摘要
目的:研究微小RNA(miRNA)干扰细胞分化抑制因子3(Id3)基因表达对人肺腺癌细胞株A549细胞体外增殖和凋亡作用的影响。方法:构建含靶向Id3 mRNA的miRNA所对应寡核苷酸的重组干扰载体pcDNATM6.2-GW/EmGF-PmiRId3(pcDNA/miRId3),通过脂质体转染技术将miRNA干扰质粒pcDNA/miRId3和含Id3基因的重组表达载体pEGFP/Id3共转染A549细胞。荧光显微镜观察EGFP表达情况;流式细胞术(FCM)分析转染24 h后A549细胞的EGFP表达效率;半定量RT-PCR和Western blot技术检测转染后A549细胞内Id3 mRNA及蛋白的表达;MTT法和Annexin V-FITC/7-AAD双染法结合FCM检测靶向Id3基因的miRNA转染A549细胞后对增殖和凋亡的影响。结果:RT-PCR和Western blot结果表明,将pEGFP/Id3和pcDNA/miRId3共转染A549细胞24 h后,A549细胞中外源性Id3 mRNA和Id3蛋白质的表达量均明显受到抑制。MTT和Annexin V/7-AAD双染法结果表明,pEGFP/Id3转染A549细胞24 h后,对A549细胞增殖功能有明显抑制作用,细胞早期凋亡率明显增加,与pEGFP对照组相比均呈统计学差异(P<0.01),但pEGFP/Id3与pcD-NA/miRId3共转染A549细胞后,细胞增殖率和凋亡率均未发生明显变化。结论:外源性Id3基因在A549细胞中的表达能够抑制细胞增殖并诱导凋亡,同时导入靶向Id3 mRNA的miRNA干扰质粒pcDNA/miRId3,可逆转外源性Id3表达对细胞增殖的抑制和致凋亡作用,重组MiRNA表达载体的构建及应用为研究Id3致A549细胞增殖抑制和诱导凋亡的作用机制奠定了实验基础。
AIM： To investigate the effect of microRNA-mediated exogenous Id3 gene silencing on proliferation and apoptosis of human lung adenocarcinoma A549 cells in vitro.METHODS： A recombinant miRNA expression vector（pcDNA6.2-GW/EmGFPmiR-Id3,pcDNA/miRId3） which targets human Id3 gene was constructed.After 24 h of transfection,the transfection efficiency was monitored by inverted fluorescence microscopy.EGFP expression efficiency in A549 cells was analyzed by flow cytometry（FCM）.Id3 expression vector pEGFP/Id3 and pcDNA/miRId3 were cotransfected into A549 cells by liposome-mediated method.After 24 h of transfection,the transfection efficiency was monitored by inverted fluorescence microscopy.Semi-quantitative RT-PCR and Western blot were used for identifying Id3 mRNA and protein expression respectively in A549 cells after transfection.Cell proliferation rate and apoptosis ratio were evaluated by MTT assay and Annexin V/7-ADD staining followed by FCM to observe the down-regulatory effect of Id3 expression by miRNA-mediated RNA interference（RNAi）.RESULTS： pcDNA/miRId3 and pEGFP/Id3 were successfully transfected into A549 cells.RT-PCR and Western blot results showed that after 24 h of cotransfection of pEGFP/Id3 and pcDNA/miRId3 in A549 cells,the exogenous expression of Id3 both at mRNA and protein levels were significantly reduced compared with the pEGFP/Id3 group.MTT assay and Annexin V/7-AAD staining showed that after 24 h of transfection with pEGFP/Id3,the proliferation rates were significantly reduced and apoptotic cell ratios were significantly higher than those of pEGFP-transfected cells.Whereas there were not any significant differences in proliferation rates or apoptotic cell ratios between pcDNA/miRId3＋pEGFP/Id3 cotransfected group and pEGFP or miRNA negative controls.CONCLUSION： Exogenous expression of Id3 in A549 cells could inhibit proliferation and induce apoptosis of A549 cells.Cotransfection of pcDNA/miRId3 and pEGFP/Id3 into A549 could reverse the Id3-induced proliferation inhibition and
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第9期852-855,857,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
江苏省"科教兴卫"医学重点人才基金课题(RC2007117)
南京军区医药卫生科研项目重点课题(07Z032)
