RESUMO
Rationale: Sma mothers against decapentaplegic homologue 4 (Smad4) is a key mediator of the transforming growth factor ß (TGF-ß) pathway and plays complex and contradictory roles in hepatocellular carcinoma (HCC). However, the specific role of Smad4 in hepatocytes in regulating hepatocarcinogenesis remains poorly elucidated. Methods: A diethylnitrosamine/carbon tetrachloride-induced HCC model was established in mice with hepatocyte-specific Smad4 deletion (AlbSmad4-/-) and liver tumorigenesis was monitored. Immune cell infiltration was examined by immunofluorescence and fluorescence activated cell sorting (FACS). Cytokine secretion, glycolysis, signal pathway, and single-cell RNA sequencing were analysed for mechanism. Results: AlbSmad4-/- mice exhibited significantly fewer and smaller liver tumor nodules, less fibrosis, reduced myeloid-derived suppressor cell infiltration and increased CD8+ T cell infiltration. Smad4 deletion in hepatocytes enhanced C-X-C motif ligand 10 (CXCL10) secretion, promoting tumor necrosis factor-α (TNF-α) production in CD8+ T cells. The loss of Smad4 activated the CXCL10/mammalian target of rapamycin (mTOR)/lactate dehydrogenase A (LDHA) pathway, which increased glycolytic activity in CD8+ T cells. HCC patients with high Smad4 expression exhibited decreased CD8+ T cell infiltration and altered glycolysis. Conclusion: Our results demonstrate that Smad4 in hepatocytes promotes hepatocarcinogenesis and is a potential and candidate target for the prevention and therapy of HCC.
Assuntos
Linfócitos T CD8-Positivos , Carcinogênese , Carcinoma Hepatocelular , Quimiocina CXCL10 , Hepatócitos , Neoplasias Hepáticas , Receptores CXCR3 , Proteína Smad4 , Animais , Proteína Smad4/metabolismo , Proteína Smad4/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Hepatócitos/metabolismo , Hepatócitos/imunologia , Camundongos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Receptores CXCR3/metabolismo , Receptores CXCR3/genética , Carcinogênese/imunologia , Carcinogênese/genética , Transdução de Sinais , Camundongos Knockout , Humanos , Camundongos Endogâmicos C57BL , MasculinoRESUMO
BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) has two main histological subtypes: large and small duct-type iCCA, which are characterized by different clinicopathological features. This study was conducted with the purpose of expanding our understanding of their differences in molecular features and immune microenvironment. METHODS: We selected 132 patients who underwent radical surgery at our department between 2015 and 2021 for clinical and survival analyses. Whole-exome sequencing was performed to analyse mutational landscapes. Bulk RNA sequencing and single-cell RNA sequencing data were used for pathway enrichment and immune infiltration analyses based on differentially expressed genes. The function of PPP1R1B was analysed both in vitro and in vivo and the gene mechanism was further investigated. RESULTS: We found that large duct-type iCCA had worse overall survival and recurrence-free survival rates than small duct-type iCCA. Mutations in ARID1A, DOT1L and ELF3 usually occur in large duct-type iCCA, whereas mutations in IDH1 and BAP1 occur in small duct-type iCCA. Among the differentially expressed genes, we found that PPP1R1B was highly expressed in large duct-type iCCA tumour tissues. Expression of PPP1R1B promoted cell proliferation, migration and invasion and indicated a worse prognosis. A combination of USF2 with the promoter of PPP1R1B can enhance gene expression in iCCA, which may further affect the expression of genes such as AHNAK, C4BPA and activating the PI3K/AKT pathway. CONCLUSIONS: Our findings extend our understanding of large and small duct-type iCCA. In addition, PPP1R1B may serve as a potential marker and therapeutic target for large duct-type iCCA.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Colangiocarcinoma/mortalidade , Colangiocarcinoma/cirurgia , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/mortalidade , Neoplasias dos Ductos Biliares/cirurgia , Masculino , Feminino , Pessoa de Meia-Idade , Mutação , Microambiente Tumoral/genética , Idoso , Proliferação de Células/genética , Linhagem Celular Tumoral , Sequenciamento do Exoma , Regulação Neoplásica da Expressão Gênica , Animais , Análise de Sobrevida , Prognóstico , Biomarcadores Tumorais/genética , Movimento Celular/genética , CamundongosRESUMO
WW domain-containing E3 ubiquitin protein ligase1 (WWP1) is reported to be upregulated in many types of human cancers; however, its expression and function in intrahepatic cholangiocarcinoma (ICC) remain unknown. Here, in this study we investigated the expression pattern, clinical prognosis, tumor biological functions, and molecular mechanisms of WWP1 in ICC. The expression of WWP1 in patient tissues was detected by western blotting, immunohistochemistry (IHC), and immunofluorescence. CCK-8, colony formation, EdU, transwell, and xenograft models were used to explore the role of WWP1 in the proliferation and metastasis of ICC. Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation, and immunofluorescence were performed to detect the potential mechanisms. Our study revealed that WWP1 was highly expressed in ICC, and high levels of WWP1 were associated with poor prognosis. Functionally, WWP1 overexpression enhanced the proliferation and metastasis of ICC cells and vice versa. Mechanistically, MYC could be enriched in the promoter region of WWP1 to facilitate its expression. Then, WWP1 targets Nedd4 family interacting protein1 (NDFIP1) and reduces NDFIP1 protein levels via ubiquitination. Downregulation of NDFIP1 in ICC cells rescued the effects of silenced WWP1 expression. WWP1 expression was also negatively correlated with the protein level of NDFIP1 in patient tissues. In conclusion, WWP1 upregulated by MYC promotes the progression of ICC via ubiquitination of NDFIP1, which reveals that WWP1 might be a potential therapeutic target for ICC.
