虫草素通过MEX3A抑制肝癌细胞增殖的机制研究

Mechanism of Inhibiting Hepatocellular Carcinoma Cell Proliferation by Cordycepin via MEX3A

[目的]探讨虫草素通过肌肉增生蛋白3A(muscle excess 3A,MEX3A)抑制肝癌细胞增殖的分子机制。[方法]以0、50、100、150、200及250μmol·L^(-1)浓度的虫草素分别处理Huh-7肝癌细胞24、48及72 h,采用细胞增殖检测(cell counting kit-8,CCK-8)法检测细胞活力,筛选出虫草素最佳干预浓度及时间。使用最佳干预浓度虫草素对Huh-7肝癌细胞进行干预,并进行高通量转录组测序,对测序结果进行生物信息学分析,筛选差异基因。设立空白对照组、MEX3A沉默组、MEX3A过表达组、虫草素组、虫草素+MEX3A沉默组和虫草素+MEX3A过表达组,以CCK-8法检测各组细胞活力,流式细胞术检测各组细胞的凋亡率及S期细胞比例,集落形成实验检测各组细胞的增殖情况。最后采用免疫印迹法检测各组细胞周期信号通路的关键蛋白细胞周期蛋白依赖性激酶4(cyclin-dependent kinase 4,CDK4)、细胞周期蛋白依赖性激酶6(cyclin-dependent kinase 6,CDK6)、细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖性激酶2(cyclin-dependent kinase 2,CDK2)及细胞周期蛋白E1(Cyclin E1)表达的变化情况。[结果]虫草素最佳干预浓度为200μmol·L^(-1),干预时间为48 h。高通量测序后转录组基因差异分析显示,虫草素组与空白对照组共存在564个差异基因,下调明显的基因集包括MEX3A基因。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析发现,明显下调的基因高度富集在细胞周期等促肝癌细胞增殖的信号通路。与空白对照组比较,虫草素可明显抑制Huh-7细胞中MEX3A的mRNA及蛋白表达(P<0.001)。虫草素干预后Huh-7细胞活力降低、克隆数减少、凋亡增加、S期细胞比例减少(P<0.01)。回复实验显示,虫草素+si-MEX3A使肝癌细胞的活力和克隆数进一步减少,凋亡率进一步增加(P<0.01),而S期细胞比例进一步减少(P<0.01)。免疫印迹结果显示,虫草素干预组的CDK4、CDK6、Cyclin D1、CDK2和Cyclin E1蛋白表达明显降低(P<0.01),虫草素+MEX3A沉默组上述蛋白表达进一步降低(P<0.01)。[结论]虫草素通过降低MEX3A表达调控细胞周期相关蛋白,从而抑制肝癌细胞的增殖。

[Objective]To investigate the molecular mechanism of inhibition of hepatocellular carcinoma cell proliferation by cordycepin via muscle excess 3A(MEX3A).[Methods]Cordycepin of 0,50,100,150,200 and 250µmol·L^(-1) concentration was set up to treat Huh-7 HCC cells for 24,48 and 72 h,respectively.cell counting kit-8(CCK-8)method was used to detect cell viability,and the optimal intervention concentration and time of cordycepin were selected.The optimal intervention concentration and time of cordycepin were used to intervene Huh-7 liver cancer cells and high-throughput transcriptome sequencing was performed,and the sequencing results were further bioinformatics analysis to screen differential genes.Blank control group,small interfering-MEX3A(si-MEX3A)group,overexpression MEX3A(OE-MEX3A)group,cordycepin group,cordycepin+si-MEX3A group and cordycepin+OE-MEX3A group were set up.The cell viability of each group was detected by CCK-8 method,the apoptosis rate and the proportion of S-phase cells were detected by flow cytometry,and the proliferation of cells in each group was detected by colony formation assay.Western blot was used to detect the expression changes of key proteins of cell cycle signaling pathway,including cyclin-dependent kinase 4(CDK4),cyclin-dependent kinase 6(CDK6),Cyclin D1,cyclin-dependent kinase 2(CDK2)and Cyclin E1 in each group.[Results]The optimal intervention concentration and time of cordycepin were 200µmol·L^(-1) and 48 h.Transcriptome gene difference analysis after high-throughput sequencing showed that there were 564 different genes between cordycepin group and blank control group,and the significantly down-regulated gene set included MEX3A gene.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis found that significantly down-regulated genes were highly enriched in cell cycle signaling pathways that promoted HCC cell proliferation.Compared with blank control group,cordycepin could significantly inhibit the mRNA and protein expression of MEX3A in Huh-7 cells(P<0.001).After cordycepin intervention,the activity of Huh-7 cells decreased,the number of clones decreased,apoptosis increased,and the proportion of S-phase cells decreased(P<0.01).The rescue experiment showed that cordycepin+si-MEX3A further reduced the cell viability and number of clones of Huh-7 cells(P<0.01),further increased the apoptosis rate(P<0.01)and further decreased the proportion of S-phase cells(P<0.01).Western blot showed that the expressions of CDK4,CDK6,Cyclin D1,CDK2 and Cyclin E1 in cordycepin group were significantly decreased(P<0.01),and cordycepin+si-MEX3A further decreased the expression of these proteins(P<0.01).[Conclusion]Cordycepin can inhibit the proliferation of hepatocellular carcinoma cells by decreasing MEX3A expression and regulating cell cycle related proteins.