靶向调控SIRT1基因表达对子宫内膜癌细胞生物学行为的影响及其机制研究

Effect of target SIRT1 on biological behaviors of endometrial carcinoma cells and its mechanism

目的靶向调控沉默信息调节子1(SIRT1)基因表达对子宫内膜癌(EC)细胞生物学行为的影响及其机制。方法构建慢病毒介导的SIRT1基因小发夹RNA(shRNA),慢病毒感染EC细胞株并验证SIRT1基因沉默效果;检测RL95-2细胞的生物学行为及p53、p21蛋白表达。结果 shSIRT1-1组、shSIRT1-2组SIRT1 mRNA、SIRT蛋白表达显著低于shControl。shSIRT1-1组、shSIRT1-2组RL95-2细胞出现明显生长抑制,shSIRT1-1组、shSIRT1-2组细胞凋亡率显著高于shControl组;shSIRT1-1组、shSIRT1-2组中细胞克隆直径> 10 mm的细胞数、迁移细胞数、侵袭细胞数显著低于shControl组;shSIRT1-1组、shSIRT1-2组p53、p21蛋白相对表达量均显著高于shControl组。结论慢病毒介导的RNA干扰技术靶向沉默SIRT1基因可能通过p53/p21途径抑制RL95-2细胞增殖、非锚定依赖性、迁移及侵袭,促进RL95-2细胞凋亡。

Objective To explore the effect of target silent information regulator 1(SIRT1) on biological behaviors of endometrial carcinoma(EC) cells and its mechanism.Methods The lentiviral-mediated short hairpin RNA(shRNA) of SIRT1 gene was constructed.EC cell line RL95-2 were infected with lentivirus, and SIRT1 gene silencing effects was verified.The biological behaviors of RL95-2 cells and the expression of p53 and P21 was tested.Results Sequence test showed that shRNA insertion sequence of SIRT1 was correct, and the obtained plasmids were named shSIRT1-1, shSIRT1-2 and shControl.Titer of shSIRT1-1 and shSIRT1-2 was 2.7×107 Tu/ml and 3.0×107 Tu/ml, respectively.Virus packaging efficiency met the needs of RNA interference.SIRT1 mRNA and SIRT protein expression were significantly lower in shSIRT1-1 group and shSIRT1-2 group than in shControl group.RL95-2 cells in shSIRT1-1 and shSIRT1-2 groups showed significant growth inhibition, and the apoptosis rate were significantly higher in shSIRT1-1 group and shSIRT1-2 group than in shControl group.Numbers of cells with cell clone diameters > 10 mm, migrating cells and invasive cells were significantly smaller in shSIRT1-1 and shSIRT1-2 than shControl group.The p53 and p21 protein relative expression levels were significantly higher in shSIRT1-1 group and shSIRT1-2 group than shControl group.Conclusion Lentiviral-mediated RNA interference for targeted silencing of SIRT1 gene may inhibit the proliferation, unanchored dependence, migration and invasion of RL95-2 cells, and promote RL95-2 apoptosisvia p53/p21 pathway.