The Investigation Of Inhibitory Functions Of Survivin Dominant Negative Mutant And MiR-24 On Lung Cancer Cells And The Related Mechanisms

Objective:Plasmids which express Cys84-Ala survivin mutant and miR-24 were transfected respectively into NCI-H441,a human lung papillary adenocarcinoma cell line.Changes of transfected cells’ biological functions and probable mechanisms were detected,so as to provide new methods for targeted therapy of lung cancer.Materials and Methods:Part One:NCI-H441 cells were divided into treatment(Cys84-Ala Survivin)and empty vector(pCDNA3)groups,and blank control.Using trypan blue exclusion assay to detect cell growth,alamar blue assay to detect cell proliferation,visualizing cell apoptosis by Annexin-V and TUNEL assays,caspase-8 and caspase-9 fluorometric assays and Magic red assay to show enzyme activities of caspase-8/-9 and caspase-3/-7,visualizing apoptosis inducing factor(AIF)expression by immunostaining.Part Two:NCI-H441 cells were divided into treatment(miR-24)and empty vector groups,and blank control.Using trypan blue exclusion assay to detect cell growth,alamar blue assay to detect cell proliferation,western blotting assay to detect survivin expression level,caspase-8 and caspase-9 fluorometric assays and Magic red assay to show enzyme activities of caspase-8/-9 and caspase-3/-7.Results:Part One:1.The result of trypan blue exclusion assay showed that the living cell numbers of treatment group 48,72,and 96 hours after transfection were significantly decreased compared to empty vector group or blank control group(P<0.001).The cell growth of treatment group was inhibited from 24 hours after transfection,and reach to a rock-bottom at 72 hours after transfection.After that,the growth speed of treatment cells increased a little bit,while the total number was still lower than empty vector and blank control group.2.Cell proliferation of treatment group was inhibited at 48,72 and 96 hours after transfection showed by alamar blue assay,and there were significant differences between treatment group and empty vector or blank control groups(P<0.001).3.With Annexin V apoptosis assay,we found that apoptotic index of treatment group at 24,48,72 hours after transfection were significantly higher than those of empty vector or blank control groups(P<0.001);Tunnel assay showed that apoptotic index of treatment group at 48 and 72 hours after transfection were significantly higher than those of empty vector and blank control groups(P<0.001).4.The caspase-9 activities were much higher in treatment group at 48 and 72 hours after transfection than those of empty vector or blank control groups(P<0.001);Magic red assay showed that caspase-3/-7 activities were induced in treatment group at 48 and 72 hours after transfection.5.Through immunostaining rmethod,we found that apoptotic inducing factor,AIF,localized at cytoplasm,and no translocation from cytoplasm to nuclear was found in treatment group at all time points.Part Two:1.The result of trypan blue exclusion assay showed that the living cell numbers of treatment group 72,96 and 120 hours after transfection were significantly decreased compared to empty vector group or blank control group(P<0.001).Cell growth of treatment group was inhibited from 24 hours to 120 hours after transfection.2.Through alamar blue assay,we found that cell proliferation of treatment group was significant lower at 72,96 and 120 hours after transfection than that of empty vector or blank control groups(P<0.01).3.Survivin protein expression was decreased in treatment group at 48,72 and 96 hours after transfection.4.The caspase-8 activities were much higher in treatment group at 72 and 96 hours after transfection than those of empty vector or blank control groups(P<0.05).Magic red assay showed that caspase-3/-7 activities were induced in treatment group at 72 and 96 hours after transfection.Conclusion:1.Cys84-Ala survivin mutant significantly inhibited growth of NCI-H441,a human lung papillary adenocarcinoma cell line,and decreased its proliferation.2.Cys84-Ala survivin mutant induced cell apoptosis of NCI-H441 through caspase-9 but not caspase-8 dependent pathway,and its effect also involved caspase-3 and caspase-7.3.The apoptosis inducing effect of Cys84-Ala survivin mutant was not associated with AIF.4.miR-24 inhibited growth of NCI-H441 and decreased its proliferation.5.miR-24 downregulated survivin protein expression,which indicated survivin gene was probably a direct or mediate target of miR-24.6.miR-24 induced activity of caspase-8,caspase-3 and caspase-7,but had no impact on caspase-9 activity,which implied its inhibition on NCI-H441 was related to extrinsic apoptotic pathway while had no concern with intrinsic apoptotic pathway.