RESUMO
Acute pancreatitis (AP) is a common gastrointestinal disease with substantial morbidity and mortality. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP. The present study aimed to investigate the effects of tRNA-derived fragments (tRFs) and the microRNA regulatory network on pancreatic acinar intracellular trypsinogen activation (PAITA) and identify novel key targets in AP. Taurolithocholic acid 3-sulfate (TLC-S)-treated AR42J cells were used to establish a PAITA model. Twenty differentially expressed tRFs and 35 DE microRNAs were identified in PAITA through gene sequencing. Based on these genes, we established the tRF-mRNA and microRNA-mRNA regulatory networks by using bioinformatics methods. The networks revealed 29 hub microRNAs (e.g., Let-7 family, miR-21-3p.) and 19 hub tRFs (e.g., tRF3-Thr-AGT, i-tRF-Met-CAT) in PAITA. GO analysis showed that the functions of the two networks were similar and mainly enriched in RNA splicing, mRNA processing, and so on. tRF3-Thr-AGT, targeting Btg2, Cd44, Zbp1, etc., was significantly decreased in PAITA. Moreover, the trypsinogen activation level was increased significantly in the tRF3-Thr-AGT deficiency groups, but rescued by tRF3-Thr-AGT mimics. The results revealed that downregulated tRF3-Thr-AGT was involved in PAITA. This study provides potential novel targets for researching the underlying mechanisms of AP.
Assuntos
Células Acinares/metabolismo , MicroRNAs/metabolismo , Pancreatite/metabolismo , RNA de Transferência/metabolismo , Tripsinogênio/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática/genética , MicroRNAs/genética , Pancreatite/genética , Pancreatite/patologia , RNA de Transferência/genética , Ratos , Tripsinogênio/genéticaRESUMO
Endogenous transfer RNA-derived small RNAs (tsRNAs) are newly identified RNAs that are closely associated with the pathogenesis of multiple diseases, but the involvement of tsRNAs in regulating acute pancreatitis (AP) development has not been reported. In this study, we screened out a novel tsRNA, tRF3-Thr-AGT, that was aberrantly downregulated in the acinar cell line AR42J treated with sodium taurocholate (STC) and the pancreatic tissues of STC-induced AP rat models. In addition, STC treatment suppressed cell viability, induced pyroptotic cell death and cellular inflammation in AP models in vitro and in vivo. Overexpression of tRF3-Thr-AGT partially reversed STC-induced detrimental effects on the AR42J cells. Next, Z-DNA-binding protein 1 (ZBP1) was identified as the downstream target of tRF3-Thr-AGT. Interestingly, upregulation of tRF3-Thr-AGT suppressed NOD-like receptor protein 3 (NLRP3)-mediated pyroptotic cell death in STC-treated AR42J cells via degrading ZBP1. Moreover, the effects of tRF3-Thr-AGT overexpression on cell viability and inflammation in AR42J cells were abrogated by upregulating ZBP1 and NLRP3. Collectively, our data indicated that tRF3-Thr-AGT suppressed ZBP1 expressions to restrain NLRP3-mediated pyroptotic cell death and inflammation in AP models. This study, for the first time, identified the role and potential underlying mechanisms by which tRF3-Thr-AGT regulated AP pathogenesis.