中药复方敷和备化方对CD133^(+)HepG2肝癌干细胞增殖及相关自噬蛋白影响的机制

Mechanism underlying the effect of Fuhebeihua Recipe on the proliferation of CD133^(+)HepG2 hepatoma stem cells and related autophagy proteins

背景:肝癌干细胞是肝癌不断发生发展和高异质性产生的关键细胞,目前尚无针对肝癌干细胞相关自噬通路为靶点的中药复方研究,因此中药复方抑制肝癌干细胞自噬的研究十分具有必要性。目的:探讨中药复方制剂敷和备化方对CD133^(+)HepG2肝癌干细胞PI3K/AKT/mTOR通路及相关自噬蛋白影响的机制。方法:免疫磁珠法分选出CD133^(+)HepG2细胞,用体积分数为2%,4%,8%,16%敷和备化方含药血清作用CD133^(+)HepG2细胞,CCK-8法检测细胞增殖活力,选出最佳干预体积分数的敷和备化方含药血清。将CD133^(+)HepG2细胞分为血清对照组、空白对照组、雷帕霉素组和敷和备化方组,分别用含体积分数16%空白大鼠血清的DMEM/F12培养基、无血清DMEM/F12培养基、含0.05%雷帕霉素的DMEM/F12培养基、含体积分数16%敷和备化方含药血清的DMEM/F12培养基培养。流式细胞术检测各组CD133^(+)HepG2肝癌干细胞百分比;RT-PCR法检测各组细胞中PI3K、AKT、mTOR、LC3-Ⅱ、Beclin-1的mRNA表达水平;Western blot法检测各组细胞中PI3K、p-AKT、AKT、p-mTOR、mTOR、LC3-Ⅱ的蛋白表达水平;免疫荧光法检测各组细胞中LC3、LC3-Ⅱ的表达水平。结果与结论:①敷和备化方含药血清有抑制CD133^(+)HepG2细胞增殖的作用,其中体积分数为16%敷和备化方含药血清干预72 h抑制作用最明显(P<0.05);②与血清对照组、空白对照组相比,敷和备化方组和雷帕霉素组CD133^(+)HepG2细胞百分比下降(P<0.05),PI3K、AKT、p-mTOR、mTOR、LC3-Ⅱ的蛋白表达水平均明显下调(P<0.05);PI3K、AKT、mTOR、LC3-Ⅱ、Beclin-1的mRNA表达水平均明显下调(P<0.05);LC3、LC3-Ⅱ的荧光信号强度均明显下调(P<0.05);③与雷帕霉素组相比,敷和备化方组CD133^(+)HepG2细胞中的PI3K、p-AKT、AKT、p-mTOR、mTOR、LC3-Ⅱ蛋白表达水平均明显下调(P<0.05),PI3K、AKT、mTOR、LC3-Ⅱ、Beclin-1的mRNA表达水平均明显下调(P<0.05),LC3、LC3-Ⅱ的荧光信号强度均明显下调(P<0.05);④结果表明,敷和备化方能抑制肝癌干细胞自噬和增殖,其机制与抑制PI3K/AKT/mTOR信号通路有关。

BACKGROUND:Hepatoma stem cells are key cells for the continuous development and high heterogeneity of liver cancer.At present,there is no research on Chinese medicine compound that targets hepatoma stem cell-related autophagy pathways.Therefore,it is very necessary to study Chinese medicine compound prescription to inhibit hepatoma stem cell autophagy.OBJECTIVE:To investigate the effect and mechanism of Fuhebeihua Recipe on PI3K/AKT/mTOR pathway and related autophagy proteins in CD133^(+)HepG2 hepatoma stem cells.METHODS:CD133^(+)HepG2 cells were selected by immunomagnetic bead method.CD133^(+)HepG2 cells were treated with different volume fractions of Fuhebeihua Recipe-containing serum of 2%,4%,8%,and 16%.The cell proliferation activity was detected by CCK-8 assay.The best volume fraction of Fuhebeihuo Recipe-containing serum was selected.CD133^(+)HepG2 cells were divided into blank control group,serum control group,rapamycin group,and Fuhebeihua Recipe group.Cells were separately cultured with DMEM/F12 containing serum of 16%,serum-free DMEM/F12,DMEM/F12 containing 0.05%rapamycin,and DMEM/F12 with Fuhebeihua Recipe-containing serum of 16%.The percentage of CD133^(+)HepG2 hepatoma stem cells was detected by flow cytometry in each group.The mRNA expression levels of PI3K,AKT,mTOR,LC3-Ⅱ,and beclin-1 were detected by RT-PCR.The protein expression levels of PI3K,p-AKT,AKT,p-mTOR,mTOR,and LC3-Ⅱ were detected by western blot assay.The expression levels of LC3 and LC3-Ⅱ were detected by immunofluorescence in each group.RESULTS AND CONCLUSION:(1)The Fuhebeihua Recipe-containing serum inhibited the proliferation of CD133^(+)HepG2 cells,and the inhibitory effect was the most obvious when the volume fraction was 16%(P<0.05).(2)Compared with serum control group and blank control group,the percentage of CD133^(+)HepG2 cells reduced(P<0.05);protein expression levels of PI3K,AKT,p-mTOR,mTOR,and LC3-Ⅱ were significantly decreased(P<0.05);mRNA expression levels of PI3K,AKT,mTOR,LC3-Ⅱ,and Beclin-1 were significantly down-regulated(P<0.05);the fluorescence signal intensities of LC3 and LC3-Ⅱ were significantly downregulated(P<0.05)in the Fuhebeihuo Recipe group and rapamycin group.(3)Compared with the rapamycin group,the protein expression levels of PI3K,p-Akt,AKT,p-mTOR,mTOR,and LC3-Ⅱ in CD133^(+)HepG2 cells were significantly down-regulated(P<0.05);the mRNA expression levels of PI3K,AKT,mTOR,LC3-Ⅱ,and Beclin-1 were significantly decreased(P<0.05);the fluorescence signal intensities of LC3 and LC3-Ⅱ were significantly decreased in the Fuhebeihuo Recipe group(P<0.05).(4)It is concluded that Fuhebeihua Recipe can inhibit autophagy and proliferation of hepatoma stem cells,and its mechanism is related to the inhibition of PI3K/AKT/mTOR signaling pathway.