Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer-related death worldwide.Accumulating researches have highlighted the ability of exosome-encapsulated microRNAs (miRNAs or miRs) as potential circulating biomarkers for lung cancer. The current study aimed to evaluate the significance of mesenchymal stem cells (MSCs)-derived exosomal miR-204 in the invasion, migration, and epithelial-mesenchymal transition (EMT) of NSCLC cells. Initially, the expression of miR-204 in human NSCLC tissues and cells was determined by RT-qPCR, which demonstrated that miR-204 was downregulated in NSCLC tissues and cells. Next, Krüppel-like factor 7 (KLF7) was predicted and validated to be a target of miR-204 using dual-luciferase reporter gene assay. NSCLC A549 cells were treated with MSCs-derived exosomes, after which the migration and invasion of A549 cells were detected and expression of EMT-related proteins (E-cadherin, N-cadherin, and Vimentin), KLF7, p-AKT/AKT, and HIFresults of gain-and loss-of-function assays revealed that miR-204 overexpression in MSCs-derived exosomes inhibited KLF7 expression and the AKT/HIF-impaired cell migration, invasion, as well as EMT. In conclusion, the key findings of the current study demonstrate that exosomal miR-204 from MSCs possesses anticarcinogenic properties against NSCLC via the KLF7/AKT/HIF-
Background
The RING finger (RNF) proteins are a large group of ubiquitin ligases whose aberrant expression is often associated with disease progression. This study examines the function of RNF protein 182 (RNF182) in lung adenocarcinoma (LUAD) cells and its impact on p65 and programmed death ligand 1 (PDL1) regulation.
Methods
Expression of RNF182, p65, and PDL1 in LUAD tissues and cells was measured using immunohistochemistry, reverse transcription quantitative polymerase chain reaction (RT‐qPCR), and/or western blot (WB) assays. LUAD cells were induced to overexpress RNF182 and p65, followed by cell counting kit‐8, colony formation, Transwell, and flow cytometry assays to evaluate the cells’ malignant phenotype. Coimmunoprecipitation and WB assays were used to verify RNF182's effect on p65 ubiquitination. Chromatin immunoprecipitation‐qPCR and luciferase assays were used to analyze p65's transcriptional regulation of PDL1. Coculture of LUAD with CD8+ cytotoxic T cells was performed to detect lactate dehydrogenase release and interferon‐γ and interleukin‐2 concentrations. LUAD cells were implanted in mice to analyze tumorigenicity.
Results
RNF182 was poorly expressed, while p65 and PDL1 were highly expressed in LUAD tissues and cells. RNF182 overexpression suppressed the malignant properties of LUAD cells, and it promoted p65 ubiquitination and protein degradation. p65 activated PDL1 transcription. Overexpression of RNF182 suppressed the PDL1 expression, increased the cytotoxicity in LUAD cells cocultured with CD8+ T cells, and suppressed the tumorigenesis of cancer cells in vivo. However, these tumor‐suppressive effects of RNF182 on LUAD cells were blocked by p65 restoration.
Conclusion
This research demonstrates that RNF182 induces p65 ubiquitination to suppress PDL1 transcription and immunosuppression in LUAD.
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