靶向抑制Hedgehog信号通路对肝细胞癌增殖、凋亡和转移的影响

摘要

目的 观察靶向抑制Hedgehog信号通路对肝细胞癌增殖、转移的影响.方法 构建Smo shRNA质粒,建立Smo稳定低表达的肝癌QGY-7701细胞系.采用流式细胞术、CCK8及Transwell实验检测Smo基因沉默前后QGY-7701细胞周期、凋亡、增殖及转移能力的变化;通过裸鼠皮下种植模型研究Smo基因沉默对肝癌细胞体内生长的抑制作用,电镜下观察肝癌细胞超微结构的变化.结果 实时PCR(RT-PCR)和蛋白印迹(Western blot)检测显示成功构建shR-Smo稳定基因转染的肝癌细胞系.流式细胞周期实验显示,与对照组比较,shR-Smo组G0/G1期细胞增加,S期细胞减少;凋亡、CCK8及Transwell实验显示Smo基因沉默能够显著增加肝细胞癌凋亡百分率到(5.46±1.46)％,增殖活性显著下降,迁移率减少到(7.82±2.14)％;裸鼠皮下种植模型显示Smo基因沉默能够明显抑制体内肝癌细胞的生长,电镜发现Smo基因沉默后肝癌细胞中溶酶体明显增多.结论 阻断Hedgehog信号通路可降低肝细胞癌恶性程度,靶向抑制Hedgehog信号通路有望成为肝细胞癌基因治疗的新选择.%Objective To observe Hedgehog signaling pathways of liver cancer cell growth and the influence of the metastatic potential targeted inhibit Hedgehog.Methods Construction of Smo shRNA plasmid,The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened after lipofection.The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened,The cell cycle,apoptosis,invasion and metastasis of QGY-7701 cells were detected by Western blot,flow cytometry,CCK8 and transwell assay.Subcutaneous implantation of hepatocarcinoma cells in nude mice.Study on the growth and metastasis of hepatocarcinoma cells with low expression of Smo in.The ultrastructural changes of hepatoma cells with low expression of Smo were observed under electron microscope.Results RT-PCR and Western blot showed stable shR-Smo cell line was successfully constructed.Cell cycle test showed that compared with the control group,G0/G1 cells increased in shR-Smo,cells in S phase decreased;apoptosis,CCK8 and Transwell tests showed that Smo-gene silencing could significantly increase the apoptosis percentage of the hepatic cancer cells to (5.46％ ± 1.46％),proliferation activity decreasedand and the migration rates reduced to (7.82％ ±2.14)％;nude mice model showed that Smo-gene silencing could inhibit the growth of hepatocellular carcinoma cells in vivo,electron microscopy revealed that lysosomes increased significantly in Smo-gene silence cells.Conclusions Blocking Hh signaling pathways,liver cancer cells in vitro malignant degree of decline.Hedgehog in treating liver cancer have hidden meaning.