasion of HNSCC tumor cells,and inhibited cell senescence and autophagy,while m⁶A might affect the expression of miR-940.