RESUMO
This study delves into the unexplored realm of castration-resistant prostate cancer (CRPC) by investigating the role of TRIM28 and its intricate molecular mechanisms using high-throughput single-cell transcriptome sequencing and advanced bioinformatics analysis. Our comprehensive examination unveiled dynamic TRIM28 expression changes, particularly in immune cells such as macrophages and CD8+ T cells within CRPC. Correlation analyses with TCGA data highlighted the connection between TRIM28 and immune checkpoint expression and emphasized its pivotal influence on the quantity and functionality of immune cells. Using TRIM28 knockout mouse models, we identified differentially expressed genes and enriched pathways, unraveling the potential regulatory involvement of TRIM28 in the cGAS-STING pathway. In vitro, experiments further illuminated that TRIM28 knockout in prostate cancer cells induced a notable anti-tumor immune effect by inhibiting M2 macrophage polarization and enhancing CD8+ T cell activity. This impactful discovery was validated in an in situ transplant tumor model, where TRIM28 knockout exhibited a deceleration in tumor growth, reduced proportions of M2 macrophages, and enhanced infiltration of CD8+ T cells. In summary, this study elucidates the hitherto unknown anti-tumor immune role of TRIM28 in CRPC and unravels its potential regulatory mechanism via the cGAS-STING signaling pathway. These findings provide novel insights into the immune landscape of CRPC, offering promising directions for developing innovative therapeutic strategies.
Assuntos
Linfócitos T CD8-Positivos , Proteínas de Membrana , Camundongos Knockout , Neoplasias de Próstata Resistentes à Castração , Proteína 28 com Motivo Tripartido , Proteína 28 com Motivo Tripartido/metabolismo , Proteína 28 com Motivo Tripartido/genética , Animais , Camundongos , Humanos , Masculino , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
AIM: To study recombinant plasmid pEGFP-AFP-TK delivered by nano-magnetic fluids targeting killed AFP positive HepG2 cells in vitro. METHODS: Constructed recombinant plasmid pEGFP-AFP-TK which was delivered by nano-magnetic fluids into AFP positive HepG2 cells and AFP negative SMMC-7721.The fluorescence was detected in order to evaluate the transfection rate, and RT-PCR and Western blot were used to detect the expression of TK gene after transfection. MTT assay was used to evaluate the effect of TK on the proliferation and bystander effect of HepG2 cells in ganciclovir (GCV). Flow cytometry was used to analysis the apoptosis of HepG2 cells. RESULTS: Nano-magnetic fluids delivered plasmid pEGFP-AFP-TK into HepG2 cells and TK gene was successfully expressed, and the transfection efficacy of nano-magnetic fluids was superior to that of Lipofectamine. RT-PCR and Western blot results demonstrated that TK gene was expressed in HepG2 cells after being transfected with nano-magnetic fluids/pEGFP-AFP-TK. MTT and flow cytometry results showed TK gene exerts cell-killing and bystander effect. CONCLUSION: Nano-magnetic fluids could successfully deliver pEGFP-AFP-TK into HepG2 cells and induce expression of TK gene, which is promising gene vector for liver cancer gene therapy. AFP enhancer can specifically enhance the expression of target TK gene within the AFP positive cell, and induce bystander effect and apoptosis in the AFP positive HepG2 cell with with GCV.
Assuntos
Ganciclovir/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Timidina Quinase/metabolismo , alfa-Fetoproteínas/metabolismo , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Efeito Espectador , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo , Terapia Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Magnetismo , Microscopia de Fluorescência , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina Quinase/genética , Transfecção/instrumentação , Transfecção/métodos , alfa-Fetoproteínas/genéticaRESUMO
AIM: To prepare PEG-PEI Fe(3)O(4) nano-magnetic fluid and invest its characteristics. METHODS: PEG-PEI Fe(3)O(4) nano-magnetic fluid was prepared by chemical co-deposition as gene delivery vector. Then DNA of plasmid pAFP-TK was wrapped into PEG-PEI Fe(3)O(4) nano-magnetic fluid. At the same time, we invest its characteristics with MTT assay. RESULTS: PEG-PEI Fe(3)O(4) nano-magnetic fluid-DNA complexes could protect the DNA from degradation of DNaseI, serum and sonication. AFP positive cells transfected by pAFP-TK were highly sensitive to the GCV treatment, and the survival of cells declined obviously. HSV-TK/GCV suicide gene therapy system had direct cell toxicity. CONCLUSION: This new target strategy of pAFP-TK/GCV suicide gene therapy system in which PEG-PEI Fe(3)O(4) nano-magnetic fluid are used as gene delivery vectors explores a promising area for AFP positive HCC and associated carcinoma therapy.
