叶下珠复方Ⅱ号对肝癌Hep3B细胞裸鼠移植瘤生长和miR-122及IGF-1R表达的影响

Effect of Compound Phyllanthus urinariaⅡon Growth and Expression of miR-122 and IGF-1R of Hepatocellular Carcinoma Hep3B Cells in Nude Mice

目的:观察叶下珠复方Ⅱ号对Hep3B2.1-7人肝癌细胞(简称Hep3B细胞)裸鼠移植瘤生长的抑制作用并探讨其机制。方法:建立肝癌Hep3B细胞裸鼠移植瘤模型,并随机分为模型组、叶下珠复方Ⅱ号高、低剂量组(57.5、28.75 g·kg^(-1))和5-氟尿嘧啶(5-FU)组(0.025 g·kg^(-1)),每组8只。叶下珠复方Ⅱ号高、低剂量组均灌胃给药,每日1次;模型组予同体积生理盐水灌胃;5-FU组采用腹腔注射,隔日1次。给药28 d后处死取移植瘤,以免疫组化(IHC)法检测瘤体组织细胞核抗原(PCNA)表达,以脱氧核糖核酸断裂原位末端标记(TUNEL)法检测瘤体组织细胞凋亡,以实时荧光定量聚合酶链式反应(Real-time PCR)检测移植瘤组织miR-122和胰岛素样生长因子-1受体(IGF-1R)的mRNA表达,以蛋白免疫印迹法(Western blot)检测瘤体组织CCAAT增强子结合蛋白α(C/EBPα)、肝细胞核因子-4α(HNF-4α)和IGF-1R的蛋白表达。结果:叶下珠复方Ⅱ号高、低剂量组和5-FU组的抑瘤率分别为74.90%、63.62%和64.15%,与模型组比较,5-FU组、叶下珠复方Ⅱ号各组的移植瘤瘤重均显著降低(P<0.01),5-FU组、叶下珠复方Ⅱ号各组肿瘤生长体积显著降低(P<0.01),5-FU组、叶下珠复方Ⅱ号各组移植瘤组织中细胞增殖PCNA阳性细胞变少、染色变浅,5-FU组、叶下珠复方Ⅱ号各组移植瘤组织凋亡细胞均明显增加(P<0.05,P<0.01),5-FU组、叶下珠复方Ⅱ号各组miR-122 mRNA表达均显著增加(P<0.01),5-FU组、叶下珠复方Ⅱ号各组IGF-1R mRNA表达显著下调(P<0.01),5-FU组、叶下珠复方Ⅱ号各组裸鼠移植瘤组织C/EBPα及HNF-4α的蛋白表达均显著上调(P<0.01),叶下珠复方Ⅱ号高剂量组的IGF-1R蛋白表达明显下调(P<0.05);与叶下珠复方Ⅱ号低剂量组比较,叶下珠复方Ⅱ号高剂量组的凋亡细胞显著增多(P<0.01),叶下珠复方Ⅱ号高剂量组miR-122 mRNA表达显著上调(P<0.01),叶下珠复方Ⅱ号高剂量组的C/EBPα和HNF-4α蛋白表达均显著上调(P<0.01)。结论:叶下珠复方Ⅱ号对Hep3B细胞裸鼠移植瘤生长具有明显的抑制作用,作用机制可能与增强转录因子HNF-4α和C/EBPα的表达从而增强miR-122的表达以抑制其靶基因IGF-1R的表达有关。

Objective:To investigate the inhibitory effects and mechanism of the compound Phyllanthus urinariaⅡ(CPUⅡ)on the growth of transplanted hepatocellular carcinoma Hep3B2.1-7(Short for Hep3R)cells in nude mice.Method:After the establishment of a xenograft model of hepatocellular carcinoma Hep3B cells in mice,the model mice were randomly divided into a model group,a high-dose CPUⅡgroup(57.5 g·kg^(-1)),a low-dose CPUⅡgroup(28.75 g·kg^(-1)),and a 5-fluorouracil(5-FU)group(0.025 g·kg^(-1)),with eight mice in each group.The mice in the high-and low-dose CPUⅡgroups were treated with drugs by gavage,once per day,and those in the model group were treated with the same volume of normal saline.The mice in the 5-FU group were treated by 5-FU by intraperitoneal injection,once every other day.After 28 days of administration,mice were sacrificed,and transplanted tumors were collected.Immunohistochemistry(IHC)was used to detect the expression of proliferating cell nuclear antigen(PCNA)of tumor tissues.Terminaldeoxynucleotidyl transferase-mediated nick end labeling(TUNEL)was used to detect cell apoptosis of tumor tissues.The mRNA expression of miR-122 and insulin-like growth factor 1 receptor(IGF-1R)in tumor tissues was detected by Real-time quantitative PCR(Real-time PCR).The protein expression of CCAAT/enhancerbinding proteinα(C/EBPα),hepatocyte nuclear factor-4α(HNF-4α),and IGF-1R in tumor tissues was detected by Western blot.Result:The tumor suppression rates of the high-and low-dose CPUⅡgroups and the 5-FU group were 74.90%,63.62%,and 64.15%,respectively.Compared with the model group,the CPUⅡgroups and the 5-FU group showed reduced weight(P<0.01)and volume of tumors(P<0.01),decreased PCNA positive cells,shallow staining,increased apoptosis cells of transplanted tumor tissues(P<0.05,P<0.01),increased expression of mRNA expression of miR-122(P<0.01),down-regulated mRNA expression of IGF-1R(P<0.01),and up-regulated protein expression of C/EBPαand HNF-4αin nude mouse transplanted tumor tissues(P<0.01).The expression of IGF-1R protein in the high-dose CPUⅡgroup was down-regulated(P<0.05).Compared with the low-dose CPUⅡgroup,the high-dose CPUⅡgroup showed increased apoptotic cells(P<0.01),up-regulated mRNA expression of miR-122(P<0.01),and increased expression of C/EBPαand HNF-4αproteins(P<0.01).Conclusion:CPUⅡhas an obvious inhibitory effect on the growth of transplanted hepatocellular carcinoma Hep3B cells in nude mice.The mechanism of action is related to enhancing the expression of transcription factors HNF-4αand C/EBPα,thereby promoting the expression of miR-122 and inhibiting the expression of its target gene IGF-1R.