SiRNA Targeting Against HIF-2α Gene Mediated By RAAV2 Increase The Sensitivity Of Pancreatic Carcinoma Cells To Gemcitabin

Objective To investigate the effect of HIF-2α-siRNA mediated by rAAV2 on proliferation,apoptosis and enhancing chemotherapy sensitivity of pancreatic carcinoma cells.Methods rAAV2-EGFP-U6- HIF-2α-siRNA was transfected into human pancreatic carcinoma cell line PANC-1. As a reporter gene, green fluorescence protein(GFP) was evaluated by flow cytometry and fluorescent microscopy to estimate the efficiency of transfection. The PANC-1 cells were cultured under normoxic and hypoxic conditions respectively. RT-PCR and immunocyto-chemistry method were used to detect HIF-2αmRNA and protein expression. MTT assay was used to measure the inhibition rate of PANC-1 cells treated with gemcitabin in different concentrations for 48 hours.While MTT assay was used to determine proliferation rate of PANC-1 cells treated with gemcitabin and rAAV2-EGFP-U6- HIF-2α-siRNA in hypoxic conditions. Detection of apoptosis was manipulated by Annexin V-FITC/PI double staining.Results rAAV2-EGFP-U6-HIF-2α-siRNA was successfully transfected into PANC-1 cells. The efficiency of transfection by flowcytometry was 40%.Expressions of HIF-2αmRNA and protein were significantly inhibited in PANC-1 cells of experimental group,compared with negative control group or blank control group.All concentrations (0.25μg/mL -4μg/mL) of gemcitabin can inhibit the proliferation and show a dose dependent manner, the diference was significantly(F=859.054,P<0.05).In the same concentration of gemcitabin ,the inhibitation rate of PANC-1 cells under normoxic condition was higher than that under hypoxic condition(F=219.471,P<0.05). The inhibitation rate of union group was higher than rAAV2-EGFP-U6-HIF- 2α-siRNA group and gemcitabin group,the difference was significant(P<0.05). The apoptosis rate of union group was highest(F=39.279,P<0.05),compapered with rAAV2-EGFP-U6-HIF-2α-siRNA group and gemcitabin group under hypoxic condition.Conclusions Transfecting rAAV2-EGFP-U6-HIF-2α-siRNA into PANC-1 cells can inhibit the expressions of HIF-2αmRNA and protein,inhibit PANC-1 cells proliferation, induce apoptosis and enhance the sensitivity of PANC-1 cells to gemcitabin.