CAJ | 학술논문

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.
목적:탐토RNAi(RNA interference)기술억제유선암MCF-7세포중AKT1화PI3K P85아기적표체대MCF-7세포증식화침습등적영향.방법:장포함AKT1、PI3K P85량충siRNA개방열독광적단발협RNA(shRNA)중조선병독질립표체재체rAd5-siAKT1-siPI3K전염지유선암MCF-7세포.응용real-time PCR화Western blotting검측전염후목적기인mRNA화단백적표체수평,병용Western blotting검측목적기인피침묵후PCNA、cyclin D1화P53적표체정황.응용MTT법、류식세포술、2-D화3-D Matrigel실험검측MCF-7세포전염전후적세포증식주기화침습능력.결과:중조선병독질립표체재체rAd5-siAKT1-siPI3K개도적파향AKT1, PI3K P85 shRNA가이유효억제목적기인AKT1화PI3 Kp85적mRNA화단백표체;하유상관인자PCNA、cyclin D1적표체역하조,P53표체칙상조.MTT법결과현시rAd5-siAKT1-siPI3K조세포생장억제솔>50%,여미전염조화rAd5-siCtrl전염조비교,출현명현적G_1/G_0세포주기조체;2-D화3-D Matrigel실험현시,미전염조화rAd5-siCtrl전염조세포정정상형태,이rAd5-siAKT1-siPI3K 전염조세포첩벽생장능력명현감저,세포단괴명현축소.결론:파향AKT1、PI3K P85아기적shRNA기술가이억제MCF-7세포중AKT1、PI3K P85아기적표체,억제MCF-7세포적체외증식.
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.