| Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death in the world and a highly inflammatory-related cancer type.Regulated cell death(RCD)play a tumor inhibition role in the "inflammation-carcinogenesis" pathway of HCC.Ferroptosis is a new type of RCD,which can circumvent the anti-apoptotic signals of the carcinogenesis process and is beneficial to inhibit the progression of HCC.The morphology of ferroptotic cells is manifested by changes in mitochondrial size,mitochondrial membrane,and mitochondrial cristae.The retrograde signal of tumor suppressor p53 is a signal of early mitochondrial dysfunction and is involved in the regulation between cell survival and death.Whether it is associated with early morphological changes in ferroptosis is unclear.Biochemical characteristics of ferroptotic cells include decreased glutathione peroxidase 4(GPX4),peroxidation of polyunsaturated fatty acid-containing phospholipids,and increased redox-active iron.Among them,GPX4 is a selenium-containing protein and its selenocysteine has a higher reactivity and efficiency in redox reactions,which is closely related to the redox status of tumor cells and drug resistance of tumor cells.In addition,different intracellular localizations of GPX4 can play different roles,recent study used computer simulation models and found that GPX4 localized by mitochondria can regulate mitochondrial signal molecules to mediate ferroptosis,but there is no experimental evidence.As a common post-translational modifications(PTMs)in tumor cells,phosphorylation/dephosphorylation modification can regulate intracellular protein distribution and its function,and it is also important for RCD signaling.However,whether PTMs participate in the change of GPX4 mitochondrial localization and whether this change regulates the occurrence of ferroptosis through mitochondrial dysfunction signals(p53 retrograde signals)remains to be studied.Objective:To establish a model of ferroptosis-induced HCC cells and find out that the expression and distribution of GPX4 are involved in regulating the distribution and translocation of mitochondrial p53.And to explore a new mechanism of mitochondrial p53 retrograde signal-mediated ferroptosis.The results provided clues for screening GPX4 functional dephosphorylation modification sites and exploring the molecular regulatory mechanism of PP2A in hepatocarcinogenesis,as well as providing experimental basis for targeting GPX4-mediated ferroptosis chemoprevention strategies for liver cancer.Methods:(1)GEO and TCGA analysis:GEO database(GSE76427,GSE38476)and TCGA database(TCGA-LIHC)were used to analyze ferroptosis-related genes,protein phosphatase 2A(PP2A)subunits genes mRNA level in adjacent non-tumor tissues(ANTTs)and primary tumors(PTs)of HCC patients and the correlation between the two.(2)In vitro:Immortalized human normal liver L02 cells were used as control cells,and quantitative real-time polymerase chain reaction(qRT-PCR)and WB were used to detect GPX4 gene mRNA level and protein expression in HCC cells HepG2,MHCC97H,Huh-7,QGY7703 and Hep3B cells to screen in vitro cells.HCC cells HepG2,MHCC97H,Huh-7 and Hep3B cells were treated with Sora(10 μmol/L,24 h)to establish cell models with different sensitivity levels of ferroptosis,Fer-1(1 μmol/L,24 h)was administered to establish a ferroptotic suppression model and detect related indicators.① Transmission electron microscope(TEM),mitochondrial membrane potential(JC-1 probe labeling method),and laser confocal microscope immunofluorescence(IF)were used to detect changes in mitochondrial morphology and function.②MTS color reaction method to detect cell proliferation/functional activity.③Flow cytometry(FCM)was used to detect the percentage of lipid peroxidation(LPO)cells labeled with LPO probe C11 BODIPY.④Western blot(WB)was used to detect total and different subcomponents of PP2A-B55β，GPX4,p53 protein expression.⑤Proximity Ligation Assay(PLA),IF detection were used to detect the near change of the spatial position of B55β and GPX4 protein.⑥Mitochondrial co-IP(Co-IP)was used to detect the distribution and interaction of GPX4 and p53 proteins on mitochondria.⑦Transfection of TP53 overexpression plasmid and TP53-ΔNLS plasmid in Hep3B cells to establish a p53 complement model to verify the role of p53 protein nuclear translocation.Construction of GPX4 hyperphosphorylated(D)or dephosphorylated(A)HepG2 and Huh-7 cell lines to verify the functional role of GPX4 phosphorylation sites.Construction of B55βoverexpression of HepG2 cells was used to verify the regulation of B55β on GPX4.(3)In vivo:Female BALB/c nude mice(SPF grade,4-6 weeks old)were injected subcutaneously with HepG2-PPP2R2B cells to establish a B55β overexpression nude mice model,and HepG2-pBabe cells injection was used to establish a control nude mice model to verify the effect of B55β on ferroptosis.From the 15th day of the inoculation,the nude mice were given saline(NC)and sorafenib(Sora)treatments in the tail vein every other day for a total of 5 times.The growth of orthotopic tumors in nude mice was routinely observed and measured,and body weight was recorded in every 2 days.Nude mice were sacrificed on the 24th day after inoculation,and the volume and quality of the tumor tissue were stripped to determine the following indicators:WB to detect ferroptosis-related proteins(B55β,GPX4 and p53)in total cells or in cytoplasm,mitochondrial and nuclear components.