Role Of FUNDC1 In Gastric Cancer And The Mechanism Of Its Effect On Iron Death By Regulating HSPB1

Objective: Gastric Cancer(GC)is the fifth most common cancer and the third leading cause of cancer-related death worldwide.Currently,surgery is the preferred treatment for GC.However,the associated costs and postoperative pain impose a significant burden on these patients.In addition,disease recurrence is also common in GC patients,so other potential therapeutic options need to be discovered and developed.Increasing knowledge of ferroptosis in different cancer types has provided new perspectives for cancer therapy.Ferroptosis is a form of iron-dependent cell death.It is characterized by intracellular lipid peroxide accumulation and REDOX imbalance.Ferroptosis,a form of regulatory cell death induced by excessive lipid peroxidation,has recently emerged as a key tumor suppressor mechanism.Glutathione peroxidase 4(GPX4)and ferroptosis inhibitor protein 1(FSP1)constitute two major ferroptosis defense systems.Ferroptosis has attracted great interest in the cancer research community in recent years,in part because it is a unique mode of cell death that is mechanically and morphologically distinct from other forms of cell death,such as apoptosis,and thus holds great potential for cancer therapy.heat shock protein beta-1(HSPB1)is a negative regulator of ferroptosis in cancer cells.At present,it is known that HSPB1 inhibits ferroptosis by reducing the production of iron-mediated lipid reactive oxygen species through the regulation of HSF1 and protein kinase C-mediated phosphorylation.More studies on HSPB1 in the field of ferroptosis are unknown,and it is necessary to enrich the research of ferroptosis.Mitochondria are major sites of energy metabolism,providing most of the energy required for normal cellular function.They are dynamic organelles that continuously undergo fission,fusion,and mitophagy to maintain their homeostasis and function.However,dysregulation of mitochondrial dynamics and mitophagia leads to reduced ATP production and DNA mutations that ultimately lead to cell death.FUNDC1 is involved in mitochondrial fission,clearance of unfolded proteins,iron metabolism in mitochondria,crosstalk between mitochondria and endoplasmic reticulum(ER)and mitophagy.Studies have shown that FUNDC1 is associated with the progression of ischemic diseases,cancer,and metabolic diseases.In summary,this study first clarified the expression of FUNDC1 in gastric cancer cell lines and human gastric cancer tissues,and clarified its functions such as proliferation,cloning,and apoptosis in gastric cancer cells.Secondly,FUNDC1 is a mitochondrial autophagy receptor,and mitochondria and autophagy are closely related to ferrozois,which clarifies the relationship between FUNDC1 and ferrozois in gastric cancer and the regulatory direction of the mechanism of ferrozois(lipid oxidation,iron content and reactive oxygen species).Finally,FUNDC was explored to regulate ferrozois by regulating the expression of HSPB1 and interacting with it.Methods: 1.Prediction of FUNDC1 expression in gastric cancer by bioinformatics analysis,and then real-time fluorescence quantitative PCR(q RT-PCR),Western blot assay(Western The expression of FUNDC1 in human gastric cancer tissues and gastric cancer cell lines was analyzed by Immunohistochemistry(IHC)and blot,and the location of FUNDC1 in cells was determined by Immunofluorescence(IF).Fu97 and MKN45 cell lines were selected for Small interfering RNA(si RNA),plasmid DNA transfection and lentivirus stabilization treatment for subsequent experiments.3.Through cellular functional experiments: Cell counting Kit-8(CCk-8)assay,Ed U assay,Colony formation assay and Annexin V/PI double staining were used to verify the effect of FUNDC1 on cell proliferation and cell death in gastric cancer cells.Wound Healing assay and endothelial cell tube formation assay were used to verify the effect of FUNDC1 on angiogenesis.The autophagy function of FUNDC1 in gastric cancer cells was determined by mitochondrial membrane potential(MMP)assay,WB assay for mitochondrial membrane proteins(Tom20,et al.)and autophagy markers(LC3B,Atg32,et al.).A series of experiments including CCK-8,ferroptosis inducer/inhibitor treatment,lipid peroxide staining(Liperfluo),intracellular ROS detection,intracellular divalent iron ion detection,malondialdehyde(MDA)detection,and GSH detection were used to verify the relationship between FUNDC1 and ferroptosis.7.The correlation between FUNDC1 and key molecules of ferroptosis was predicted by bioinformatics,and verified by q RT-PCR and WB experiments.8.HSPB1 was identified as a key FUNDC1 molecule in regulating ferroptosis,and the role of FUNDC1-HSPB1 axis in regulating ferroptosis was further verified by rescue experiments.9.By immune co-precipitation(Co-Immunoprecippitation,Co IP)validation FUNDC1-HSPB1 interaction,and by immunofluorescence experiments verify the orientation relationship.10.The effect of FUNDC1 on three phosphorylation sites of HSPB1 was detected by WB experiment to further verify the mechanism of FUNDC1 on HSPB1.Results: Bioinformatics analysis showed that FUNDC1 was highly expressed in gastric cancer tissues,and experimental validation of both clinical specimens and cell lines confirmed the high expression of FUNDC1 in gastric cancer.In vitro experiments demonstrated that reduced expression of FUNDC1 inhibited proliferation,clone formation and promoted apoptosis in human gastric cancer cell lines;knockdown of FUNDC1 inhibited angioplasty while overexpression induced mitochondrial autophagy in gastric cancer cells.2.The sensitivity of FUNDC1 to two iron death inducers,Erastin and RSL3,was different: downregulation of FUNDC1 expression promoted Erastin-induced cell death while insensitive to the effect of RSL3;downregulation of FUNDC1 resulted in increased cell activity,lipid oxidation,ROS and iron ion content and decreased GSH levels under Erastin induction;indicating that FUNDC1 plays a role in inhibiting iron death in gastric cancer.3.Correlation between FUNDC1 and key target genes of iron death by raw letter analysis The relationship between HSPB1 and FUNDC1 was determined,and a positive correlation was demonstrated by analysis of clinical samples;down-regulation of FUNDC1 expression promoted Erastin-induced cell death but was insensitive to the effect of RSL3;after down-regulation of FUNDC1,cell activity,lipid oxidation,ROS and iron ion content were increased and GSH levels were decreased under Erastin induction;after up-regulation of HSPB1,cell activity,lipid oxidation,ROS and iron ion content were increased and GSH levels were decreased.Co IP assay and Erastin-induced fluorescence localization assay of both further illustrated the regulation of iron death through binding action.Conclusion: 1.FUNDC1 is highly expressed in human gastric cancer tissues,and down-regulation of FUNDC1 expression inhibits the proliferation and clone formation of gastric cancer cells;promotes apoptosis of gastric cancer cells;inhibits angiogenesis and induces autophagy;2.Down-regulation of FUNDC1 expression in gastric cancer cells promotes the occurrence of iron death in gastric cancer cells,i.e.FUNDC1 acts as a negative regulator of iron death in gastric cancer cells;3.FUNDC1 and HSPB1 are positively correlated and highly expressed in human gastric cancer tissues,and FUNDC1 acts as a suppressor of iron death by inducing HSPB1 translocation to mitochondria,and then the two combine to regulate HSPB1 expression;4.FUNDC1 induces HSPB1 translocation binding independent of P62.