CISD2 Promotes Resistance To Sorafenib-induced Ferroptosis By Regulating Beclin1-dependent Autophagy In Hepatocellular Carcinoma Cells

Objective: Sorafenib is the first target drug for advanced hepatocellular carcinoma(HCC)patients in China.However,many HCC patients do not respond well to sorafenib or develop drug resistance after several months of treatment.The drug resistance of HCC patients to sorafenib mostly appeared after 6 months of treatment.Previous studies on tumor targeted therapy mainly focused on apoptosis,necrosis,autophagic death and other forms of cell death.However,tumor cells can avoid apoptosis and necrosis by increasing the phenotype of drug resistance and promoting the expression of Ras and other signaling pathways,and the effect of drugs based on apoptosis,autophagy and other mechanisms in clinical trials is not ideal,which indicates that tumor cells may promote their own resistance to chemotherapy drugs through other forms.Therefore,it is of great significance to understand the death mode of tumor cells for antagonizing drug resistance.Current studies have shown that there is a close relationship between tumor resistance and ferroptosis.Ferroptosis is a severe form of death,which is caused by the imbalance of reactive oxygen species(ROS)induced by excessive accumulation of iron ions in cells,and then leads to the peroxidation and rupture of membrane lipid structure of cell membrane and organelle.Ferroptosis is accompanied by the loss of antioxidant enzyme function,such as glutathione peroxidase 4(GPX4),The expression of GPX4 was inhibited.High expression genes with prominent phenotype in tumor resistance,such as GPX4,nuclear factor erythroid-2-related factor 2(NRF2),are the core negative regulators of ferroptosis,and ferroptosis has been gradually applied to the research of experimental compounds and clinical drugs.In addition,the inherent high iron content of tumor cells reacts with abundant polyunsaturated fatty acids,which provides a favorable material reserve for ferroptosis.Therefore,the use of ferroptosis targeted treatment of tumor resistance has certain advantages.Currently,a variety of drugs including sorafenib can induce ferroptosis in tumor cells.Exploring the mechanism of ferroptosis and its role in tumor cells is expected to provide new and effective strategies for targeted therapy of HCC.The iron sulfur domain(C-X-C-X2-(S/T)X3-P-X-C-D-G-(S/A/T)-H,CDGSH)protein family plays a key role in the regulation of iron ion,reactive oxygen species homeostasis and autophagy.CDGSH iron sulfur domain 2(CISD2),as a member of this protein family,has been proved to be related to the occurrence and development of many cancers.However,the role of CISD2 in HCC drug resistance has not been reported.Studies have shown that autophagy plays an important role in tumor cell homeostasis and ferroptosis,but the balance between drug resistance and ferroptosis is the current research difficulty.This study aims to explore the role of CISD2 in sorafenib resistance and the regulatory mechanism of ferroptosis,and explore the role of CISD2 in promoting the resistance of HCC drug-resistant cells to ferroptosis by regulating autophagy,so as to provide a new strategy for targeted therapy of sorafenib resistance in HCC.Methods: 1.In this study,we analyzed the expression of ferroptosis negative regulatory genes in Gene Expression Omnibus(GEO)resistant samples.The RNA sequence data of369 HCC patients’ tumor tissues and 50 HCC patients’ adjacent tissues were compared in the Cancer Genome Atlas(TCGA)database.The survival curve of patients was analyzed by Kaplan-Meier method.The correlation between CISD2 expression in HCC and prognosis of patients was confirmed.At the cellular level,real-time quantitative polymerase chain reaction(Real-time PCR)and western blot were used to explore the expression of CISD2 in HCC cell lines.2.In order to construct the sorafenib resistant model,the concentration gradient culture method was used to construct HCC sorafenib resistant cell line.Cell counting kit-8(CCK-8)was used to detect the cell activity under different concentrations of sorafenib.Western blot was used to detect the sorafenib target extracellular regulated protein kinases(ERK)and phosphorylated ERK(p-ERK)were detected.3.CCK-8 was used to detect the changes of cell proliferation after sorafenib was treated with ferrostatin-1(Fer-1),deferoxamine(DFO),Z-Val-Ala Asp(OMe)-FMK(ZV AD-FMK)and necrosulfonamide(NSA),The level of apoptosis was detected by flow cytometry.4.In order to investigate the effect of CISD2 on drug-resistant HCC cells,we used liposome transfection technology to construct stable and low expression of CISD2 in drug-resistant HCC cells.Real-time PCR and western blot were used to detect the expression of CISD2 after transfection.CCK-8 method was used to detect the activity changes of drug-resistant cells treated with sorafenib,Fer-1,DFO,ZVAD-FMK and NSA,the apoptosis of sorafenib resistant cells was detected by flow cytometry.5.In order to investigate the effect of CISD2 knockdown on ferroptosis in resistant HCC cells,the expression of ROS and malondialdehyde(MDA)were detected by 2,7-Dichlorodi hydrofluorescein diacetate(DCFH-DA)staining and lipid peroxidation kit respectively,the expression of glutathione(GSH)was detected by micro reduced glutathione kit,and the expression of iron was detected by calcein-acetoxymethyl ester(Ca-AM)fluorescent probe and flow cytometry.6.In order to clarify the effect of CISD2 on ferroptosis of HCC