摘要
目的:探讨RNAi(RNA interference)技术抑制乳腺癌MCF-7细胞中AKT1和PI3KP85亚基的表达对MCF-7细胞增殖和侵袭等的影响。方法:将包含AKT1、PI3KP85两种siRNA开放阅读框的短发夹RNA(shRNA)重组腺病毒质粒表达载体rAd5-siAKT1-siPI3K转染至乳腺癌MCF-7细胞。应用real-timePCR和Western blotting检测转染后目的基因mRNA和蛋白的表达水平,并用Western blotting检测目的基因被沉默后PCNA、cyclinD1和P53的表达情况。应用MTT法、流式细胞术、2-D和3-DMatrigel实验检测MCF-7细胞转染前后的细胞增殖周期和侵袭能力。结果:重组腺病毒质粒表达载体rAd5-siAKT1-siPI3K介导的靶向AKT1,PI3KP85shRNA可以有效抑制目的基因AKT1和PI3Kp85的mRNA和蛋白表达;下游相关因子PCNA、cyclinD1的表达亦下调,P53表达则上调。MTT法结果显示rAd5-siAKT1-siPI3K组细胞生长抑制率>50%,与未转染组和rAd5-siCtrl转染组比较,出现明显的G1/G0细胞周期阻滞;2-D和3-DMatrigel实验显示,未转染组和rAd5-siCtrl转染组细胞呈正常形态,而rAd5-siAKT1-siPI3K转染组细胞贴壁生长能力明显减低,细胞团块明显缩小。结论:靶向AKT1、PI3KP85亚基的shRNA技术可以抑制MCF-7细胞中AKT1、PI3KP85亚基的表达,抑制MCF-7细胞的体外增殖。
Objective：To investigate the effect of RNA interference （RNAi） targeting AKT1 and PI3K P85 on the proliferation and invasion of breast carcinoma MCF-7 cells.Methods：The recombinant adenovirus expression vector,which contained short hairpin RNA （shRNA） targeting open reading frames of AKT1 and PI3K P85 （rAd5-siAKT1-siPI3K）,was transfected into human breast carcinoma MCF-7 cells.AKT1 and PI3K P85 mRNA and protein expressions were detected by real-time PCR and Western blotting analysis.The expressions of PCNA,cyclinD1,and P53 were also detected by Western blotting analysis.The proliferation and apoptosis of MCF-7 cells were measured by MTT,flow cytometry and 2-dementinal and 3-dementional matrigel assay.Results：Recombinant adenovirus vector rAd5-siAKT1-siPI3K dramatically down-regulated AKT1 and PI3K P85 mRNA and protein expressions in MCF-7 cells;the downstream factors PCNA and cyclin D1 were also down-regulated,while P53 was up-regulated.Growth of MCF-7 cells was inhibited by over 50% in rAd5-siAKT1-siPI3K group as measured by MTT assay,and cell cycle was arrested in G1/G0 phase compared with untransfected and rAd5-siCtrl transfected groups.Cell growth on matrigel matrix showed normal cell shapes,while the cells in rAd5-siAKT1-siPI3K transfected group were detached from the matrix or grew in scattered clustering patterns,forming only small aggregates.Conclusion：shRNA targeting AKT1 and PI3K P85 can significantly down-regulate the expression of AKT1 and PI3K P85 in breast carcinoma MCF-7 cells,and inhibit the growth of MCF-7 cells in vitro.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2010年第1期51-56,共6页
Chinese Journal of Cancer Biotherapy
基金
国家重点基础研究发展规划(973计划)资助项目(No.2009CB918903)
国家自然科学基金资助项目(No.30670802)
天津市应用基础与前沿计划重点项目(No.09JCZDJC19700, No.10JCYBJC12500)~~
