Decorin基因过表达对HEK293T细胞微管相关蛋白LC3和自噬基因beclin1表达的影响

摘要

目的:构建人核心蛋白聚糖(DCN)重组质粒,研究其对微管相关蛋白LC3和自噬基因beclin1表达的影响.方法:从人胚肺二倍体成纤维细胞(2BS)提取总RNA,经RT-PCR获得decorin的全长cDNA,克隆至T-载体pUM-T并测序,亚克隆至真核表达载体pcDNA3.1/V5-His,再次测序正确后以磷酸钙共沉淀法瞬时转染HEK293T细胞,利用RT-PCR、Western Blot方法检测空载体组和重组质粒转染组微管相关蛋白LC3和自噬基因beclin1转录水平及翻译水平的表达.结果:经DNA测序证实人DCN cDNA片段正确插入真核表达载体中.Western Blot结果显示经磷酸钙共沉淀法转染的HEK 293T细胞中目的基因高表达.RT-PCR和Western Blot结果显示,与空载体组相比,重组质粒转染组LC3转录水平显著降低,beclin1和LC3-Ⅱ的翻译水平均显著降低.结论:成功构建人DCN重组质粒,其在HEK 293T中过表达可以显著降低微管相关蛋白LC3和自噬基因beclin1表达,为进一步研究DCN与自噬的相互作用奠定了基础.%Objective:To investigate the effects of decorin on regulating the autophagy of HEK 293T cells. Methods: HEK 293T cells were transiently transfected with recombinant plasmid decorin by calcium phos-phate.After 72 h,the expression of human decorin in HEK 293T cells was detected by Western Blot.Then,the effects of decorin on the expression of autophagy related gene (including LC3 and beclinl) were analyzed by RT-PCR and Western blot.Results:The recombinant plasmid DNA decorin was successfully constructed.The fusion protein can express in HEK 293T cell.Compared with control vector group,the mRNA expression of LC3 was significantly down-regulated in the group of overexpression of decorin(P<0.05),meanwhile,the mRNA level of beclinl showed no significant difference between the two groups (P>0.05).The Western Blot analysis revealed that LC3-II and beclinl decreased in the group of overexpression of decorin compared with control group(P<0.01,P<0.05,respectively),while there were no difference of LC3-I protein levels.Conchlsion:Overexpression of decorin could decrease the expression of autophagy associated genes of HEK 293T cells.lt may play an important role in the autophagy.