drug-resistant cells by regulating autophagy,we used gene set enrichment analysis(GSEA)website to analyze CISD2 in HCC database samples.Western blot was used to detect microtubule associated proteins 1A / 1B light chain 3(LC3)after CISD2 knockdown,western blot was used to detect the effect of CISD2 knockdown on mammalian target of rapamycin(m TOR)pathway related proteins,including phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),the protein expression levels of serine / threonine protein kinase(AKT),phosphorylated AKT(p-AKT),m TOR and phosphorylated m TOR(p-m TOR)were measured.Trypan blue dye was used to detect the changes of cell death level after CISD2 knockdown in 3-methyladenine(3-MA)or bafilomycin A1(Baf A1)treatment.Flow cytometry was used to detect the expression levels of ROS and iron ions in HCC drug-resistant cells after inhibition of autophagy.The expression levels of MDA and GSH were detected by Kit.7.Real-time PCR was used to detect the expression of autophagy related genes after CISD2 knockdown.Western blot was used to detect the expression of Beclin1 after CISD2 knockdown.The stable and low expression of Beclin1 was constructed by liposome transfection.The expression of Beclin1 was detected by Realtime PCR and western blot.After co-treatment with CISD2 inhibitor pioglitazone(Pg)and knockdown of Beclin1,the expression of autophagy related protein LC3 II / I was detected by western blot,the number of LC3 fluorescent spots was detected by immunofluorescence,and the expression of MDA was detected by lipid peroxidation kit.Result: 1.The expression of ferroptosis negative regulatory gene in GEO sorafenib resistant samples showed that CISD2 was relatively high expression,TCGA database showed that CISD2 was significantly up regulated in HCC,Kaplan-Meier survival analysis showed that the survival rate of CISD2 high expression patients was lower than that of low expression patients.The Real-time PCR and western blot results showed that the expression of CISD2 in HCC cells was higher than that of normal hepatocytes.2.The activity of the drug-resistant cells constructed by concentration gradient method was improved with different concentrations of sorafenib,and the expression of p-ERK in drugresistant cells could not be inhibited by sorafenib.3.The effect of sorafenib on HCC cell apoptosis was not as good as that of staurosporine(STS),which was not inhibited by ferroptosis blockers Fer-1 and DFO,but not by ZVAD-FMK and NSA.4.The results of flow cytometry showed that the apoptosis effect of CISD2 was not obvious,the low CISD2 promoted the increase of ROS expression,MDA expression of lipid peroxide and iron ion accumulation in the drug-resistant cells induced by sorafenib,but the expression of GSH did not change significantly.5.GSEA function enrichment analysis showed that CISD2 was mainly involved in the regulation of autophagy in HCC.Western blot showed that the expression level of LC3 II / I was significantly increased after the knockdown of CISD2,and the fluorescence spots of LC3 increased significantly.Western blot showed that pPI3 K,p-AKT and p-m TOR protein did not change significantly after the knockdown of CISD2.6.Trypan blue staining showed that 3-MA and Baf A1 inhibited cell death,among which 3-MA played a stronger role.Western blot results showed that the expression level of LC3 II / I was significantly inhibited in CISD2 knockdown group,and the spot light of LC3 was significantly inhibited.Flow cytometry analysis showed that MDA were significantly inhibited in 3-MA treatment group.The expression level of ROS was significantly reduced,and the level of iron ion was significantly reduced,but the change of GSH expression level was not affected by 3-MA treatment.7.The results of Real-time PCR and western blot showed that knockdown CISD2 promoted the expression of Beclin1,which was associated with autophagy.Western blot showed that the expression level of LC3 II / I was significantly increased after the combination of sorafenib and Pg,but the expression level of LC3 II / I was decreased by combined treatment with Beclin1 sh RNA.The immunofluorescence results showed that the expression level of LC3 II / I was decreased after the combination of sorafenib and Pg,LC3 fluorescence spots increased significantly,and LC3 fluorescence spots decreased after Beclin1 was knockdown;Compared with the combination of sorafenib and Pg,Beclin1 knockdown significantly reduced cell mortality,and the expression level of MDA in drug-resistant cells was lower than that of CISD2 alone.Conclusion: 1.CISD2 is highly expressed in HCC and is associated with poor prognosis.2.Sorafenib mainly promoted ferroptosis in HCC cells.3.Knockdown of CISD2 promoted ferroptosis in sorafenib resistant cells.4.CISD2 knockdown promotes autophagy of sorafenib resistant cells and promotes ferroptosis through autophagy.5.CISD2 participates in resistance to sorafenib induced ferroptosis by regulating Beclin1.6.CISD2 inhibitor combined with sorafenib can significantly inhibit the activity of HCC drug-resistant cells,which is expected to provide a certain strategy for the targeted treatment of HCC.This study confirmed that CISD2 mediates sorafenib resistance of HCC cells by inhibiting ferroptosis,and inhibition of CISD2 can promote ferroptosis of drug-resistant cells through Beclin1 dependent autophagy.CISD2 is expected to become a therapeutic target for antagonizing drug resistance of HCC,providing a theoretical basis for clinical diagnosis and treatment of HCC.