RsL3 Induces Ferroptosis In Multiple Myeloma Cells And Its Mechanism

Objective:To investigate whether RSL3 can induce ferroptosis in multiple myeloma cell line RPMI8226 and its mechanism.Methods:1.RPMI 8226 were treated with RSL3 and cell viability was assayed using CCK-8 kit;RPMI 8226 were treated with the indicated inhibitors(Fer-1;necrostatin-1;z-vad-fmk;CQ)with 0.5 u M RSL3 for 24 h and cell viability was assayed using CCK-8 kit.2.The lipid reactive oxygen species in each group was assayed by Fluorescence microscope and flow cytometry.The concentration of Fe2+ in each group was assayed using colorimetric method.3.The relative mRNA expression levels of the NCOA4 and FTH1 in each group were analyzed by Real-time qPCR;The relative protein expression levels of NCOA4 and FTH1 in each group were analyzed by Western Blot.4.RNA was extracted by the TRIzol method and the samples were sent to Shenzhen Huada Technology Co.,Ltd.for whole transcriptome sequencing(RNA-Seq).DNBSEQ platform was used to detect differentially expressed genes.cluster Profiler software was used to analyze the Gene Ontology(GO)function enrichment and KEGG pathway enrichment.String database(http://string-db.org/)was used to analyze the protein-protein interaction.Results:1.The cell viability was significantly decreased by RSL3(P <0.05);The growth inhibition effect of RSL3 on RPMI 8226 cells could partially reversed by Fer-1 and necrostatin-1.The effect of ferroptosis inhibitor Fer-1 is the most significant(P <0.05).2.The lipid reactive oxygen species were increased following RSL3treatment(p<0.05);The concentration of Fe2+ did not remarkably change following RSL3 treatment(p>0.05)3.RT-qPCR and Western Blot showed NCOA4 and FTH1 were expressed in RPMI8226 cells in each group.Compared with blank control group(0u M RSL3),mRNA and protein expression level of NCOA4 were down-regulated and mRNA and protein expression level of FTH1 were up-regulated under the stimulation of different concentrations of RSL3(P <0.05).4.Based on padj<0.05,compared with the control group,RNA-seq revealed 5 differentially expressed genes,namely FTH1、HMOX1、AKR1C2、B4GALNT1.all of them were up-regulated genes.According to Gene Ontology(GO)enrichment analysis,the cellular component were mainly concentrated in chromaffin granule、autolysosome and secondary lysosome;the molecular function were mainly concentrated in oxidoreductase activity、bile acid binding and ferric iron binding;the biological process were mainly concentrated in cellular iron ion homeostasis、iron ion homeostasis and intrinsic apoptotic signaling pathway in response to DNA damage.According to KEGG enrichment analysis,differentially expressed genes mainly enriched in ferroptosis、mineral absorption、Glycosphingolipid biosynthesis 、Porphyrin and chlorophyll metabolism and so on.Conclusion:1.RSL3 can induce ferroptosis in multiple myeloma cell.2.Ferritinophagy is involved in ferroptosis induced by RSL3 in multiple myeloma cells.3.RSL3 may exert related functions by regulating iron metabolism genes such as FTH1 and HMOX1.