蛋白激酶Cδ失活对骨肉瘤干细胞增殖、凋亡及相关信号通路的影响

摘要

目的探讨短发夹RNA（ shRNA）靶向沉默人蛋白激酶Cδ（ PKCδ）对骨肉瘤干细胞增殖、凋亡及相关信号通路的影响。方法根据Genbank数据库中PKCδ序列，设计、合成互补并特异性编码其 shRNA序列（ PKCδ⁃shRNA⁃1和PKCδ⁃shRNA⁃2），克隆至pRNA6�1⁃Neo质粒载体后采用Western blotting检测PKCδ蛋白水平，筛选抑制效果最好的shRNA片段转染骨肉瘤干细胞（转染组），设阴性对照组（转染shRNA无意义随机阴性对照序列）和空白对照组（不行转染处理）。采用噻唑蓝（ MTT）法检测各组转染24、48、72、96 h后的增殖抑制率，流式细胞术检测各组转染后的细胞凋亡和细胞周期情况，实时荧光定量PCR检测凋亡相关基因的表达，采用Western blotting检测各组转染96 h后PI3K／Akt及Wnt／β⁃catenin信号通路中相关蛋白的水平。结果转染PKCδ⁃shRNA⁃1和PKCδ⁃shRNA⁃2后PKCδ蛋白水平均降低（ P＜0�05），且PKCδ⁃shRNA⁃1的抑制效果优于PKCδ⁃shRNA⁃2，故后续实验选择PKCδ⁃shRNA⁃1；与其余两组相比，转染组的增殖抑制率、凋亡率及促凋亡基因Bax、Bad的mRNA水平均升高，而Bcl⁃2水平降低，且在细胞周期上，G0／G1期细胞比例升高，但S期和G2／M期细胞比例均降低，以上差异均有统计学意义（ P＜0�05）；转染组PI3K／Akt通路中的PTEN水平升高、Akt水平降低，Wnt／β⁃catenin通路中β⁃cate⁃nin、C⁃myc和Cyclin D1水平均降低（ P＜0�05）。结论通过shRNA抑制PKCδ基因表达可抑制骨肉瘤干细胞增殖并诱导凋亡和细胞周期阻滞，对PI3K／Akt和Wnt／β⁃catenin信号通路激活有一定抑制作用，可能对骨肉瘤的防治有一定价值。%Objective To investigate the effect of short hairpin RNA ( shRNA) targeting protein kinase Cδ( PKCδ) on the proliferation, apoptosis and related signaling pathways of human osteosarcoma stem cells. Methods According to the PKCδsequence in Genbank database, the specified shRNA series for PKCδ ( PKCδ⁃shRNA⁃1 and PKCδ⁃shRNA⁃2) were designed, synthesized, and then cloned into pRNA6�1⁃Neo plasmid vector. The Western blotting was used to detect the level of PKCδat 24, 48, 72, and 96 h af⁃ter transfectioin. The shRNA with the best inhibition effect was chosen to transfect osteosarcoma stem cells ( Transfection group) , and the stem cells transfected with the negative control sequence ( Negative control group ) and without any transfection ( Blank control group) were prepared. The proliferation inhibition rates were measured by Thiazolyl blue ( MTT) at 24, 48, 72, and 96 h after trans⁃fectioin. The flow cytometry was used to measure the apoptosis and cell cycle. Fluorescent quantitative PCR was used to detect the ex⁃pression of related apoptotic genes. Western blotting was used to detect the levels of related protein in PI3K/Akt and Wnt/β⁃catenin pathways at 96 h after transfection. Results The level of PKCδdecreased after the transfection of PKCδ⁃shRNA⁃1 and PKCδ⁃shRNA⁃2. Given the better inhibition effect of PKCδ⁃shRNA⁃1, PKC⁃shRNA⁃1 was used in the subsequent experiment. Compared with other two groups, the proliferation inhibition rate, apoptosis rate and mRNA levels of Bax and Bad were increased, while the Bcl⁃2 level was decreased in Transfection group ( P<0�05) . The proportion of G0/G1 phase was increased, but the S phase and G2/M phase cells were decreased in Transfection group versus other groups ( P<0�05) . In PI3K/Akt pathway, the level of PTEN was increased, but the level of Akt was decreased. In Wnt/β⁃catenin pathway, the levels of β⁃catenin, C⁃myc and Cyclin D1 were decreased in the transfected group( P<0�05) . Conclusion ShRNA inhibits the proliferation and induces apoptosis and cell cycle arrest of osteosarcoma stem cells by inhibiting the expression of PKCδ gene.