| To investigate the effect of proliferation,metastasis,invasion and apoptosis on pancreatic carcinoma cell strain PANC-1 by knockdown of NF-κB P65 expression through RNA interference.Methods:There were three groups in this study,RelAsiRNA group,negative control group and blank control group.Designed and synthesized small interference RNA directed against human NF-κB P65 by"RNAi express",at the same time designed negative control siRNA.Chemically synthesized small interference RNA directed against human NF-κB P65 was transfected into pancreatic carcinoma cell strain PANC-1 by using cationic liposome Lipofectamine?2000.The expression of NF-κB P65,cyclinD1,ICAM-1 gene was detected by using RT-PCR.The DNA binding activity of NF-κB was detected by the chemicon non-radioactive NF-κB P65 transcription factor assay kit.The effect of cell proliferation was studied by MTT.Clony assay was used to measure the proliferation of PANC-1 cells in groups.Matrigel transwell invasion assay was used to study the invasive ability of cells in study groups.Flow cytometry was used to measure the apoptosis and cell cycle of transfected cells.Nuclear DNA staining by Hoechst33258 and transmission electron microscope were used to ascertain whether cell apoptosis could be induced by down-regulating the expression of P65 gene in PANC-1 cell.Results: 1,The results of RT-PCR indicated that chemically synthesized siRNA could knock down the transcription and expression of NF-κB P65,cyclinD1,ICAM-1 gene.The difference between the RelAsiRNA group and control groups was significant.RT-PCR revealed that the expression of NF-κB P65 mRNA in RelAsiRNA group,negative control group and blank control group was 0.227±0.045,0.381±0.038 and 0.404±0.031(P<0.01);the expression of cyclinD1 mRNA was 0.791±0.086,1.099±0.108 and 1.160±0.098(P<0.01),the expression of ICAM-1 mRNA was 0.597±0.083,0.983±0.068 and 1.027±0.098(P<0.01).The expression of p65,cyclinD1 and ICAM-1 mRNA of the RelAsiRNA group was much lower than the other groups.2,After transfection,the DNA binding activity of NF-κB P65 in RelA siRNA group was much lower than the negative control group and blank control group(P<0.05).3,The results from the MTT assay indicated that 24 hours,48hours,72hours and 96hours after transfection,the proliferation of RelAsiRNA group cell was inhibited(P<0.05),The inhibition peaked at 72hours after transfection(P<0.01).4,Clony assay revealed that the cloning efficiency of RelAsiRNA group,negative control group and blank control group was (14.1±3.1)%,(24.5±2.1)% and (27.2±2.6)%(P<0.01).The cloning efficiency of the RelAsiRNA group was obviously reduced.5,Matrigel transwell invasion assay revealed that the cells of the three groups could invaded through the matrigel transwell filters,and the number of cells that invaded were 80.25±6.35,123.83±8.80 and 127.68±9.23(P<0.01) every high power field.The number of invasive cells of the RelAsiRNA group was much less than the other groups.6,The result of flow cytometry showed that the apoptotic rate of RelAsiRNA group,negative control group and blank control group was (25.4±4.5)%,(20.6±3.6)% and (16.9±3.0)%(P<0.01).The apoptotic rate of RelAsiRNA group was significantly higher than control groups and its cell number during G1 phases was (65.4±3.6)% increased about 10%,during S phases was (29.9±4.4)% decreased about 4% and G2/M phases decreased about 6%.7,Nuclear DNA staining by Hoechst33258 and transmission electron microscopy showed that knockdown of P65 expression by RNAi induced apoptosis in PANC-1 cells.Conclusion:By using cationic liposome,chemically synthesized small interference RNA directed against human NF-κB P65 could be transfected into PANC-1 cells succesfully.Extracorporeal experimental study provides basis for researching the function of NF-κB P65 gene,indicating down-regulating the expression of NF-κB may inhibit proliferation,metastasis,invasion and induce apoptosis in pancreatic carcinoma cell strain PANC-1. |
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