Study on mechanism of apoptosis and immunogenic death of cervical cancer cells induced by shikonin
Objective To investigate the molecular expression changes of cervical cancer cell apoptosis and immunogenic death induced by shikonin.Methods Through network pharmacology,bioinformatics,and WGCNA method,the molecular mechanism of shikonin induced apoptosis of cervical cancer cells was investigated;the target of shikonin was obtained from CTD database;the differential genes of cervical cancer were analyzed by GEO database and WGCNA,and the disease targets were obtained after intersection,and the therapeutic targets were obtained after mapping between drug targets;proteinprotein interaction was used to STRING database;DAVID database for gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.In vitro experiments,the survival rate of human cervical cancer SiHa cells treated with 5-80 μmol/L shikonin for 48 h was measured by CCK8 method;SiHa cells were treated with 10 μmol/L shikonin(low concentration group)and 20 μmol/L shikonin(high concentration group)for 48 h,and cell apoptosis,immunogenic death related molecules,and co-stimulatory molecules were detected by flow cytometry.In addition,20 ng/μl 5-fluorouracil was used as positive control group,and no shikonin was used as blank control group.Results A total of 1 989 differential genes of cervical cancer were screened by WGCNA,and 91 KEGG fluorouracil,884 biological processes,81 molecular functions,and 21 cell components were identified by DAVID database.PPI network showed that shikonin was closely related to TP53 and CASP3 targets in cervical cancer cells.Shiksin interfered with the core targets of cervical cancer,mainly acting on apoptosis protein pathway,negative regulatory pathway of immune system process,lymphocyte activation involved in immune response,and regulatory pathway of apoptosis execution stage.Shikonin can significantly inhibit the proliferation of SiHa cells,with IC50 value of 29.62 μmol/L.The cell apoptosis rate,and the expression levels of caspase-3,caspase-9,CRT,HSP70,PD-L1,and Galectin9 in low concentration and high concentration groups were higher than those in blank control group(P＜0.05);the cell apoptosis rates,and the expression levels of caspase-3,caspase-9,CRT,HSP70,PD-L1,and Galectin9 in high concentration group were higher than those in low concentration group(P＜0.05).Conclusion Shikotin can induce apoptosis of cervical cancer cells by acting on TP53,CASP3 and other targets,and can also induce immunogenic cell death.
ShikoninCervical cancerNetwork pharmacologyImmunogenic deathImmunodetection site
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