摘要
目的探讨瞬时受体电位C1(TRPC1)对人卵巢癌细胞ES-2增殖和凋亡的影响。方法利用荧光定量PCR检测人卵巢癌细胞ES-2、SKOV3、OV1以及OV2和正常卵巢上皮细胞IOSE80中TRPC1的表达水平。设计并合成靶向TRPC1的特异性短发卡RNA(shRNA),通过脂质体转染TRPC1表达最高的人卵巢癌细胞ES-2以构建稳定低表达TRPC1细胞株ES-2/shRNA,通过蛋白免疫印迹法和荧光定量PCR检测shRNA的干扰效率、MTT比色法检测干扰后细胞的增殖能力、流式细胞仪检测细胞凋亡情况、蛋白免疫印迹法检测磷酸化磷脂酰肌醇3-激酶(p-PI3K)、细胞周期素D1(Cyclin D1)以及B淋巴细胞瘤-2(Bcl-2)的表达水平。结果在4种卵巢癌细胞株中,ES-2的TRPC1表达水平最高。特异性靶向转染TRPC1 shRNA能够有效下调ES-2细胞中TRPC1表达水平;ES-2/shRNA细胞的增殖速度较转染空白载体的ES-2/Con及ES-2细胞明显减慢(P<0.01)。ES-2/shRNA细胞的凋亡率明显高于ES-2/Con及ES-2细胞(P<0.01)。TRPC1干扰组细胞的p-PI3K、Cyclin D1以及Bcl-2的蛋白表达强度比ES-2/Con及ES-2明显减弱。结论采用RNA干扰技术能够有效沉默ES-2细胞的TRPC1基因,并致使其细胞增殖能力下降以及细胞凋亡率升高,其中可能的机制为通过对细胞因子PI3K的调控影响人卵巢癌细胞的增殖和凋亡。
Objective To investigate the effect of transient receptor potential channel 1（ TRPC1）upon the proliferation and apoptosis of human ovarian carcinoma cell ES-2. Methods The expression levels of TRPC1 in human ovarian carcinoma cell ES-2,SKOV3,OV1 and OV2,and normal human ovarian epithelial cell ISOE80 were detected by fluorescent quantitative PCR. Specific short hairpin RNA（ shRNA） targeting TRPC1 was designed,synthesized,and then transfected into the ES-2 cells with the highest expression of TRPC1 via liposome to construct ES-2 / shRNA cell line stably and lowly expressing TRPC1. The interfering efficiency of shRNA was validated by western blot and fluorescent quantitative PCR. The proliferation and apoptosis of ES-2 were detected by using MTT assay and flow cytometry. The expression levels of phosphatidylinositol3-kinase（ p-PI3K）,Cyclin D1 and B-cell lymphoma（ Bcl-2） were measured by western blot. Results Among four human ovarian carcinoma cell lines,ES-2 expressed the highest level of TRPC1. TRPC1-targeted shRNA could effectively down-regulate the expression level of TRPC1 in ES-2 cells. Compared with those of ES-2 / Con and ES-2,the proliferation rate of ES-2 / shRNA was significantly lower（ P〈0. 01） whereas the apoptosis rate was considerably higher（ P〈0. 01）. The expression levels of p-PI3 K,Cyclin D1 and Bcl-2 in the shRNA interference group were significantly down-regulated compared with those in the ES-2 / Con and ES-2cells. Conclusions RNA interference technique can effectively silence the TRPC1 in ES-2 cells,decrease cell proliferation ability and enhance cell apoptosis,probably by regulating cytokine PI3 K to affect the proliferation and apoptosis of human ovarian carcinoma cells.
出处
《新医学》
2016年第2期97-101,共5页
Journal of New Medicine
关键词
瞬时受体电位C1
增殖
凋亡
卵巢癌
Transient receptor potential channel 1
Proliferation
Apoptosis
Ovarian carcinoma
