The Mechanism Of GGCT Negatively Regulating Ferroptosis In Papillary Thyroid Cancer

Objective:Papillary thyroid cancer(PTC)is the most common pathological subtype of thyroid cancer,accounting for 85%-90% of the total incidence.PTC is generally considered to have a favorable prognostic effect,but some PTC can change to a more aggressive phenotype,metastasize to lymph nodes or distant tissues,and dedifferentiate into a lethal thyroid cancer.With the rapid growth of the incidence and recurrence rate of thyroid cancer,it is urgent to find new therapeutic targets,as well as to explore more specific molecular mechanisms of thyroid cancer.γ-glutamine cyclotransferase(GGCT)is an enzyme involved in the glutathione(GSH)metabolism cycle.Abnormal GSH metabolism is often related to the occurrence of ferroptosis.More and more evidence has confirmed that GGCT is highly expressed in tumors,and the expression level of GGCT is related to the malignant degree of tumors.The aim of this study is to investigate whether GGCT is involved in the process of ferroptosis in PTC.The upstream and downstream regulation mechanisms of GGCT were further explored to provide theoretical support for the mechanism of the occurrence and development of PTC,and to provide theoretical basis for GGCT as a target for the diagnosis and treatment of PTC.Methods:The mRNA or protein expression levels of the target genes in the cells and tumor tissues were detected by immunohistochemistry(IHC),real-time fluorescent quantitative PCR(q RT-PCR)and Western blot.Enzyme-linked immunosorbent assay(ELISA)was used to analyze the serum level of GGCT in patients with papillary thyroid cancer before or within 24 h after radical surgery.Immunoprecipitation(IP)and liquid chromatography-tandem mass(LC-MS/MS)spectrometry were used to screen proteins that interacted with GGCT.The interaction between the two proteins was analyzed by co-immunoprecipitation(Co-IP)assay and immunofluorescence homologous double labeling assay.Dual luciferase reporter assay was used to verify the binding of miRNA to target genes.Lentiviral packaging technology was used to construct cell lines with stable knockdown or overexpression of target genes.The cell proliferation ability was analyzed by cell counting kit-8(CCK-8)and cell clone formation assay.Cell wound healing assay.and transwell assay were used to analyze the migration and invasion ability of cells.The glutathione(GSH)level in cells or tissues was analyzed by GSH detection kit.C11 BODIPY 581/591 probe combined with Flow cytometry(FCM)was used to detect the level of Lipid ROS in the cells.Malondialdehyde(MDA)lipid oxidation level detection kit was used to detect the level of MDA in cells or tissues.The morphology of mitochondria was observed by transmission electron microscopy.The effect of the target gene on the tumorigenic ability of tumor cells was studied in nude mice.The effect of the target gene on the migration and invasion of tumor cells was evaluated by lung metastasis model in nude mice.Tumor nodules formed in the lung tissue of nude mice were analyzed by H&E staining.Results:(1)The study of GGCT negatively regulating ferroptosis in PTC: GGCT is highly expressed in PTC tissues and cell lines,and the level of GGCT in serum of patients with PTC decreased significantly 24 h after radical surgery.Knockdown of GGCT inhibited cell proliferation,migration and invasion in vivo and in vitro.Ferroptosis inhibitor reversed the decrease in cell viability caused by GGCT knockdown.Further experiments showed that GGCT knockdown inhibited the synthesis of GSH and promoted the accumulation of MDA and ROS,which further promoted ferroptosis of PTC cancer.(2)Study on the molecular mechanism of GGCT regulating ferroptosis in PTC: IP combined with LC-MS/MS results showed that there was an interaction between GGCT and RPS15 A.RPS15A was highly expressed in PTC tissues and cells and was positively correlated with the expression level of GGCT.Knockdown of RPS15 A promoted the expression of p53,which in turn inhibited the expression of SLC7A11 and caused ferroptosis.Overexpression of RPS15 A reversed GGCT-induced ferroptosis.(3)The upstream regulatory mechanism of GGCT: miR-205-5p is lowly expressed in PTC.Overexpression of miR-205-5p inhibits GSH synthesis,promotes MDA and ROS accumulation,and promotes cell ferroptosis.miR-205-5p inhibited GGCT protein expression by targeting the 3’UTR of GGCT.Overexpression of miR-205-5p inhibited GGCT-mediated tumor growth and lung metastasis in vivo.Conclusion: In this study,GGCT was found to be highly expressed in PTC.Knockdown of GGCT promoted ferroptosis and inhibited cell proliferation,migration and invasion.Mechanistically,GGCT interacts with RPS15 A.Knocking down RPS15 A promotes p53 expression,inhibits SLC7A11 expression,and further inhibits GSH synthesis.The upstream mechanism of GGCT regulation showed that miR-205-5p was down-regulated in thyroid papillary carcinoma,and miR-205-5p inhibited GGCT protein expression by targeting the 3’UTR of GGCT.Overexpression of miR-205-5p inhibited GGCT-mediated tumor growth and lung metastasis in vivo.