翼核果素对人肝癌细胞HepG2裸鼠移植瘤的抑制作用及其机制探讨

Inhibitory effect and mechanism of ventilagolin on subcutaneous tumor of human hepatocellular carcinoma cells HepG2 transplanted in nude mice

目的:观察翼核果素对人肝癌细胞HepG2裸鼠移植瘤生长的抑制作用,并探讨其机制。方法:通过皮下注射肝癌HepG2细胞的方法构建荷肝癌BALB/C裸鼠模型,将50只荷肝癌裸鼠随机分为5组,每组10只,分别为模型组、氟尿嘧啶(20 mg/kg)组、翼核果素低、中、高剂量(1 mg/kg、2 mg/kg、4 mg/kg)组,隔天腹腔注射给药1次,模型组给予同体积的生理盐水,两周后处死裸鼠,剥离瘤块称重并计算抑瘤率。采用illumina HiseqTM测序平台对翼核果素(100μmol/L)Test组和Normal组的HepG2细胞进行转录组测序(RNA sequencing,RNA-seq),并对结果进行基因差异表达、基因功能和信号通路富集分析。根据RNA-seq结果,采用qPCR检测c-Jun氨基末端激酶(JNK)、肿瘤坏死因子(TNF)、Fas相关死亡结构域蛋白(FADD)、白介素-1(IL-1)、p53等基因的表达水平。结果:与模型组比较,翼核果素高剂量组瘤块重量显著减轻(P<0.01),抑瘤率显著升高。RNA-seq结果显示,翼核果素处理HepG2细胞后共有3052个基因的表达发生显著变化,其中1037个基因上调,1979个基因下调(P<0.05),这些差异基因主要富集在MicroRNAs、MAPK signaling pathway、Spliceosome、p53 signaling pathway、TNF signaling pathway、mTOR signaling pathway等通路,影响肝癌细胞的增殖、免疫、周期、凋亡及代谢等功能。qPCR检测发现,100μmol/L翼核果素能够显著提高JNK、IL-1和p53的表达,降低TNF和FADD的表达。结论:翼核果素对人肝癌细胞HepG2裸鼠移植瘤生长具有一定的抑制作用,其机制可能与翼核果素调节肝癌细胞增殖、免疫、周期、凋亡及代谢等功能有关,其中MAPK、p53和TNF信号通路可能起关键性调控作用。

Objective:To investigate the inhibitory effect and mechanism of Ventilagolin on subcutaneous tumor of human hepatocellular carcinoma cells HepG2 transplanted in nude mice.Methods:The xenograft BALB/C mice model was established by subcutaneous injection with HepG2 cells.Fifty nude mice tumor-bearing models were randomly divided into five groups,with 10 mice in each group,including the model group,the fluorouracil(20 mg/kg)group,and the low(1 mg/kg),medium(2 mg/kg)and high(4 mg/kg)dose ventilagolin group.Fluorouracil and three ventilagolin groups were given corresponding drug once every other day for 2 weeks using by intraperitoneal injection,and the model group was given the same volume of normal saline.Two weeks later,nude mice were sacrificed,the tumor mass was stripped and weighed to calculate the tumor inhibition rate.Illumina HiseqTM sequencing platform was used to perform transcriptional sequencing(RNA sequencing,RNA-seq)on HepG2 cells from the ventilagolin(100μmol/L)test group and the control group,and the results were analyzed for gene differential expression,gene function and signal pathway enrichment.qRTPCR was used to detect the expression levels of cJun N-terminal kinase(JNK),tumor necrosis factor(TNF),fas-associated death domain protein(FADD),interleukin 1(IL-1),p53 and other genes.Result:Compared with model group,the tumor weight of Ventilagolin high-dose group were significantly decreased(P<0.01)and tumor inhibition rate were significantly increased.RNA-seq results showed that there were 3025 genes differently expressed after ventilagolin treatment,of which 1037 genes were up-regulated and 1979 genes were down-regulated(P<0.01).These differentially expressed genes were mainly enriched in MicroRNAs,MAPK signaling pathway,Spliceosome,p53 signaling pathway,TNF signaling pathway,mTOR signaling pathway.And these differentially expressed genes mainly affected cell proliferation immunization,cycle,apoptosis and metabolism related functions.q RT-PCR assay found that 100μmol/L of ventilagolin could significantly increase the expressions of JNK,IL-1 and p53,and decrease the expressions of TNF and FADD.Conclusion:Ventilagolin has inhibitive effects on tumor growth in nude mice tumor-bearing models,which may related to the regulation of hepatic cell proliferation,immunization,cycle,apoptosis and metabolism related functions.MAPK,p53 and TNF pathways might play a key regulatory role in it.