Related Study On A Functional SNP In The MiR-199a Binding Stie Of The Hypoxia-Inducible Factor-1α Gene And Pancreatic Cancer Pathogenesis And Prognosis

ObjectivePancreatic cancer is the most malignant and the worst of the highest prognosis solid tumor. Hypoxia is a common microenvironment of solid tumor, and hypoxia-inducible factor-1is one of the key regulators under hypoxia conditions. HIF-1is a heterodimeric transcription factor consisting of α and β subunits. The clinical pathology data showed that about70%of pancreatic cancer has a high expression of HIF-1. Recent studies reported that rs2057482polymorphism influences the prognosis of renal cell cancer, and influences colon cancer susceptibility via the impact of Wnt signal pathway. But its relationship with pancreatic cancer has not yet been reported. We hypothesized that there were association between single-nucleotide polymorphism of HIF-1α and susceptibility of pancreatic cancer, which could provide new ways and targets for early diagnosis and treatment of patients with pancreatic cancer.Methods1. Pancreatic cancer (PC) patients and control subjects.A hospital-based case-control study was conducted in Tianjin Medical University Cancer Hospital and Institute (TMUCIH) from2001to2011. A total of291pancreatic cancer patients and247healthy controls enrolled in this study. Control subject were selected during the same period of time from healthy inhabitants of Tianjin who visited TMUCIH for physical check-up.2. Genomic DNA extraction, amplification, genotyping and immunohistochemistryPeripheral blood samples and PC specimens were obtained from pancreatic cancer patients. Genomic DNA was extracted using the DNA isolation kit. The rs2057482SNP in the gene of HIF-1α were detected by polymerase chain reaction (PCR). DNA fragments were sequenced in company. We stained HIF-1α by imminohistochemistry in150PC tumors to examine intensity and frequency of HIF-1α positive cells.3. Cell functional experiments We cloned the HIF-1α3’UTR fragments with either the C or T allele into a luciferase reporter vector. Reporter gene vectors containing either the C or T allele (or the mutant of the miR-199a binding site) and the miR-199a mimic (or control) were transiently transfected into HEK-293T cell line and PC cell line, and the relative Firefly luciferase to Renilla luciferase activity was measured.4. StatisticsHIF-1α genotypes in PC and control groups were evaluated Chi-square test and Fisher’s exact tests. The relation of genotypes and alleles to the risk of PC was determined using ORs and logistic regression [95%confidence interval (CI)]. Patient’s survival was used by Kaplan-Meier method.Results1. SNP rs2057482was more frequent in PC patients than in healthy volunteers (73.5%vs61.9%, P<0.01). Further, it was associated with higher risks for PC (OR=2.156,95%CI:1.324-3.511, P<0.05).2. Kaplan-Meier survival curves showed compared with the CT genotypes, the CC genotype was significantly associated with poor OS (P=0.01). And it is observed that the SNP was significantly associated with lymph node metastasis (P<0.05).3. The dual luciferase assay demonstrated that C/T variants of rs2057482could lead to differential regulation of the HIF-1α mRNA by miR-199a.ConclusionsA functional SNP in the miR-199a binding stie of the Hypoxia-Inducible Factor-la closely related to pancreatic cancer pathogenesis and prognosis, and it can provide a new way and target for early diagnosis and treatment of patients with pancreatic cancer.