Hypoxia plays an important role in the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), leading to pathology of RA. This study was conducted to evaluate hypoxia-induced microRNAs (hypoxamiR) in RA-FLS and its role in the function of RA-FLS. RA-FLS were cultured under normoxia (21% O2) or hypoxia (3% O2) condition, followed by a microRNA (miRNA) array analysis. The upregulation of miR-191 by hypoxia was confirmed in RA-FLS and FLS from osteoarthritis (OA) patients by quantitative real-time polymerase chain reaction (RT-PCR). Transfection of miR-191 mimic and inhibitor was used to investigate the function of miR-191 in RA-FLS. The functional targets of miR-191 were predicted by bioinfomatics and then validated by reporter gene assay. A subset of miRNAs was identified to be induced by hypoxia including miR-191. The upregulation of miR-191 was found to be specific in hypoxic RA-FLS, compared to hypoxic OA-FLS. We observed that miR-191 in RA-FLS increased cellular proliferation via promoting G1/S transition of the cell cycle and suppressed cell apoptosis induced by cell starvation. Bioinformatical analysis and experimental assays identified CCAAT/enhancer binding protein β (C/EBPβ) as a target gene of miR-191 in RA-FLS. Enforced expression of C/EBPβ rescued the cellular phenotypes induced by miR-191. In addition, an inverse correlation between the C/EBPβ level and hypoxia stimulation was found in RA-FLS, and overexpression of C/EBPβ could partly rescue the hypoxia-induced cell proliferation. We demonstrated the miR-191-C/EBPβ signaling pathway mediating the hypoxia-induced cell proliferation in RA.

Background: The amino acid transporter SLC6A14, which transports 18 of the 20 proteinogenic amino acids, is too low to be detected in healthy normal tissues but is significantly increased in some solid cancers. However, little is known about the roles of SLC6A14 in colorectal cancer (CRC). Methods: The mRNA and protein levels of SLC6A14 were detected using TCGA database, real-time polymerase chain reaction, western blot, and tissue microarrays, respectively. Amino acids concentration was determined by LC-MS/MS. Cell proliferation and apoptosis were determined using MTT assay and flow cytometry. Transwell invasion assay and wound healing assay were employed to analyze cell migration and invasion. The protein levels of Akt-mTOR signaling pathway and MMPs proteins were detected by western blot. Results: Both of the mRNA and protein levels of SLC6A14 were upregulated in CRC tissues, and the protein levels of SLC6A14 were closely related to the tumor cells differentiation: the higher the expression of SLC6A14 was, the poorer the differentiation of the tumor cells was. Further knockdown SLC6A14 with siRNA or treatment with α-MT in CRC cell lines reduced cell proliferation and migration in vitro and inhibited xenograft tumor growth in vivo. Mechanistically, SLC6A14 was demonstrated to regulate the expression and phosphorylation of Akt-mTOR, which mediates the promoting tumor growth function of SLC6A14. Blockade of SLC6A14 with α-MT inhibited the activation of mTOR signaling. Conclusion: SLC6A14 was upregulated in CRC and could promote tumor progression by activating the Akt-mTOR signaling pathway, which may serve as an effective molecular target for the treatment of CRC.

Hypoxia is associated with the pathogenesis of rheumatoid arthritis (RA). RA fibroblast-like synoviocytes (FLSs) are able to differentiate into osteoblasts and adipocytes. In this study, we aimed to investigate the role of hypoxia in the osteogenesis or adipogenesis of RA-FLSs. Bioinformatics analysis was performed to profile gene expression in the datasets of GSE21959, GSE32006, and GSE55875, and flow cytometry was performed for FLS characterization, while Alizarin Redand Oil Red O staining for osteogenic or adipogenic differentiation of FLSs, respectively. RNA interference leptin knockdown was used to determine the role of leptin in the osteogenesis and adipogenesis of RA-FLSs, and the expression of osteogenic and adipogenic markers was quantified by RT-qPCR and Western blotting. FLSs exhibited a mesenchymal stem cell (MSC)-like phenotype and we observed a limited self-renewal capacity in RA-FLSs compared to that in MSCs, but it was still greater than osteoarthritis (OA)-FLSs. Hypoxia did not change the RA-FLS MSC-like phenotype but inhibited the osteogenic differentiation and promoted the adipogenic differentiation of RA-FLSs. From the bioinformatics analysis ofGSE21959, GSE32006, and GSE55875 datasets, we found leptin, the only perturbed hypoxia-mediated upregulated gene across the three profiled datasets. Leptin knockdown in RA-FLSs reversed the hypoxia-mediated reduction of osteogenesis and hypoxia-mediated enhancement of adipogenesis by elevated expression of osteogenic markers and reduced expression of adipogenic markers, respectively. Therefore, hypoxia-leptin regulation of the osteogenic and adipogenic differentiation of RA-FLSs advances our understanding of RA pathogenesis, meanwhile also provides opportunities for future therapeutic intervention of RA.

Background: Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA. Methods: The concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC–MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo. Results: L-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1β, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice. Conclusion: CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), as the key component of the transcription initiation factor complex EIF4F, is significantly upregulated in multiple solid tumours, including lung cancer. However, the function and mechanism of EIF4G1 in the regulation of non‐small‐cell lung cancer (NSCLC) remain unclear. Here, using the clinical samples and the comprehensive survival analysis platforms Kaplan‐Meier plotter, we observed aberrant upregulation of EIF4G1 in NSCLC tissues; furthermore, high expression of EIF4G1 showed association with low differentiation of lung cancer cells and poor overall survival in NSCLC patients. Non‐small‐cell lung cancer cell line A549 and H1703 stably infected with EIF4G1 shRNA were used to determine the function of EIF4G1 in regulating cell proliferation and tumorigenesis in vitro and in vivo. The results demonstrated that EIF4G1 promoted the G1/S transition of the cell cycle and tumour cell proliferation in non‐small cell lung cancer. Mechanistically, EIF4G1 was found to regulate the expression and phosphorylation of mTOR (Ser2448), which mediates the tumorigenesis‐promoting function of EIF4G1. The inhibition of mTOR attenuated the EIF4G1‐induced development and progression of tumours. These findings demonstrated that EIF4G1 is a new potential molecular target for the clinical treatment of non‐small cell lung cancer.