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【目的】研究叶下珠复方Ⅱ号对肝癌Huh7细胞白细胞介素-6(IL-6)信号通路与微小RNA let-7a网络失衡的调节作用。【方法】以不同浓度的叶下珠复方Ⅱ号体外作用于人肝癌Huh7细胞株,采用四甲基偶氮唑盐(MTT)法检测药物对人肝癌Huh7细胞增殖的影响。设空白对照组、叶下珠复方II号高剂量组(48.0 g/L)和低剂量组(24.0 g/L),采用放射免疫法检测药物作用后肝癌Huh7细胞分泌IL-6的变化;采用茎环结构的逆转录实时荧光定量PCR方法检测肝癌Huh7细胞MicroRNA let-7a和miR-21的表达变化,采用实时荧光定量PCR方法检测药物作用后IL-6、核因子-κB亚基P65(NF-κB/P65)、转录活化因子(STAT3)、蛋白激酶2(AKT2)、PTEN、K-ras、c-myc、Bcl-2基因的mRNA表达变化,采用Westernblot法检测PTEN、STAT3、AKT2蛋白表达的变化。【结果】肝癌Huh7细胞经叶下珠复方Ⅱ号处理48 h,高、低剂量组Huh7细胞增殖率和上清液IL-6含量均显著下降,与空白对照组比较差异均具有统计学意义(P<0.01)。叶下珠复方Ⅱ号高、低剂量处理后microRNA let-7a表达量显著上升、miR-21的表达显著下降,与空白对照组比较差异均具有统计学意义(P<0.01);叶下珠复方Ⅱ号高、低剂量组与空白对照组比较,NF-κB/P65、STAT3、AKT2、K-ras、c-myc、Bcl-2基因mRNA的表达量均显著下降(P<0.01),而PTEN mRNA的表达显著增加(P<0.01),并呈明显的量效关系。叶下珠复方Ⅱ号高、低剂量组处理后PTEN的蛋白表达显著增加,STAT3、AKT2的蛋白表达显著减少,与空白对照组比较,差异均有统计学意义(P<0.01)。【结论】叶下珠复方Ⅱ号对人肝癌Huh7细胞增殖有明显的抑制作用,作用机制与抑制IL-6表达和IL-6/STAT3信号通路活化,调节IL-6信号通路与MicroRNA let-7a网络的失衡作用有关。
【OBJECTIVE】 To investigate the regulatory effect of Ye Xia Zhu Fu Fang No.2 on the imbalance of interleukin-6 (IL-6) signaling and the let-7a network of microRNA in Huh7 cells. 【Method】 The human hepatocellular carcinoma Huh7 cells were treated with different concentrations of Phyllanthus Compound Ⅱ in vitro. The effects of drugs on the proliferation of Huh7 cells were detected by MTT assay. The blank control group, Phyllanthus Compound II high-dose group (48.0 g / L) and low-dose group (24.0 g / L), the radioimmunoassay was used to detect the change of IL- RT-PCR was used to detect the expression of MicroRNA let-7a and miR-21 in hepatocellular carcinoma Huh7 cells. Real-time fluorescent quantitative PCR was used to detect the expression of IL-6, NF-κB subunit P65 The expressions of PTEN, STAT3, AKT2, PTEN, K-ras, c-myc and Bcl-2 mRNA were detected by Western blot. The change. 【Results】 The proliferation of Huh7 cells and the content of supernatant IL-6 in high and low dose Huh7 cells treated with Phyllanthus Compound Ⅱ for 48 h were significantly lower than those in blank control group (P <0.01) P <0.01). The expression level of microRNA let-7a in high and low dose of Phyllanthus Compound II was significantly increased and the expression of miR-21 was significantly decreased compared with the blank control group (P <0.01) The mRNA expression of NF-κB / P65, STAT3, AKT2, K-ras, c-myc and Bcl-2 in high and low dose group was significantly decreased compared with the blank control group (P <0.01) mRNA expression increased significantly (P <0.01), and showed a significant dose-effect relationship. The protein expression of PTEN was significantly increased and the protein expressions of STAT3 and AKT2 were significantly decreased in Phytophyllum xylinum complex Ⅱ treated group (P <0.01). 【Conclusion】 Yexiazhu Compound Ⅱ significantly inhibits the proliferation of human hepatocellular carcinoma Huh7 cells, and its mechanism of action and inhibition of IL-6 expression and activation of IL-6 / STAT3 signaling pathway, regulate IL-6 signaling pathway and MicroRNA let-7a Network unbalance effect.