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目的探讨紫杉醇诱导肝癌HepG2细胞凋亡时miRNA对基因的沉默的作用。方法用不同浓度紫杉醇处理肝癌HepG2细胞后,流式细胞术检测其细胞凋亡;通过实时定量PCR(Real-time PCR)测定miR-224和凋亡抑制因子5(api-5)mRNA的表达;用免疫组织化学二步法和免疫印迹法(Western blotting)检测API-5蛋白的表达。结果紫杉醇诱导HepG2细胞凋亡时,通过2-ΔΔCT法分析发现,除了0.5mg/L、1mg/L紫杉醇培养HepG2肝癌细胞24h miR-224不升反降外,其他浓度和处理时间miR-224表达均显示上调;0.5mg/L、1mg/L紫杉醇培养HepG2肝癌细胞24h api-5 mRNA表达稍微降低,0.5mg/L紫杉醇处理48h无增长,其他浓度和处理时间api-5 mRNA表达均上调;应用IPP v 5.1图像分析软件处理,经t检验分析发现,紫杉醇处理后API-5蛋白表达均下调(P<0.05)。结论上调的miR-224可能作用其靶基因api-5的表达,通过与api-5 mRNA的3’UTR结合阻止翻译的完成,从而对肝癌细胞的凋亡途径产生影响。
Objective To investigate the role of miRNAs in gene silencing induced by paclitaxel in HepG2 cells. Methods Hepatoma cell line HepG2 was treated with different concentrations of paclitaxel. Flow cytometry was used to detect the apoptosis of HepG2 cells. The mRNA expression of miR-224 and api-5 was detected by real-time PCR. The expression of API-5 protein was detected by immunohistochemical two-step method and Western blotting. Results Paclitaxel-induced apoptosis of HepG2 cells by 2-ΔΔCT method found that, except for 0.5mg / L, 1mg / L paclitaxel cultured HepG2 hepatoma cells 24h miR-224 does not rise and fall, other concentrations and processing time miR-224 expression The expression of api-5 mRNA in HepG2 hepatocarcinoma cells treated with 0.5 mg / L and 1 mg / L paclitaxel was slightly decreased, while no significant increase was observed at 48 h after treatment with 0.5 mg / L paclitaxel. The expression of api-5 mRNA at other concentrations and treatment time was increased. IPP v5.1 image analysis software processing, t-test analysis showed that paclitaxel treatment API-5 protein expression were down (P <0.05). Conclusion Upregulated miR-224 may affect the expression of api-5, a target gene of api-5 mRNA, and block the translation of api-5 mRNA by binding to the 3’UTR of api-5 mRNA, thereby affecting the apoptotic pathway of hepatoma cells.