| Background and ObjectiveHepatocellular carcinoma is a primary liver cancer with high mortality rates.According to cancer statistics in 2022,the incidence of liver cancer in China ranked fourth among malignant tumors,while the death rate was second.Surgical resection still is the main method for curing primary liver cancer.However,most liver cancer patients are already in the middle to late stages when diagnosed,and have lost the chance for surgical resection due to the high malignancy level.In recent years,targeted therapy with high specificity and minimal side effects has gradually become one of the important means of anti-liver cancer treatment.Currently,sorafenib and lenvatinib are the only first-line targeted drugs for treating liver cancer clinically.The complex biological mechanisms underlying liver cancer result in problems such as low response rates and drug resistance for targeted drugs.Therefore,it is urgent to elucidate the molecular mechanisms of liver cancer occurrence and development in detail,and explore new drug targets.Glycine degradation supplies one-carbon units and nucleotide synthesis,which is closely related to the rapid proliferation of tumor cells.Glycine decarboxylase(GLDC)is the key limiting enzyme in glycine breakdown metabolism.Abnormal expression of GLDC in various tumor cells regulates the occurrence and development of cancer.GLDC regulates pyrimidine synthesis and glycolysis to promote the occurrence of non-small cell lung cancer.mTORmediated acetylation modification of GLDC leads to impaired enzyme activity and degradation,thereby inhibiting the progression of glioblastoma.In addition,GLDC regulates the degradation of the cell cytoskeleton protein cofilin to inhibit liver cancer metastasis.Therefore,whether GLDC plays a promoting or inhibitory role in the tumor process depends on the microenvironment in which the tumor occurs.Due to the heterogeneity of liver cancer,the mechanism by which GLDC regulates autophagy and proliferation in liver cancer is not yet clear.This study analyzes the expression levels of GLDC in liver cancer patient tissues using bioinformatics,as well as its clinical prognosis correlation.The authors construct liver cancer cell lines with GLDC overexpression and knockdown,and study the effects of GLDC on biological functions such as cell proliferation,migration,senescence,and autophagy at the cellular level.High-throughput RNA sequencing is used to analyze downstream signaling pathways regulated by GLDC.Immunoprecipitation experiments reveal the molecular mechanism by which GLDC binds to VPS34 to induce autophagy.A nude mouse transplantation model is used to investigate the effect of GLDC on the tumorigenic ability of liver cancer cells in vivo.Methods1.Analyze the expression level of GLDC in liver cancer patients in the TCGA database using bioinformatics,as well as the correlation between GLDC expression and the clinical prognosis;2.Lentivirus infection was used to construct stable GLDC knockdown Huh7,HepG2 and Hep3B cell lines and GLDC overexpression Huh7 cell line.The knockdown and overexpression efficiency were identified by Western Blot.The effects of GLDC on the proliferation and senescence of HCC cells were detected by CCK-8 assay,plate clone formation assay,soft agar clone formation assay,and β-galactosidase staining assay;3.The effect of GLDC on the migration in HCC cells was detected by cell scratch and Transwell assay,and the effect of GLDC on the expression of epithelial-mesenchymal transition markers was detected Western blot;4.JC-1 mitochondrial membrane potential fluorescent probe assay,Western Blot and immunofluorescence assay were used to explore the effect of GLDC on mitochondrial membrane potential and autophagy;5.The downstream target genes and signaling pathways regulated by GLDC were confirmed by high-throughput RNA sequencing analysis and qRT-PCR experiment;6.Co-immunoprecipitation(co-IP)assay was performed to detect the autophagy-related proteins binding to GLDC,and the interaction between them was further confirmed through GLDC fragmentation and acetylation;7.Co-immunoprecipitation assay was performed to detect the effect of GLDC on the proteinprotein interaction of VPS34 autophagy complex;8.The effect of overexpressing GLDC on the tumor forming ability of Huh7 cells was examined in nude mice xenograft model.Results1.GLDC is lowly expressed in liver cancer tissues and is associated with poor clinical prognosisUsing TCGA database to analyze the expression level of GLDC and overall survival in patients,the results showed that the expression of GLDC in HCC patients was significantly lower than that in normal population,and lower GLDC expression was associated with poor prognosis.2.Efficiency evaluation of GLDC knockdown and overexpression cell linesGLDC knockdown Huh7,HepG2 and Hep3B cell lines and GLDC overexpression Huh7 cell line were identified by Western Blot.The results showed that the expression of GLDC was significantly changed as planned at protein levels.3.GLDC inhibits proliferation and promotes senescence of HCC cellsKnockdown of GLDC could promote the proliferation of Huh7 and Hep3B cells,as well as enhance the plate and soft agar colony formation ability of Huh7 and HepG2 cells,compared with the control group,indicating that GLDC could inhibit the proliferation of hepatoma cells.In addition,compared with the control group,β-galactosidase activity was increased in the GLDC overexpression group and decreased in the GLDC knockdown group,indicating that GLDC could promote cell senescence.RNA sequencing and GSEA enrichment analysis showed that GLDC may regulate cell cycle and lipid metabolism-related signaling pathways.4.GLDC downregulates the expression level of Vimentin and inhibits HCC cell migrationCompared with the control group,the migration number and rate of liver cancer cells were increased in the knockdown GLDC group,whereas the number of migration cells was decreased in the overexpression GLDC group,indicating that GLDC inhibits HCC cell migration.Meanwhile,the results of Western blot assay showed that GLDC downregulated the mRNA and protein levels of Vimentin,indicating that GLDC may inhibit liver cancer cell migration through downregulating Vimentin.5.GLDC increases mitochondrial membrane potential and promotes autophagy in HCC cellsKnockdown of GLDC reduced the mitochondrial membrane potential in HCC cells c ompared with the control cells.Western Blot and immunofluorescence experiments showed that silencing GLDC could down-regulate the expression of autophagy marker LC3B-II protein,implying that GLDC promoted autophagy in liver cancer cells.6.GLDC interacts with VPS34 and regulates autophagy by affecting the formation of PI3K complexImmunoprecipitation assay showed that GLDC interacted with VPS34 significantly.Treatment of cells with deacetylase inhibitors attenuated this interaction,suggesting that acetylation may mediate the interaction between GLDC and VPS34.Co-immunoprecipitation assay showed that overexpressed GLDC significantly enhanced the interaction of VPS34 with Beclinl and ATG14(PI3K complex).These results suggested that GLDC probably promotes autophagy in liver cancer cells through promoting the interaction of PI3K complex.7.Overexpressed GLDC suppresses the tumor growth of Huh7 cellsCompared with the control group,the tumor volume was smaller in the GLDC overexpression group,the weight of tumor was reduced as well,suggesting that GLDC presented a certain inhibitory effect on tumor growth in vivo.Conclusion1.GLDC is underexpressed in HCC tissues and negatively correlated with clinical prognosis;2.GLDC inhibits the proliferation and migration ability of HCC cells,and promotes autophagy and senescence of liver cancer cells;3.GLDC binds to the autophagy protein VPS34 and regulates the interaction of the autophagy PI3K complex,thereby promoting autophagy in HCC cells. |
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