【作者】 马斌；

【导师】 谢小冬； 白仲添；

【作者基本信息】 兰州大学 ， 生物学·遗传学， 2020， 硕士

【摘要】 目的:研究天然小分子化合物N-trans-feruloyloctopamine(FO)对人肝癌细胞系Huh7和HCCLM3细胞增殖、迁移及侵袭的影响,探讨FO抑制肝癌细胞生长的作用靶点及调控的关键机制。方法:体外培养肝癌Huh7和HCCLM3细胞,采用CCK-8试剂盒检测不同浓度FO对人肝癌细胞Huh7和HCCLM3增殖能力的抑制作用,确定IC50;采用细胞划痕实验和Transwell实验观察FO对Huh7和HCCLM3细胞迁移及侵袭能力的影响;通过Western Blot检测FO干预后Huh7和HCCLM3细胞中AKT、p38 MAPK、ERK1/2、p65的表达水平的影响,并进一步分析FO对人肝癌细胞EMT通路中关键蛋白Slug、Snail、N-cadherin、E-cadherin表达水平变化。用流式细胞术检测FO干预对细胞凋亡的影响;利用RNA-seq技术筛选FO干预后肝癌细胞中的差异基因,用DAVID生物信息学工具对差异基因进行GO和KEGG富集分析,进一步明确差异基因调节的功能和信号通路,结合GO和KEGG分析数据,筛选出FO调节的潜在靶基因。结果:1.细胞活性实验:不同浓度FO(0mΜ、0.1mΜ、0.5mΜ、1mΜ、2mΜ、4mΜ、10mΜ、20mΜ、50mΜ)对人肝癌细胞Huh7和HCCLM3的增殖具有抑制作用,且这种作用具有剂量依赖性(p<0.05),并且确定了FO半抑制浓度(IC50)的值分别为2.00mM、2.27mM;2.细胞表型实验:通过细胞划痕实验和Transwell实验发现FO具有明显抑制肝癌细胞侵袭的能力,用FO(2.00mM、2.27mM)干预Huh7细胞和HCCLM3细胞48h后,穿过的细胞数明显少于对照组,(p<0.05)。但是,划痕实验结果显示,用FO干预之后,细胞转移能力并没有受到明显的抑制;3.分子生物学实验:Western Blot结果显示人肝癌细胞Huh7和HCCLM3在FO(2.00mM、2.27mM)作用24h后,细胞中AKT、p38MAPK的磷酸化水平发生了明显的下降。在EMT信号通路中,E-cadherin的表达水平发生了增加,Slug的表达水平均降低,而Snail、N-cadherin的表达并没有发生明显变化(p<0.05);4.流式细胞术检测结果显示:FO对肝癌细胞的凋亡有促进作用,且这种作用具有时间依赖性。当细胞用FO处理48h时,凋亡细胞率明显高于FO处理24h的细胞凋亡率;5.RNA-seq数据显示:FO干预后,肝癌细胞中共有317个差异表达基因,其中有188个基因表达上调,129个基因表达下调。通过GO及KEGG富集分析发现,这些差异基因主要富集在细胞增殖、凋亡、细胞周期等GO条目中及PI3K-AKT、MAPK、apoptosis等信号通路中。结合GO及KEGG富集分析的结果,筛选出CDKN1A、BBC3、DDIT3、NOXA为FO潜在的靶点。结论:FO具有抑制肝癌细胞增殖和侵袭,促进凋亡的特性。FO通过调节AKT、p38 MAPK的磷酸化水平抑制肝癌细胞的增殖,通过调节E-cadherin和Slug的表达介导EMT信号通路并抑制肝癌的侵袭。FO干预后会有一组重要核心基因的表达差异及相关信号通路变化,其可以通过调节CDKN1A、BBC3、DDIT3、NOXA的表达介导肝癌细胞的PI3K-AKT、Apoptosis信号通路。这些候选基因有望成为肝癌诊断和治疗的潜在靶点。这为肝癌的靶向治疗及天然抗肿瘤药物的研究提供了理论基础和实验数据。更多还原

