Mechanism of Inhibiting Hepatocellular Carcinoma Cell Proliferation by Cordycepin via MEX3A
[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 200 µmol·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.
cordycepintranscriptome sequencingbiological information analysishepatocellular carcinomaMEX3Acell cycle signaling pathwaycell proliferationcell apoptosis
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维普
