叶下珠复方Ⅱ号对肝癌Huh7细胞miR-122表达及IGF-1R信号通路的调控作用

Regulatory Effect of Compound Phyllanthus urinsria II on miR-122 Expression and IGF-1R Signaling Pathway in Hepatoma Huh7 Cells

目的观察叶下珠复方Ⅱ号对肝癌Huh7细胞miR-122表达及胰岛素样生长因子-1受体(IGF-1R)信号通路的影响,探讨其抗肝癌作用机制。方法采用反义寡核苷酸技术(siRNA)建立miR-122表达抑制肝癌Huh7细胞株(anti-miR-122-Huh7细胞)。体外培养Huh7、anti-miR-122-Huh7细胞,分别设置对照组、叶下珠复方Ⅱ号高剂量组(3.0 mg·mL^-1)和低剂量组(1.5 mg·mL^-1)。采用噻唑蓝(MTT)比色法检测2种细胞增殖;流式细胞术检测anti-miR-122-Huh7细胞调亡;采用qRT-PCR法和Western Blot法分别检测miR-122及其转录因子C/EBPα、靶基因IGF-1R及下游AKT3、KRAS、ERK1和CyclinG1基因表达。结果与肝癌Huh7细胞比较,anti-miR-122-Huh7细胞miR-122的表达显著下降、靶基因IGF-1R蛋白表达明显升高(P<0.05),miR-122表达抑制肝癌Huh7细胞构建成功。与对照组比较,叶下珠复方Ⅱ号高、低剂量组作用于肝癌Huh7细胞后,可以显著上调miR-122的表达(P<0.05),并下调IGF-1R mRNA的表达(P<0.05);叶下珠复方Ⅱ号高、低剂量组均可显著抑制Huh7、anti-miR-122-Huh7细胞的增殖(P<0.05),且对anti-miR-122-Huh7细胞抑制作用更为明显(与Huh7细胞比较,P<0.05)。与对照组比较,叶下珠复方Ⅱ号高、低剂量组作用于anti-miR-122-Huh7细胞后,细胞晚期凋亡率及总凋亡率均显著升高(P<0.05);高剂量组的miR-122表达及miR-122的上游转录因子C/EBPα的mRNA和蛋白表达明显上调(P<0.05);高、低剂量组的IGF-1R及下游AKT3、KRAS、CyclinG1基因的mRNA、蛋白表达均显著下调(P<0.05),且ERK1蛋白表达亦明显下调(P<0.05)。结论叶下珠复方Ⅱ号可能通过提高miR-122的表达,抑制IGF-1R信号通路活化,从而抑制肝癌Huh7细胞增殖和促进其凋亡。

Objective To observe the effect of Compound Phyllanthus urinsria Ⅱ(CPU Ⅱ) on the expression of miR-122 and IGF-1 R signaling pathway in hepatoma Huh7 cells,and to explore its anti-hepatocarcinogenesis mechanism.Methods miR-122 expression inhibited liver cancer Huh7 cell line(anti-miR-122-Huh7 cells) was established using antisense oligonucleotide technology(siRNA).Huh7 cells and anti-miR-122-Huh7 cells were cultured in vitro,and the control group,high-dose CPU Ⅱ group(3.0 mg·mL^-1) and low-dose CPU Ⅱ group(1.5 mg·mL^-1) were set.The proliferation of cells were detected by Thiazolyl blue(MTT) colorimetry.Apoptosis of antimiR-122 Huh7 cells were detected by Annexin V-FITC/PI flow cytometry.The expression of miR-122 and its transcription factor C/EBPα,target gene IGF-1 R and downstream genes AKT3,KRAS,ERK1 and CyclinGl were detected by qRT-PCR and Western Blot.Results Compared with hepatoma Huh7 cells,anti-miR-122-Huh7 cells showed a significant decrease in miR-122 expression and a significant increase in target gene IGF-1 R protein expression,which indicates that the miR-122 expression inhibited liver cancer Huh7 cells were successfully established.Compared with the control group,the high-and low-dose CPU Ⅱ significantly up-regulated the expression of miR-122(P<0.05) and down-regulated the mRNA expression of IGF-1 R(P<0.05) in hepatoma Huh7 cells.The high-and low-dose CPU Ⅱ significantly inhibited the proliferation of Huh7 cells and anti-miR-122-Huh7 cells(P<0.05),and the inhibition effect of CPU Ⅱ on anti-miR-122-Huh7 cells was more obvious compared with that in Huh7 cells(P<0.05).Compared with the control group,after treating anti-miR-122-Huh7 cells with the high-and low-dose CPU Ⅱ,the late apoptotic rates and total apoptotic rates of the cells were significantly increased(P<0.05);the expression of miR-122 and the mRNA and protein expression of the upstream transcription factor C/EBPα of miR-122 in the high-dose CPU Ⅱ group were significantly up-regulated(P<0.05).The mRNA expression and protein expression of IGF-1 R and downstream genes AKT3,KRAS and CyclinGl in the high-and low-dose CPU Ⅱ group cells were significantly down-regulated(P<0.05),and the protein expression of ERK1 was significantly down-regulated(P<0.05).Conclusion CPU Ⅱ can inhibit the activation of IGF-1 R signaling pathway by increasing the expression of miR-122,thereby inhibit proliferation of Huh7 cells and promote their apoptosis.