摘要
【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 observe the regulatory effect of Compound Phyllanthus urinaria Ⅱ （CPUⅡ ） on interleukin 6 （IL-6） signaling pathway and microRNA let-7a network imbalance in human hepatoma Huh7 cells. Methods After treatment of Huh7 cells with different concentrations of CPU Ⅱ, the proliferation of Huh7 cells was tested by methyl thiazolyl tetrazolium （MTT） assay. Blank control group and high- and low-dose CPU Ⅱ groups （ cultured with CPU Ⅱ in the dosage of 48.0 and 24.0 g/L respectively） were set up. The change of IL-6 secreted by Huh7 cells was detected by using radioimmunoassay （RIA）. The microRNA let-7a and miR-21 expression levels were measured by the method of stem-loop real-time fluorescence quantitative polymerase chain reaction （PCR）. IL-6, NF-κB/P65, STAT3, AKT2, K-ras, Bcl-2 and c-myc mRNA expression levels were detected by real-time fluorescence quantitative PCR, and PTEN, STAT3 and AKT2 protein expression levels were analyzed by Western blot method. Results After culturing for 48 hours, the proliferation rate of Huh7 cells and IL-6 content in the culture supernatant were reduced, microRNA let-7a expression level was elevated, miR-21 was decreased, the mRNA expression levels of IL-6, NF-κB/P65, STAT3, AKT2, K-ras, Bcl-2 and c-myc were obviously decreased （P 〈 0. 01 ）, PTEN mRNA expression was increased in high- and low-dose CPU I! groups when compared with the blank control group （P 〈0. 01 ）, and the effects were in dose-dependent manner. High- and low-dose CPU II groups also had higher protein expression level of FFEN, and had lower STAT3 and AKT2 protein expression levels than the blank control group, the difference being significant （P 〈 0. 01 ）. Conclusion CPU II can effectively inhibit the proliferation of human hepatoma Huh7 cells in vitro, and its therapeutic mechanism may be related with the regulation of imbalance of IL-6 signaling pathway and microRNA let-7a network through the inhibition of IL-6 expression and IL-6/STAT3 signal pathway activation
出处
《广州中医药大学学报》
CAS
北大核心
2012年第6期683-690,750,共9页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
广东省科技计划重点资助项目(编号:2011A030100008)
关键词
叶下珠复方Ⅱ号
药理学
肝癌
中药疗法
信号通路
细胞培养
基因表达调控
COMPOUND PHYLLANTHUS URINARIA Ⅱ （CPU Ⅱ） /pharmacology
LIVER CANCER/TCD therapy
SIGNALING PATHWAY
CELL CULTURE
GENE EXPRESSION REGULATION