②5,5’-dithionitrobenzoic acid(DTNB)rate ratio colorimetric method was used to detect the content of reduced glutathione(GSH)in tumor tissues.③The thiobarbituric acid(TBA)color reaction method was used to detect the malondialdehyde(MDA)content in tumor tissues.④Immunohistochemistry(IHC)detected the expression and distribution of iron death-related proteins in tumor tissues.⑤ The mitochondrial morphology was detected by TEM.Results:(1)GEO and TCGA analysis:GEO database(GSE76427,GSE38476)and TCGA-LIHC database analysis showed that compared with ANTTs samples,GPX4 gene mRNA level was increased and PPP2R2B gene mRNA was decreased in PTs cases(P<0.05).the gene expression/level of GPX4 was negatively correlated with that of PPP2R2B(P<0.05).(2)In vitro:①Selection of in vitro assay cell lines:compared with immortalized human normal liver L02 cells,the level of GPX4 gene mRNA and its protein expression were decreased in HepG2 and MHCC97H cells(P<0.05),while increased in Huh-7,QGY7703,and Hep3B cells(P<0.05).Therefore,HepG2 and MHCC97H cells were selected for the next experiments.②Detection of ferroptosis model indicators:compared with the control group,ferroptosis-inducing cells:the outer membrane electron density of mitochondria increased,mitochondrial ridges decreased or disappeared,mitochondrial membrane potential damaged,and mitochondrial LPO increased.Overall cell and mitochondrial LPO increased.Cell viability decreased(P<0.05)and rescued after being combined with ferroptosis inhibitor Fer-1(P<0.05),while did not change significantly when combined with other cell death inhibitors(Z-VAD,Nec-1).B55β total protein level and mitochondrial component protein level were increased,GPX4 total protein level and mitochondrial component protein level were decreased,p53 mitochondrial component protein was decreased and nuclear component was increased.③Verification of the role of p53 protein nuclear translocation:compared with the control group,the cell viability of Hep3B cells with transfected TP53 overexpression plasmid decreased and the level of LPO increased(P<0.05).The cell viability and LPO level of Hep3B cells with transfected TP53-ΔNLS plasmid did not change significantly.④Functional verification of GPX4 phosphorylation site:compared with HepG2-pBabe cells,the cell viability of HepG2-GPX4-S2A cells decreased(P<0.05),and the overall LPO level increased(P<0.05),while compared with HepG2-GPX4-S2D cells,the translocation of mitochondrial p53 into the nucleus increased.The above phenomenon in ferroptosis-resisitant Huh-7 cells showed that compared with Huh-7-pBabe cells,the cell viability of Huh-7-GPY4-S2A cells decreased(P<0.05),and overall LPO increased(P<0.05),while compared with Huh-7-GPX4-S2D cells,the translocation of mitochondrial p53 into the nucleus increased.⑤Verification of the regulation of B55β on GPX4:PLA showed the positions of B55β and GPX4 proteins were close,and IF showed the co-localization of mitochondria of B55β and GPX4 proteins was reduced,which suggested that the interaction between B55β and GPX4 may affect the expression of GPX4 protein in mitochondria.Compared with HepG2-pBabe cells,the levels of B55β protein increased and the levels of GPX4 protein decreased in HepG2 cells with B55β overexpression after Sora treatment.(3)In vivo:Compared with the NS-treated group of nude mice inoculated with HepG2-pBabe cells,the drug-treated group of nude mice inoculated with HepG2-pBabe cells,the NS-treated group with nude mice inoculated with HepG2-PPP2R2B and the drug-treated group with nude mice inoculated with HepG2-PPP2R2B tumor volume increased slowly,tumor mass and volume decreased,B55β protein level increased and GPX4 protein level decreased,GSH content decreased(P<0.05),and MDA content increased(P<0.05).It is suggested that B55β can inhibit tumor growth through ferroptosis.Conclusion:This study clarifies that GPX4Ser2 dephosphorylation can regulate the translocation of mitochondrial p53 to the nucleus through its mitochondrial distribution and participate in the induction of ferroptosis,and PP2A participated in this process upstream,and a new molecular mechanism involved in mediating ferroptosis signals via mitochondrial-to-nuclear organelle communication was elucidated.The results provide experiments for screening GPX4 functional(de)phosphorylation modification sites and elucidating the molecular mechanism of protein phosphatase(PP2A)regulation in liver carcinogenesis and provides experimental basis for targeted chemoprevention of ferroptosis-dependent tumor. |
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