体外靶向PI3K基因RNA干扰对卵巢癌SKOV3细胞Akt表达的影响

Effects of PI3K-gene-targeted RNA interference on the expression of Akt in SKOV3 cells in vitro

目的采用RNA干扰技术沉默卵巢癌SKOV3细胞中PI3K/Akt信号转导通路中关键基因PI3Kp110α,观察其对PI3K/Akt信号通路下游信号蛋白Akt的影响,以期为卵巢癌的基因治疗提供可靠的理论依据。方法体外培养人卵巢癌SKOV3细胞,脂质体介导靶向P13Kp110α基因的小干扰RNA(siRNA)转染卵巢癌SKOV3细胞,采用荧光定量PCR法检测干扰后PI3K、Akt mRNA的表达,免疫荧光双标法观察RNA干扰48 h后对PI3K、Akt蛋白的影响。结果 Real-time PCR结果示RNA干扰组PI3K、Akt mRNA相对含量显著低于空白载体组和对照组,差异具有统计学意义(P<0.05),而空白载体组与对照组表达差异无统计学意义(P>0.05);免疫荧光细胞化学结果示RNA干扰组的PI3K、Akt蛋白阳性率较对照组与空白载体组明显减少,而对照组与空白载体组比较无明显差异。结论 siRNA可沉默卵巢癌SKOV3细胞P13Kp110α基因的表达,同时减轻PI3K蛋白及下游信号蛋白Akt的表达,以PI3Kp110α为靶点的RNA干扰技术可望成为卵巢癌基因治疗的新策略。

【Objectives】 To observe the effects of RNA interference technique on the Akt protein which is the downstream signal protein of the PI3K/Akt signal pathway after silenced PI3Kp110α gene,the key gene of the PI3K/Akt signal pathway,in SKOV3 cells.Then provide reliable theoretical basis for the gene therapy of ovarian cancer.【Methods】 Human ovarian carcinoma SKOV3 cells in vitro were transfected by PI3K gene-targeted small interference RNA（siRNA） mediated by liposome.The mRNA of both PI3K and Akt were determined by RT-PCR and the effects on the expression of the PI3K and Akt proteins were examined by the immuno-fluorescence double staining method in 48 hours after RNA interference.【Results】 The RT PCR showed that the relative quantitation of PI3K and Akt mRNA in the RNA interference group were lower than that in the control group or the empty vector group（P 0.05）.And there was no significant difference between the empty vector group and the control group（P 0.05）.The immuno-fluorescence double staining showed that the positive rates of both the PI3K protein and the Akt protein in the RNA interference group were lower than control group or the empty vector group.But there was no significant difference between the empty vector group and the control group.【Conclusion】 The siRNA could silence the expression of PI3K gene in SKOV3 cells and reduce the expression of PI3K protein and Akt protein at the same time,PI3Kgene-targeted RNA interference might be a new therapeutic method for gene treatment of ovarian cancer.