Global germ line loss of fat mass- and obesity-associated (FTO) gene results in both the reduction of fat mass and lean mass in mice. The role of FTO in adipogenesis has been proposed, however, that in myogenesis has not. Skeletal muscle is the main component of body lean mass, so its connection with FTO physiologic significance need to be clarified. Here, we assessed the impact of FTO on murine skeletal muscle differentiation by in vitro and in vivo experiments. We found that FTO expression increased during myoblasts differentiation, while the silence of FTO inhibited the differentiation; in addition, skeletal muscle development was impaired in skeletal muscle FTO-deficient mice. Significantly, FTO-promoted myogenic differentiation was dependent on its m6A demethylase activity. Mechanically, we found that FTO downregulation suppressed mitochondria biogenesis and energy production, showing as the decreased mitochondria mass and mitochondrial DNA (mtDNA) content, the downregulated expression of mtDNA-encoding genes and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) gene, together with declined ATP level. Moreover, the involvement of mTOR-PGC-1α pathway in the connection between FTO and muscle differentiation is displayed, since the expression of FTO affected the activity of mTOR and rapamycin blocked FTO-induced PGC-1α transcription, along with the parallel alteration pattern of FTO expression and mTOR phosphorylation during myoblasts differentiation. Summarily, our findings provide the first evidence for the contribution of FTO for skeletal muscle differentiation and a new insight to study the physiologic significance of RNA methylation.

Background Fibrinogen-like protein 1 (FGL1)—Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown. Methods The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8 + T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker’s expression and clinical significances were studied. Results We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3 + cells but not PD-L1. CD8 + T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3 + cells and low levels of CD8 + T cells were correlated with poor disease outcome. Moreover, LAG-3 + cells deteriorated patient stratification based on the abundance of CD8 + T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC + ) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC − ). Furthermore, PD-L1 TC − in combination with high densities of LAG-3 + cells showed the worst prognosis, and PD-L1 TC + patients with low densities of LAG-3 + cells had the best prognosis. Conclusions LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3 + cells and CD8 + T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.

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Background/Aims: MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. Downregulated microRNAs and their roles in cancer development have attracted much attention. A growing body of evidence showed that microRNA-133a (miR-133a) has inhibitory effects on cell proliferation, migration, invasion, and metastasis of osteosarcoma. Methods: MiR-133a expression in human osteosarcoma cell lines and human normal osteoblastic cell line hFOB was investigated by real-time PCR (RT-PCR). The role of miR-133a in human osteosarcoma growth and invasion was assessed in cell lines in vitro and in vivo. Then, luciferase reporter assay validated IGF-1R as a downstream and functional target of miR-133a, and functional studies revealed that the anti-tumor effect of miR-133a was probably due to targeting and repressing of IGF-1R expression. Results: MiR-133a was lower expressed in human osteosarcoma cell lines than human normal osteoblastic cell line hFOB and its effect on inhibiting proliferation, invasion and metastasis is mediated by its direct interaction with the IGF-1R. Furthermore, the tumour-suppressive function of miR-133a probably contributed to inhibiting the activation AKT and ERK signaling pathway. Conclusion: MiR-133a suppresses osteosarcoma progression and metastasis by targeting IGF-1R in human osteosarcoma cells, providing a novel candidate prognostic factor and a potential anti-metastasis therapeutic target in osteosarcoma.

