Antisense Oligonucleotide Targeting Survivin Induces Apoptosis Of Pancreatic Cancer Cells

【Objective】:To investigate the inhibitory effect of survivin antisense oligonucleotide (ASODN) on proliferation of PANC-1 cell.【Methods】:The antisense oligodeoxynucleotides(ASODN) and sense oligodeoxynucleotides (SODN) were complementary to survivin sequences.PANC-1 cells were divided into six groups:control group,oligofectamine group,200nmol/L SODN group,50-800nmol/L ASODN group.Survivin ASODN was designed and transfected into PANC-1 cells mediated by Oligofectamine carrier,the percentage of fluorecent cells was counted by flow cytometry(FCM) after transfection for 24h,the highest percentage of fluorescent cells is as the the best transfection efficiency,so the number of cells,the concentration of ASODN and Oligofectamine;Cell morphological change was observed with inversphase microscope;MTT assay Was used to detect cytotoxicity;Cell morphological changes were examined by transmission electronmicroscopy;The cell-cycle and apoptic rate were analyzed by flow cytometry;Immunohistochemical staining techniques were used(ABC method),and the express of survivin were observed under light microscopy,examined and analysed by computer image.【Results】:1.The percentage of fluorescent cells was 31.9%when the transfecting concentration of ASODN was 50 nmol/L,and 37.4%to 100 nmol/L,41.4%to 150 nmol/L,52.6%to 200 nmol/L,24.2%to 250nmol/L,11.4%to 400nmol/L, 16.1%to 600nmol/L and 15.5%to 800nmol/L,respetively;2.The percentage of fluorescent cells was 12%when the inoculated cells was 2×l0⁴/well,and 50.8%to 2×10⁵/well,11.2%to 2×10⁶/well,respectively;3.The percentage of fluorescent cells was 58.8%when 2μl of liposome was used during transfection,34.0%when 3μl,23.6%when 4μl,respectively;4.Observing with inversphase microscope,we find changes of cells in dose-dependent manner after transfection,such as reducing of cell volume, anomaly form,shrinking,shrinking of nucleolus,cytoplast roughness and fewer cell division.However the untreated cells are in integrity form and well growth;5.The PANC-1 cells are treated by 200nmol/L surviving ASODN after 24h,36h,48h,the inhibition rate were 30.67%,42.67%,61.3%respectively and show time-dependent manners,the difference was significant(P＜0.05);6.The changes of PANC-1 cells apoptosis were observed by AO and EB;7.early apoptotic morphology was demonstrated by electron microscopy;8.The higher apoptosis rate 38.1%could be induced in PANC-1 cells by survivin ASODN as compared with that induced by the sense oligodeoxynucleotide(4.16%,P＜0.05);9.After transfection,the expression of survivin protein in PANC-1 significiantly decreased.【Conclusion】:Within the 12-well plate,the optimal transfection conditions of ASODN to PANC-1 cells were as follows:the cell count of 2×10⁵/well,concentration of ASODN 200 nmol/L,and cationic liposome OligofectamineTM^TM2μl,respectively. Transfection efficiency could be improved after transfection optimization,and this would help set foundation for further investigation.Survivin ASODN Can inhibit the proliferation of pancreatic cancer cells and induce their apoptosis.