The Expression Of The SCF Mediated By HIF-1Participate In Pancreatic Cancer Cells Adapt To Hypoxia

ObjectivePancreatic cancer is a malignant solid tumor which mainly comes from pancreatic ducts. In recent years, the incidence of pancreatic cancer is increasing stable at home and abroad and increased about15%every10years. The prognosis of pancreatic cancer is the worst in the digestive system tumor and the survival rate of5years is only3%. Despite improvement of therapeutic methods including Surgery, radiotherapy and chemotherapy, the malignant tumor remains a poor prognosis. Hypoxia plays a central role in the development and behavior of pancreatic carcinoma which can activate a series of genes to adapt hypoxic condition, and change the biological behaviour of tumor cells, such as proliferation inhibition apoptosis, the formation of blood vessels, energy metabolism, invasion and metastasis, et al. These reactions increased the difficulty of clinical treatment. But the adjustment mechanism in this environment remains to be defined.The important response to hypoxia is the hypoxia-inducible factor (HIF)-1expression which can recognizes and binds to the hypoxia-response elements (HRE;5’-RCGTG-3’) of hypoxia-inducible promoters and change the biological actions of tumor cells by adjusting its target genes transcription activity. Research has shown that low oxygen micro environment can induce the expression of the SCF.SCF is stem cell factor which express in a wide variety of tumor cells and play an important role in proliferation and angiogenesis of tumors. Therefore, we speculate SCF maybe involved in the adaptive mechanism of pancreatic carcinoma in hypoxia environment, we examined the influence of hypoxia on expression of HIF-1α and SCF in pancreatic cancer cell and the regulatory mechanism by the related experimental technologys. Finally, we also examined the role of SCF expression on pancreatic cancer cells.Methods1. The expression of SCF, HIF-la were examined by immunohidtochemistry in tumor tissues of95pancreatic cancer patients.2. Choose the cell lines by Operation flow cytometry and western blot.1) MIAPaCa-2、BxPC-3、PANC-1、Capan-1as object and examed c-kit (CD117) expression by flow cytometry.2) In normoxia,we evaluate the basic level of HIF-1expression by western blot.3. Evaluate expressio of HIF-1and SCF in pancreatic carcinoma under hypoxia.1) We choose BxPC-3、PANC-1as c-kit positive cells and evaluate protein levels by western blot.2) Knockdown of HIF-1α by RNA interference and evaluate SCF, HIF-1α, VEGF and β-actin mRNA and protein levels by realtime PCR and Western blot.4. Explore the regulatory mechanism of HIF-1and SCF under hypoxia.1) Chromatin immunoprecipitation assay (ChIP) was performed to detect the binding of HIF-1to SCF promoter in BxPC-3cell;2) HIF-1expression plasmids and firefly luciferase reporters carring SCF promoters were co-transfected into BxPC-3cell and regulation of SCF gene was evaluated by luciferase assay.ResultsSCF、HIF-1have high expression in the pancreatic cancer tissue and there is a positive correlation between them. We found that PANC-1and BxPC-3have high expressed c-kit. BxPC-3has less or no HIF-1expression under normoxia but PANC-1has more. Hypoxia increased the expression of SCF and HIF-1on mRNA and protein levels in PANC-1、BxPC-3cancer cells; Knockdown of Hypoxia inducible factor-1(HIF-1) by RNA interference decreased the expression of SCF. HIF-1could directly bind to the hypoxia-response element located in the promoter region of SCF and up regulate the transcription of this promoter.ConclusionIn conclusion, HIF-1is a key transcription factor for SCF expression in pancreatic cancer cells; SCF may be an important factor for the growth and angiogenesis of pancreatic cancer; HIF-1α and SCF/c-kit signal system might be a potential target for the treatment of pancreatic carcinoma.