目的：探讨RNAi（RNA interference）技术抑制乳腺癌MCF7细胞中AKT1和PI3K P85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、PI3K P85两种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time PCR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、cyclin D1和P53的表达情况。应用MTT法、流式细胞术、2D和3D Matrigel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〖STBX〗AKT1, PI3K P85 shRNA可以有效抑制目的基因AKT1和PI3 Kp85的mRNA和蛋白表达；下游相关因子PCNA、cyclin D1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G1/G0细胞周期阻滞；2D和3D Matrigel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、PI3K P85亚基的shRNA技术可以抑制MCF7细胞中AKT1、PI3K P85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 G1/G0 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.

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）