Effects Of Antisense Oligonucleotides And SiRNAs Targeted Protein Kinase C α On A549 Cell Line

Objective:The protein kinase C(PKC),a family of serine/threonine protein kinases,is involved in cell proliferation,differentiation and malignant transformation.It has been demonstrated that PKCαis overexpressed in lung carcinoma cells,and PKCαblocking can inhibit lung carcinoma cell proliferation.In recent years,PKCαhas been identified to be a potential therapeutic target for the lung cancer.The purpose of the present study was to investigate the effects of antisense oligonucleotides(ASODN) mediated by polyethyleneimine(PEI) and small interfering RNA(siRNA) for selective surpressing PKCαexpression on lung carcinoma A549 cells.MethodsPartⅠPKCαASODN and random oligonucleotides(RODN) were transfected into A549 cells,the proliferation and the clone formation of A549 cells were detected by CCK-8 and clone formation assay,respectively.The morphology was examined by fluorescence microscope and transmission electron microscope.The percentage of hypodiploid cells of A549 cells treated with PKCαASODN for 48 h was determined with propidium iodide(PI) staining by flow cytometry.Early apoptosis was detected with Annexin V-FITC and PI dual parameter by flow cytometry.Furthermore,the expression of PKCαwas analyzed by RT-PCR and Western blot.PartⅡBased on siRNA design guidelines,PKCαsiRNAs were designed by various siRNA design software.In order to screen out more effective PKCαsiRNAs,six candicated PKCαsiRNAs and siRNA controls were chemical synthesized by Shanghai Jima company.PKCαsiRNA was transfected into A549 cells by Interferin^TM siRNA transfect reagent,the subsequent alterations in PKCαmRNA level were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR),and advanced MTT assays were employed to examine the changes of cell proliferation.Then,the effects of the effective PKCαsiRNAs on A549 were further investigated.The clone formation assay was performed.The morphology was examined by fluorescence microscope after hoechst 33258 staining.The percentage of hypodiploid cells of A549 cells treated with PKCαsiRNAs for 48 h was determined with propidium iodide(PI) staining and early apoptosis was detected with Annexin V-FITC and PI dual parameter by flow cytometry.In addition, the PKCαprotein level of A549 cells treated with PKCαsiRNAs for 72 h was analyzed by Western blot.ResultsPartⅠCompared with control group,PEI group and PEI-RODN group,the proliferation and clone formation of A549 cells treated with ASODN targeted PKCαwere significantly inhibited(P＜0.05).Nuclear fragmentation,and condensation were observed in A549 cells treated with ASODN targeted PKCα.Percentages of hypodiploid cells and early apoptosis rates were significantly higher in the ASODN targeted PKCαgroup than those in control, PEI and PEI-RODN group(P＜0.05).The expression of PKCαmRNA and protein in A549 cells treated with ASODN targeted PKCαwere significantly lower than those in control group(P＜0.05).PartⅡSix candicated PKCαsiRNAs were designed and named No.1,No.2,No.3,No.4,No.5, No.6 PKCαsiRNA,respectively.Compared with control,interferin^TM control,negative control,PKCαmRNA levels were all significantly downregulated and the cell proliferation were inhibited except No.5 in A549 cells treated with candicated PKCαsiRNAs(50nmol/L) for 48 h(P＜0.05).The three sequences,No.3,No.6 and No.1 PKCαsiRNA,which were more effectively inhibited A549 cell growth and PKCαmRNA level,were identified among 6 candidate PKCαsiRNAs using RT-PCR and proliferatary inhibition test.Compared with control,interferin ^TMcontrol and negative control,the clone formation of A549 cells treated with 1 nmol/L No.3,No.6 or No.1 PKCαsiRNA was significantly inhibited(P＜0.05). Percentages of hypodiploid cells and early apoptosis rates were significantly higher in No.3, No.6 or No.1 PKCαsiRNA(50nmol/L) treatment groups than those in control interferin ^TMcontrol and negative control group(P＜0.01).The expression of PKCαprotein in A549 cells treated with 50 nmol/L No.3,No.6 or No.1 PKCαsiRNA for 72 h was significantly lower than those in control,interferin^TM control and negative control group(P＜0.05).Conclusion1.The PKCαASODN mediated by PEI downregulates the expression of PKCαmRNA and protein,induces apoptosis and inhibits proliferation and clone formation in A549 ceils.2.No.1,No.2,No.3,No.4,No.5,No.6 PKCαsiRNAs can all downregulate expression of PKCαmRNA and inhibit cell proliferation in A549 cells.No.3,No.6,No.1 PKCαsiRNA are more effective than the other PKCαsiRNAs,they can induce apoptosis, inhibit clone formation and downregulate PKCαprotein expression in A549 cells.3.The effective concentration of siRNA is far lower than that of ASODN.These results suggest antisense oligonucleotides and siRNAs targeted PKCαmight be useful gene therapeutic strategy for lung carcinoma.