【Abstract】 objective: To investigate the effect of natural compound N-transferuloyloctopamine(FO)on the proliferation,migration and invasion of hepatocellular carcinoma cell lines Huh7 and HCCLM3,and to explore the role of FO in inhibiting the growth of liver cancer cells and its molecular mechanisms.Methods: Hepatocellular carcinoma cells Huh7 and HCCLM3 were cultured in vitro,and CCK-8 assays were used to verify the inhibitory effect of different concentrations of FO on the proliferation of HCC cells and the IC50 was calculated;The effects of FO on the migration and invasion ability of hepatocellular carcinoma cells were observed using wound-healing and Transwell assays;Western Blot was used to verify the phosphorylation levels of AKT,p38 MAPK,ERK1/2,and p65 in HCC and the expression levels of key proteins Slug,Snail,Ncadherin,and E-cadherin in the EMT pathway after FO was treated;Flow cytometry was performed to detect the role of FO on apoptosis,and using RNAseq technology to screen differentially expressed genes in Huh7 cells after FO treated;GO and KEGG enrichment analysis of differentially expressed genes using DAVID bioinformatics tools to further clarify the functions and signaling pathways of differential gene regulation,combined with GO and KEGG analysis data,to screen potential target genes for FO regulation.Results: 1.Different concentrations of FO(0mM,0.1mM,0.5mM,1mM,2mM,4mM,10 mM,20mM,50mM)can significantly inhibit Huh7 and HCCLM3 cells,this effect is dose-dependent(p<0.05).The IC50 of Huh7 and HCCLM3 cells were determined to be 2.00 mM and 2.27 mM,respectively.2.Transwell assay showed that FO could significantly reduce the invasion of hepatocellular carcinoma cells.When Huh7 and HCCLM3 cells were treated with FO(2.00 mM,2.27mM)for 48 h,the number of cells crossing the matrix membrane was significantly less than NC group(p<0.05).However,wound-healing assay showed that FO can delay the metastasis of liver cancer cells,but this inhibitory effect is not statistically significant.3.Western Blot data showed that phosphorylation of AKT and p38 MAPK was inhibited.Moreover,in the EMT signal pathway,the expression of E-cadherin was up-regulated and Slug was decreased,but the expression of Snail and Ncadherin did not change.When hepatocellular carcinoma cells were treated with FO for 24 hours(p< 0.05).4.Flow cytometry showed that FO could promote the apoptosis of HCC cells,and this effect was time-dependent.Compared with the treatment with FO for 24 hours,the apoptosis rate was significantly increased in 48 hours.5.RNA-seq data showed that there were 317 differentially expressed genes in Huh7 cells after FO was treated,of which 188 genes were up-regulated and 129 genes were down-regulated.GO and KEGG enrichment analysis found that the main GO terms enriched by these DEGs include: cell proliferation,apoptosis,cell cycle,etc.The main enriched KEGG pathways include: PI3K-AKT,MAPK,and apoptosis.Combined the data of GO and KEGG enrichment analysis,CDKN1 A,BBC3,DDIT3,and NOXA were screened as potential hub genes of FO.Conclusion: In HCC,FO can inhibit proliferation,invasion,migration and promote apoptosis.FO inhibits the proliferation of liver cancer cells by regulating the phosphorylation levels of AKT and p38 MAPK,regulates the expression of Ecadherin and Slug to inhibit the invasion of liver cancer cells by mediating the EMT signaling pathway.In addition,transcriptome sequencing results revealed that a group of important key genes and signaling pathways changed after FO intervention.Through our comprehensive bioinformatics analysis,we found that FO can mediate the PI3K-AKT and apoptosis signal pathway of liver cancer cells by regulating the expression of CDKN1 A,BBC3,DDIT3,and NOXA.These candidate genes are expected to be potential targets for the diagnosis and treatment of liver cancer.This provides theoretical basis and experimental data for the research of targeted treatment of liver cancer and natural antitumor drugs.更多还原