The Study Of Biological Characteristics Of Oct4and Nanog Of Pancreatic Cancer Stem Cells

ObjectiveTo study the gene expression of Oct4and Nanog that related toembryonic stem cell in human pancreatic cancer stem cells（PCSCs）and study the proliferation and drug-resistence ability of pancreaticcancer stem cell in vitro. Silence Oct4and Nanog expression in PCSCsby specific shRNA, study the biological characteristics of PCSCs andthe related mechanisms.MethodsPancreatic cancer stem cells of CD44+CD24+ESA+phenotypesisolated by a FACS Aria II in human cell line Panc-1. Detect theexpression of Oct4and Nanog gene expression in Panc-1cells andPCSCs cells by immunofluorescence and RT-PCR. To study theproliferation ability of cancer stem cell, we draw a growth curve bythe ways of CCK8staining. To detect the ability of drug-resistence ofPCSCs, we stimulated both cells by gemcitabine and study thedifference of their IC50. Nanog and Oct4in PCSCs silenced byLentiviral vector-GFP(LV) mediated shRNA. Observe themigration,invasion, proliferation and the drug sensitivity of PCSCsinhibited by Oct4and Nanog by monoclonal formation as well as theCCK8assay, transwell chamber model. Investigate related mechanismsby FQRT-PCR and Western blot and NOD/SCID mice tumorigenicityexperiments was done to study the tumorigenic potential of PCSCsinfluenced by Oct4and Nanog. ResultsThe isolated PCSCs is0.1%-0.8%of all cells. Oct4and Nanoggenes have a higher expression in sorted cells than unsorted ones.The△CT of Nanog in PCSCs and Panc-1cell is7.945±0.652. The△CT of Oct4in PCSCs and Panc-1cell is8.141±1.378; Both twogenes were expressed in nucleus and were restrained by gemcitabine.The48th hour’s IC50of Panc-1cell is982μg/mL, IC50of PCSCsis1981μg/mL. The72th hour’s IC50of Panc-1cell is670μg/mL,IC50of PCSCs cell is1484μg/mL.LV mediated shRNA have stronginhibitory effects for Nanog and Oct4expression(P<0.05).Compared tothe control group,The proliferation, monoclonal formation, invasionability and migration of PCSCs decreased significantly if silencing Oct4and Nanog expression, while enhance drug sensitivity and induceapoptosis(P<0.05). Tumorigenicity experiments showed PCSCstumorigenic capacity decreased significantly than that of control groupif Oct4and Nanog gene silenced.(P<0.05).Conclusions1. Cancer stem cell perform a stem cell-like characteristic of highlydrug-resistence and declined proliferation.2. Oct4and Nanog gene have a higher expression in PCSCs thanin Panc-1cells.3. LV mediated shRNA effectively reduce the expression of Oct4and Nanog gene in PCSCs.4. Silence Nanog and Oct4can inhibit the biological characteristicsof PCSCs，while enhance drug sensitivity and induce apoptosis.