Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis.miRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117IHC+/KITmutation GISTs and 19 CD117IHC-/wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3.Of the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117IHC+/KITmutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3'-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients.miR-148b-3p functions as an important regulator of KIT expression and a potential prognostic biomarker for GISTs.
Transforming growth factor-β (TGF-β), a well-known cytokine with pleiotropic biological functions, has an important role in the regulation of cellular proliferation. Rapamycin has specific antagonistic activity on the function of the mammalian target of the rapamycin signaling pathway. The cooperation of TGF-β and rapamycin on the proliferation of Michigan Cancer Foundation (MCF)-7 human breast cancer cells is unclear. The present study demonstrated that TGF-β had a growth-arresting effect on MCF-7 cancer cells. TGF-β stimulation resulted in the upregulation of several cyclin-dependent kinase inhibitors, including p14ARF, p15INK4b, p16INK4a and p21WAF1/CIP1. The present study also demonstrated that rapamycin enhances the antiproliferative effect of TGF-β. The combination of rapamycin and TGF-β induced apoptosis of MCF-7 tumor cells. These findings advance the current understanding of the biological effects of TGF-β and rapamycin.
Histiocytosis is an uncommon disease characterized by excessive accumulation of histiocytes. Here, we report a rare case of non-Langerhans-cell histiocytosis in a 51-year-old woman who presented with severe symptoms of pericardial effusion. Radiologic investigation also detected multiple bone (lower limbs, vertebrae, ribs, and ilium) lesions. Resected pericardium showed abundant mono- or multi-nucleated non-foamy histiocytes (CD68+/CD163+/S-100+/CD1α−/langerin−) in a fibroinflammatory background. The histiocytes demonstrated emperipolesis of lymphocytes, a hallmark feature of Rosai-Dorfman disease (RDD). However, molecular analysis revealed a BRAF V600E mutation of the proliferating histiocytes, highlighting the neoplastic features frequently observed in another non-Langerhans-cell histiocytosis known as Erdheim-Chester Disease (ECD). We consider this case to be a unique presentation of ECD harboring some RDD-like cells with emperipolesis, but not a case of RDD with a BRAF mutation concerning its clinical manifestation (involvement of the heart and bones) and neoplastic features. Key words: Histiocytosis, Erdheim-Chester disease, Rosai-Dorfman disease, Emperipolesis
Abstract Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy.
Complement hyperactivation and thrombotic microangiopathy are closely associated with severe COVID-19. Endothelial dysfunction is a key mechanism underlying thrombotic microangiopathy. To address the relationship between endothelial injury, complement activation and thrombotic microangiopathy of severe COVID-19, we wonder whether, and if so, what and how SARS-CoV-2 factors make endothelial cells (ECs) sensitive to complement-mediated cytotoxicity. We revealed that multiple SARS-CoV-2 proteins enhanced complement-mediated cytotoxicity to ECs via inhibiting membrane complement regulatory proteins (CRPs) and enhancing the deposition of complement recognizing component FCN1. By screening with CRISPR/Cas9-gRNA libraries, we identified that ADAMTS9, SYAP1 and HIGD1A as intrinsic regulators of CD59 on ECs that were inhibited by the SARS-CoV-2 M, NSP16 and ORF9b proteins. IFN-γ, GM-CSF and IFN-α upregulated CD55 and CD59, which IFN-γ antagonized the inhibition of CD55 and CD59 by the three SARS-CoV-2 proteins. So, the deficiency of IFN-γ weakened the protection of CRPs against complement-mediated injury which may be enhanced during infection. Our findings illustrated the regulation of protection against complement-mediated attack on self-cells by SARS-CoV-2 infection and immune responses, providing insights for endothelial injury, thrombotic microangiopathy and potential targets for treating severe COVID-19.
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