| Research Background A new form of cell death,ferroptosis,was recently discovered.Ferroptosis results from iron-dependent lipid peroxide accumulation and is characterized mainly by cell volume shrinkage and increased mitochondrial membrane density without typical apoptotic and necrotic manifestations.Ferroptotic cell death can be induced by two classes of small-molecule substances: Class 1 ferroptosis inducers include erastin,sulfasalazine(SAS)and buthionine sulfoximine,which can inhibit system XC_and reduce the intracellular glutathione content,causing an oxidation-reduction imbalance in cells;Class 2 ferroptosis inducers include Ras selective lethal 3 compound(RSL3),DPI7,DPI10,DPI12,DPI13,etc.,which can directly inhibit glutathione peroxidase 4(GPX4),and ultimately lead to an accumulation of lipid peroxides.In addition,ferroptosis can be induced by various drugs(e.g.sorafenib,artemisinin and its derivatives).HCC is a common type of cancer that is ranked sixth in prevalence and third in mortality worldwide.It is a leading cause of death among hepatic cirrhosis patients.Recently It is reported that Sorafenib is a strong inducer of ferroptosis but not of apoptosis,which guides us a new direction of research.System XC_transports intracellular glutamate to the extracellular space,while it transports extracellular cystine into the cell,which is then transformed into cysteine for glutathione(GSH)synthesis.Cellular uptake of cysteine is a key step of GSH synthesis,and GSH generation and maintenance is critical for protecting cells from the damage caused by oxidative stress responses.GPX4 is an enzyme that decomposes H2O2 and organic H2O2 into water or corresponding alcohols,and GSH is an essential cofactor in its activation.Therefore,by depleting the intracellular GSH pool,the ferroptosis inducers reduce GPX4 activity and elevate cytoplasmic and lipid ROS levels,ultimately leading to cell ferroptosis.It is reported that voltage-dependent anion channels,p53,HO-1 and other regulatory pathways of ferroptosis like The MVA pathway,the HSF1-HSPB1 pathway and the p62-Keap1-NRF2 pathway are all involved in the course of ferroptosis.However,ferroptotic regulatory networks have not been known clearly.Further researches are needed to explore the networks.The aim of this study is to explore the mechanism of ferroptosis in HCC cells.First we used ferroptotic inducers to induce ferroptosis,under this ground we investigated the roles of several signal factors in ferroptosis.Then we selectively interfered the expression of certain factors to explore the influence on other related factors.Finally,the relevant inferences were verified again in animal experiments.By exploring the course of ferroptosis occurring in HCC cells,this study provides novel ideas for clinical treatment of HCC,and also has important implications for the study of sorafenib resistance.Chapter Ⅰ Sigma 1 Receptor Protects against Ferroptosis in Hepatocellular Carcinoma CellsObjective: To explore the role of S1 R in HCC cells under ferroptosis.Methods: Firstly,we detected the expression of S1 R under treatment of soranfeib in HCC cells,and observed the translocation of S1 R by confocal microscopy.Then we explored which was the upstream gene to S1 R in ferroptosis.Furthermore,we detected cell viability and the levels of GSH,Fe2+ and MDA,and the expression of GPX4 HO-1 and NRF2 when downregulating S1 R.Finally,we verified the inferences in animal experiments again.Results: The expression of S1 R was upregulated under treatment with sorafenib,and we for the first time found that most of S1 Rs were transferred away from nucleus after sorafenib treatment.knockdown of NRF2,but not of p53 or HIF1α,significantly induced S1 R m RNA expression.Inhibition of S1R(by RNAi or antagonists)increased sorafenib-induced HCC cell death.Knockdown of S1 R blocked the expression of GPX4.Iron metabolism and lipid peroxidation increased in the S1 R knockdown groups treated with sorafenib compared to the control counterpart.Conclusion: We demonstrated for the first time that S1 R protects HCC cells against sorafenib and subsequent ferroptosis.Inhibition of S1 R by RNAi and antagonists markedly increased the anticancer activity of sorafenib by modulating the expression of GPX4,iron metabolism and ROS.Chapter Ⅱ Micro RNA-214 Regulates Ferroptosis in Hepatocellular Carcinoma Cells via ATF4Objective: To explore the role of Micro RNA-214 in HCC cells under ferroptosis.Methods: Firstly,we verified that erastin caused ferroptosis,and constructed stable transfected Micro RNA-214 cell lines to detect the effect of overexpression and knockdown of Micro RNA-214 on cell viability and clonogenic survival assay.Then we detected the effect of Micro RNA-214 overexpression on lipid metabolism in ferroptosis.Furthermore,we detected the effect of Micro RNA-214 overexpression on ATF4 and its downstream molecular expression,and then verified it by dual-luciferase assay,and detected cell viability,clonogenic survival,MDA and ROS levels.Finally,we verified the inferences in animal experiments again.Results: The cell viability and clonal formation rate of ov-pre-mir-214 group were significantly decreased,while those of anti-mir-214-3p group were significantly increased.The ov-pre-mir-214 group further increased the levels of malondialdehyde,Fe2+ and ROS in HCC cells under treatment with erastin,and decreased the intracellular concentration of GSH.Erastin significantly promoted the expression of ATF4 and HSP5 A in HCC cells,while ov-pre-mir-214 inhibited the increasement.Compared with ATF4 3 ’UTR + mir-214 mimics group,the luciferase activity of ATF4 3’ UTR + NC mimics group was significantly decreased,while that of ATF4 3 ’UTR mutant was recovered.Overexpression of ATF4 increased cell viability and clonal formation rate of HCC cells under treatment with erastin,while decreasing MDA concentration and ROS levels.The ov-pre-mir-214 group reduced tumorigenic volume of nude mice under treatment with erastin,and the m RNA levels of mir-214-3p in the tumor was increased,while the m RNA and protein expression of ATF4 were decreased.Conclusion: MIR-214 was involved in the erastin-induced ferroptosis in HCC cells,and overexpression of MIR-214 aggravated the ferroptosis process.MIR-214 affects the process of ferroptosis via regulating malondialdehyde,Fe2+,GSH and ATF4. |