N-Carbamylglutamate (NCG), an effective precursor of arginine (ARG), can enhance ARG synthesis, increase intestinal growth, and improve reproductive performance. However, the antioxidant effect of NCG remains largely unknown. This study aims to survey the effects of ARG and NCG supplementation on the antioxidant statuses of the liver and plasma in rats under oxidative stress. Rats were fed for 30 days with one of the three iso-nitrogenous diets: basal diet (BD), BD plus 1% ARG, and BD plus 0.1% NCG. On day 28, half of the rats fed with BD were intraperitoneally injected with 12 mg per kg body weight of diquat (diquat group) and the other half was injected intraperitoneally with sterile 0.9% NaCl solution (control group). The other diet groups also received an intraperitoneal injection of 12 mg per kg body weight of diquat, as follows: diquat + 1% ARG (DT + ARG), and diquat + 0.1% NCG (DT + NCG). Rat liver and plasma samples obtained 48 h after diquat injection were analyzed. Results indicated that diquat significantly affected the plasma conventional biochemical components (relative to the controls), which were partially alleviated in both the DT + ARG and DT + NCG groups (P < 0.05). Diquat also significantly decreased the glutathione (GSH) content (by 30.0%), and decreased anti-superoxide anion (ASA; by 13.8%) and anti-hydroxyl radical (AHR; by 38.9%) abilities in the plasma, and also decreased catalase (CAT) activity both in the liver (by 17.5%) and plasma (by 33.4%) compared with the control group. By contrast, diquat increased the malondialdehyde (MDA) content (by 23.0%) in the plasma (P < 0.05) compared with the control group. Relative to those of the diquat group, higher CAT activity and GSH content were noted in the plasma of the DT + ARG group and in the liver of both DT + ARG and DT + NCG groups (P < 0.05). Furthermore, the DT + ARG group exhibited significantly enhanced plasma ASA activity (P < 0.05). The DT + NCG group showed significantly improved total antioxidant capacity (T-AOC) in the liver and plasma (P < 0.05). Increased GSH content and elevated ASA and AHR activities were also found, but the MDA content in the plasma was depleted (P < 0.05). Compared with the DT + ARG group, the DT + NCG group showed increased liver and plasma T-AOC, enhanced plasma AHR activity, increased liver ASA activity, and decreased plasma MDA content (P < 0.05). Overall, supplementation of 1% ARG and 0.1% NCG can partially protect the liver and plasma from oxidative stress. Furthermore, compared with 1% ARG, 0.1% NCG more effectively alleviated oxidative stress.

BackgroundHepatocellular carcinoma (HCC) develops in a complex microenvironment characterized by chronic inflammation. In recent years, cholesterol metabolic abnormalities have been implicated the importance in cancer cell physiology. This study was designed to investigate the relationship between inflammation and cholesterol accumulation in HCC cells.MethodsHuman HCC cells HepG2 and Huh7 were cultured and stimulated with lipopolysaccharide (LPS) for 24 h. The changes of HCC cells related to cholesterol metabolism including intracellular cholesterol concentrations, cholesterol uptake, and the expression of cholesterol-related genes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), LDL receptor (LDLR), sterol regulatory element-binding transcription factor 2 (SREBF2), and proprotein convertase subtilisin/kexin 9 (PCSK9) were comparatively analyzed. Simultaneously, the effects of nuclear factor-kappa B (NF-κB) signaling pathway on cholesterol metabolism were clarified by knocking-down of nuclear factor kappa-B kinase subunit alpha (IKKα) and TGF-beta-activated kinase 1 and MAP3K7-binding protein 3 (TAB3) via RNAi and microRNA (miR)-195. Subsequently, the roles of cholesterol accumulation in LPS induced pro-inflammatory effects were further investigated.ResultsPro-inflammatory factor LPS significantly increased intracellular cholesterol accumulation by upregulating the expression of HMGCR, LDLR, and SREBF2, while downregulating the expression of PCSK9. These effects were revealed to depend on NF-κB signaling pathway by knocking-down and overexpression of IKKα and TAB3. Additionally, miR-195, a regulator directly targeting IKKα and TAB3, blocked the effects of cholesterol accumulation, further supporting the critical role of pro-inflammation NF-κB signaling in regulating cholesterol accumulation. Intriguingly, the accumulation of cholesterol conversely exerted an augmented pro-inflammation effects by further activating NF-κB signaling pathway.ConclusionsThese results indicated that pro-inflammation effects of NF-κB signaling could be augmented by a positive feedback via enhancing the cholesterol accumulation in liver cancer cells.

The present work aimed at investigating the effects of spermine supplementation and extended spermine administration on the intestinal morphology, enzyme activity, and serum antioxidant capacity of suckling piglets.

Spermine is a ubiquitous cellular component that plays vital roles in the maintenance of nucleic acids, regulation of kinase activities, protein synthesis, control of ion channel activities and renewal of the gut epithelium.