Hypoxia-inducible Factor(HIF)-1? Transactivates The Human Leptin Receptor(Ob-R) Gene Promoter In Human Pancreatic Cancer

ObjectiveIt is known to everyone that pancreatic cancer is a kind of highly malignant tumor of digestive system,whose 5-year survival rate of less than 5%,the mortality rate is very high.Although surgical operation and chemotherapy has been greatly improved,during the nearest 20 years,survival rate of patients who had operation excision is only 17%.Therefore,explore the development mechanisms and internal molecular biology of pancreatic cancer,witnessed how effective therapeutic targets and chemotherapy drugs were is the current problems needed to resolve.Leptin is a protein secreted by fat cells(16kD),elevated circulating leptin suppress the appetite and increase heat production to counteract obesity,in the traditional view,leptin is hormone to inhibit obesity.However,recent studies showed that leptin also has a very important role in the progress,metabolism,neuroendocrine,immune regulation of many malignant tumors.Somasundar reported,leptin promoted cell proliferation of breast cancer,esophageal cancer and prostate cancer,but inhibit pancreatic cancer cell proliferation,however,so far its molecular mechanism has not yet proved.Leptin play a physiological function through a combination of leptin receptor(Ob-R),so the molecular function and biological process of leptin and leptin receptor is almost the same.Severe hypoxia is an outstanding characteristic of pancreatic cancers,suggesting a poor blood supply in pancreatic cancer tissues.The study was designed to test the hypothesis that leptin receptor(Ob-R)is a direct target of the hypoxia-inducible factor HIF-1??a key transcriptional factor for the cellular response to hypoxia in human pancreatic cancer.Methods1.The transcriptional regulation of HIF-la to Ob-R in pancreatic cancer.(1)Eleven paired tissue mRNAs were extracted from carcinoma tissue and the corresponding para-carcinoma tissues,quantitative real-time PCR was used to investigate the mRNA expression of Ob-R and HIF-la in these tissues.(2)Cell culture:human pancreatic cancer cells(Mia-PaCa-2?ASPC-1)were maintained in appropriate medium supplemented with 10%fetal bovine serum(GIBICO),L-glutamine.Cells were grown at 37? in a humidified atmosphere of 95%air and 5%CO2.(3)Western blot analysis:messenger:protein levels were determined by western blot after transfection,respectively.(4)Real-time RT-PCR detection:messenger RNA(mRNA)levels were determined by quantitative Real-time PCR after transfection,respectively.(5)Chromatin immunoprecipitation assay(ChIP):ChIP assay was performed using a ChIP assay kit(Upstate Biotechnology,Waltham?USA)according to the manufacturer's instructions,then PCR was used to detect the expression of Ob-R.(6)Transient transfection experiments and reporter assays:To observe the effect of transcriptional regulation of HIF-la on the Ob-R gene,we constructed Ob-R luciferase promoter vectors and HIF-la cDNA fragments and cotransfected them into ASPC-1,Mia-PaCa-2 cells.2.Functional proofs of HIF-1?-induced regulation of Ob-R.(1)Cell culture:the same as the first part.(2)Colorimetric MTT assay:Pancreas cell lines(Mia-PaCa-2,ASPC-1)were serum-deprived over night,and exposed to leptin(Ong/ml,50ng/ml,100ng/ml,250ng/ml and 500ng/ml)for 24,48 and 72h respectively.Then the cells were incubated with 20?p,1 MTT at 37? for 4h.150?1 of DMSO was added to the cells after removal of MTT,the absorbance was measured at 570nm with a microplate reader.(3)Measure cell apoptosis:after cells were treated by leptin(0,100,250,500ng/ml)for 24 hours,PI and AnnexinV-FIFC were used to dye,flow cytometry was used to measure cell apoptosis.(4)Measure cell cycles:after cells were treated by leptin(0,100,250,500ng/ml)for 24 hours,75%alcohol was added for another 24 hours,then PI was used to dye,flow cytometry was used to measure cell cycles.(5)EdU proliferation assay:cells were incubated with 10?M EdU for 2h before fixation,permeabilization,EdU staining,which were carried out following the supplier's protocol.The proportion of nucleated cells incorporating EdU was observed by fluorescence microscopy.Functional proofs of HIF-1?-induced regulation of Ob-R by MTT and EdU proliferation assay.ResultsHypoxia or overexpression of HIF-la in pancreatic cancer cells both strengthened the protein and mRNA expression levels of Ob-R expression.Silencing the expression of HIF-la by RNA interference significantly inhibits hypoxia-induced increase of Ob-R expression.The analysis of Ob-R gene promoter found three hypoxia-responsive elements in the region upstream of the transcription start site.Using ChIP assay,we revealed that HIF-la directly bound to hypoxia-responsive element located at-832 to-828 of Ob-R gene promoter,which is essential for HIF-la-induced expression.Luciferase analysis revealed a 3.43-fold(ASPC-1)and 3.76-fold(Mia-PaCa-2)induction of luciferase activity with HIF-la cotransfection.Apoptosis rate of the cells treated with different concentrations of leptin was higher than control cells,the highest one was 1.71 times(P<0.05),suggesting leptin treatment promote pancreatic cancer cell apoptosis.Pancreatic cancer cell was blocked at G1 phase after leptin treatment.In both pancreatic cancer ASPC-1 and Mia-PaCa-2 cells,leptin inhibited cell proliferation(P<0.05).However,leptin-induced inhibition effect of proliferation weakened after silencing of HIF-1?through siRNA interference.ConculsionIn pancreatic cancer cells,HIF-la regulated OB-R expression through directly binding to the HRE region of the Ob-R promoter,which may be a valuable therapeutic target.