Correlation Of The Variant Of SNPs At MicroRNA Complementary Site In The PD-L1 3’-Untranslated Region To Non-small Cell Lung Cancer

Objective: Lung cancer is one of the most common malignant tumor which threatens the health of human beings around the world.Non-small cell lung cancer(NSCLC)accounts for 85% of all cases.It is well recognized that genetic inheritance and environmental factors including smoking and industrial pollution are all involved in the development of lung cancer.In addition to environmental exposure,genetic polymorphism plays an important role in the hereditary susceptibility among individuals.The immune reaction via T cells provides a vital role in the process of the tumorigenesis and eradication.PD-L1 is one of the most popular co-inhibitory signal molecule.It can mediate tumor immune escape by inhibiting T cells proliferation and inducing T cells to be exhausted.And here we conducted a study to evaluate the relevance between these SNPs located in the 3′-UTR of PD-L1 with NSCLC risk,which will provide a noval potential biomarker for the early diagnosis and treatment of NSCLC.Methods: Based on a case-control study,we enrolled 320 NSCLC patients and 199 healthy controls from the First Affiliated Hospital of Soochow University to collect peripheral venous blood.SNPs with MAF over 10% which located at microRNA complementary site in the PD-L1 3′-UTR are selected via bioinformatic prediction.ASA-PCR was used to determine the genotypes of these SNPs.We will figure out whether there exists an inner relationship between the SNPs and the risk of NSCLC.After we identified functional SNPs,we uesd the dual-luciferase reporter asssay system to analyze the role of SNPs which play in the process of affecting the interaction of miRNA and the complementary sites.Results:(1)Three SNPs of the microRNA-binding sites in the 3′-UTR region of the PD-L1 gene including rs2297136,rs4143815,rs4742098 are selected from the databases.Statistical analysis demonstrated that the AA、GG、AG genotype frequency of rs2297136 are 25.9%、70.6%、3.5% and 42.2%、50.2%、7.5% respectively in case and control group.Individuals carrying a genotype of AG and G carrier show a significant evaluation in risk of NSCLC compared to those carrying AA genotype(OR 2.287,95%CI 1.558-3.358,p< 0.001;OR 1.599,95%CI 1.027-2.488,p = 0.037),respectively.For the SNP rs4143815,The CC、GG、GC genotype frequency are38.4%、16.3%、45.3% and37.8%、20.7%、41.5% respectively.No significant differences was found between patients and healthy controls(p = 0.439).As for the SNP rs4742098,The AA、GG、AG genotype frequency are 20.9%、20.0%、59.1% and 25.6%、29.2%、45.2% respectively.Only the genotype AG is related to a significantly higher risk of NSCLC compared to the A allele homozygotes(OR=1.599,95%CI=1.027-2.488).(2)The analysis show that compared with the AA homozygote,the GG genotype is associated with the lymph node metastasis and distant metastases(p= 0.049,p = 0.035).However,we did not note the statistically significant association of the rs4143815 genotypes with clinical characteristics.For the rs4742098,there are strong associations between the variant genotypes and depth of tumor infiltration.In comparision with the genotype AA,AG genotype、GG genotype and G carrier are associated with the depth of tumor infiltration(p = 0.009,p = 0.006,p=0.003).(3)For luciferase reporter assays,it indicate that the SNPs in the 3′-UTR region of the PD-L1 gene can affect the interaction of mi RNA and the complementary sites.In terms of the SNP rs2297136,the expression of G-allele-specific psiCHECK2 construct is significantly suppressed by miR-296-5p(p<0.05),however no significant function is observed in A-allele-specific psiCHECK2 construct.When it turns to the SNP rs4742098,the expression of A-allele-specific psiCHECK2 construct is inhibited by miR-138(p<0.05),the role in G-allele-specific psiCHECK2 construct is invalid.Conclusion: The SNPs in the 3′-UTR region of the PD-L1 gene including rs2297136,rs4742098 have significant relevance with the risk of NSCLC and the clinicopathological features.These results support the hypothesis that genetic variants can interrupt the interaction between miRNA and PD-L1,which indicate that the mi RNA-mediated regulation of the target gene can be involved in the development of tumorigenesis.