Functional Genetic Variants In The TNF-α Induced Inflammation/apoptosis Related Genes Contribute To Cervical Cancer Susceptibility And Clinical Outcomes

Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in women. More than85%of these cases and deaths occur in developing countries, including China. A large body of molecular epidemiology research supports the hypothesis that persistent infection with an oncogenic human papillomavirus (HPV) type and chronic inflammation are the primary cause of cervical cancer, deemed as a necessary cause for the disease. However, only few HPV infected women develop cervical cancer, suggesting that there is an inter-individual variation in genetic susceptibility to cervical cancer. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variations among human beings, which may be the underlying molecular mechanisms of cervical cancer susceptibility.Tumor necrosis factor-a (TNF-a), one of the important cytokines, can activate caspase family genes and induce apoptosis. While, in a certain kind of cells, it can activate the nuclear transcription factor-KB pathway, recruit proinflammatory cytokines, such as interleukin6(IL6), and thus inhibit apoptosis. Moreover, the stimulation of TNF-a may increase the expression of TNF-a induced protein8(TNFAIP8), inhibit the activity of caspases and keep cells away from apoptosis. Previous data have shown that HPV E6and E7may be involved in the TNF-a induced inflammation/apoptosis pathway, thus contributing to cervical carcinogenesis.This is the first study to explore genetic variants in the TNF-a induced inflammation/apoptosis related genes and their associations with cervical cancer susceptibility and clinical outcomes. We hypothesize that (1) genetic variants (i.e., SNPs) in the TNF-a induced inflammation/apoptosis related genes are associated with cervical cancer susceptibility individually or jointly, and gene-gene/gene-enviornment interactions play important roles;(2) functional genetic variants (i.e., SNPs) regulate cervical cancer susceptibility by a certain biological mechanism;(3) genetic variants (i.e., SNPs) in the TNF-a induced inflammation/apoptosis related genes are associated with clinical outcomes in cervical cancer patients. Part Ⅰ Associations between genetic variants in the TNF-α induced inflammation/apoptosis related genes and cervical cancer susceptibilityTo explore the associations between SNPs in the TNF-α induced inflammation/apoptosis related genes and cervical cancer susceptibility, we performed a large case-control study with1,584cervical cancer cases and1,756cancer-free female controls. We found that the TNFAIP8-rs11064GG genotype was associated with an increased risk of cervical cancer, compared with AA/AG genotypes (adjusted OR=2.16,95%CI=1.16-4.03). A significantly increased risk of cervical cancer was associated with IL6-rs2069837AG/GG and CASP7-rs10787498GT genotypes (adjusted OR=1.27and1.19,95%CI=1.08-1.49and1.00-1.41, respectively). Additionally, we also observed an association between CASP7-rs4353229C>T and cervical cancer risk (OR=1.20,95%CI=1.02-1.40for TT vs. CC/CT).Combined genotype data showed that cervical cancer risk was increased in a dose-response manner with the increasing of risk genotypes in the TNF-α induced inflammation/apoptosis related genes. The haplotype Grs2069837Crs2069840in IL6was associated with a substantially altered risk for cervical cancer, compared with the haplotype Ars2069837Crs2069840·By using the logistic regression model, we observed the evidence for multiplicative interactions between two of CASP7SNPs,between CASP7and IL6, between SNPs and environmental factors. Further multifactor dimensionality reduction (MDR) also showed high-order interactions among SNPs in the TNF-α induced inflammation/apoptosis related genes.Taken together, although lacked of information on individual HPV infection status, our study provides evidence that SNPs in the TNF-α induced inflammation/apoptosis related genes individually or jointly contribute to cervical cancer susceptibility in Chinese Han women and that gene-gene/gene-environment interactions play important roles in cervical carcinogenesis. Part II Functional analysis of genetic variants significantly associated with cervical cancer susceptibilityIn this study, we investigated biological effects of four SNPs significantly associated with cervical cancer risk on regulating cervical carcinogenesis. Based on the HapMap genotyping and GENEVA gene expression databases, we compared levels of mRNA expression among different genotype groups of these SNPs and found that the TNFAIP8-rs11064allele G was associated with a high level of TNFAIP8mRNA. The tissue microarray and immunohistochemistry assays also confirmed that the TNFAIP8-rs11064allele G was associated with significantly increased levels of the TNFAIP8protein expression (adjusted OR=3.57,95%CI=1.09-11.72). Further in vitro functional experiments showed a significantly decreased level of the relative luciferase activity in the psiCHECK2:rs11064A group co-transfected with miR-22, compared with that in the psiCHECK2:rs11064G group (Student’s t test, P<0.001for HeLa, SiHa and SW480; P=0.009for A549, respectively), indicating that the TNFAIP8-vs11064variant G allele weakened the binding affinity of miR-22to TNFAIP83’-UTR in all four cancer cell lines tested.Taken together, the TNFAIP8-rs11064SNP may function by affecting the affinity of miR-22binding to the3’-UTR of TNFAIP8and regulating the mRNA and protein expression of TNFAIP8, thus contributing to cervical cancer susceptibility. Part Ⅲ Genetic variants in the TNF-α induced inflammation/apoptosis related genes predict clinical outcomes in cervical cancerTo investigate effects of SNPs in the TNF-α induced inflammation/apoptosis related genes on prognosis of cervical cancer, we recruited148cervical cancer patients from the Part Ⅰ case set, who were followed up for at least two years. We tested in vitro platinum resistance (i.e., cisplatin, carboplatin, nedaplatin and oxaliplatin) and TNFAIP8expression by using the MTT and immunohistochemistry methods, respectively. Although no significant association was found between SNPs and platinum resistence, cervical cancer cells with high expression levels of TNFAIP8showed more resistance to cisplatin and nedaplatin than those with low TNFAIP8expression levels(Wilcoxon test, P=0.043and0.009, respectively). Cox hazards proportional regression analyses indicated higher tumor stage, larger tumor size, lympho-vascular space invasion, TNFAIP8L1-rs1060555GG genotype and high TNFAIP8expression to be predictors for tumor recurrence. In addition, lympho-vascular space invasion and high TNFAIP8expression was found to predict cervical cancer death.These finding suggest that variations in TNF-a induced inflammation/apoptosis related genes, especially those in the TNFAIP8family genes, play important roles in the development of cervical cancer and may be predictive biomarkers for prognosis of cervical cancer.