TRIM21-mediated ubiquitylation of TAT suppresses liver metastasis in gallbladder cancer.

Liver metastasis is common in patients with gallbladder cancer (GBC), imposing a significant challenge in clinical management and serving as a poor prognostic indicator. However, the mechanisms underlying liver metastasis remain largely unknown. Here, we report a crucial role of tyrosine aminotransferase (TAT) in liver metastasis of GBC. TAT is frequently up-regulated in GBC tissues. Increased TAT expression is associated with frequent liver metastasis and poor prognosis of GBC patients. Overexpression of TAT promotes GBC cell migration and invasion in vitro, as well as liver metastasis in vivo. TAT knockdown has the opposite effects. Intriguingly, TAT promotes liver metastasis of GBC by potentiating cardiolipin-dependent mitophagy. Mechanistically, TAT directly binds to cardiolipin and leads to cardiolipin externalization and subsequent mitophagy. Moreover, TRIM21 (Tripartite Motif Containing 21), an E3 ubiquitin ligase, interacts with TAT. The histine residues 336 and 338 at TRIM21 are essential for this binding. TRIM21 preferentially adds the lysine 63 (K63)-linked ubiquitin chains on TAT principally at K136. TRIM21-mediated TAT ubiquitination impairs its dimerization and mitochondrial location, subsequently inhibiting tumor invasion and migration of GBC cells. Therefore, our study identifies TAT as a novel driver of GBC liver metastasis, emphasizing its potential as a therapeutic target.

NONO promotes gallbladder cancer cell proliferation by enhancing oncogenic RNA splicing of DLG1 through interaction with IGF2BP3/RBM14.

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

Retinoic acid enhances ovarian steroidogenesis by regulating granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway.

INTRODUCTION: Ovarian steroidogenesis not only affects the embryonic development and pregnancy outcome, but also associates with many diseases in mammals and women. Exploring the nutrients and mechanisms influencing ovarian steroidogenesis is critical to maintaining the optimal reproductive performance, as well as guaranteeing body health. OBJECTIVES: This research aimed to explore the effect of retinol metabolism on ovarian steroidogenesis and the underlying mechanisms. METHODS: Comparative transcriptomic analysis of ovaries from normal and low reproductive performance sows were performed to identify the main causes leading to low fertility. The metabolites regulating steroid hormones synthesis were investigated in ovarian granulosa cells. Gene interference, overexpression, dual-luciferase reporter assays, chromatin immunoprecipitation and transcriptome analysis were further conducted to explore the underlying mechanisms of Aldh1a1 mediating ovarian steroidogenesis. RESULTS: Transcriptome analysis of ovaries from normal and low reproductive performance sows showed the significant differences in both retinol metabolism and steroid hormones synthesis, indicating retinol metabolism probably influenced steroid hormones synthesis. The related metabolite retinoic acid was furtherly proven a highly active and potent substance strengthening estrogen and progesterone synthesis in ovarian granulosa cells. For the first time, we revealed that retinoic acid synthesis in porcine and human ovarian granulosa cells was dominated by Aldh1a1, and required the assistance of Aldh1a2. Importantly, we demonstrated that Aldh1a1 enhanced the proliferation of ovarian granulosa cells by activating PI3K-Akt-hedgehog signaling pathways. In addition, Aldh1a1 regulated the expression of transcription factor MESP2, which targeted the transcription of Star and Cyp11a1 through binding to corresponding promoter regions. CONCLUSION: Our data identified Aldh1a1 modulates ovarian steroidogenesis through enhancing granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway. These findings provide valuable clues for improving ovarian health in mammals.

Effects of vitamin K supplementation on reproductive performance and bone metabolism-related biochemical markers in lactation sows.

OBJECTIVE: This study was conducted to evaluate the effects of vitamin K (VK) supplementation on reproductive performance and bone metabolism-related biochemical markers in sows. METHODS: Twenty-four Large White×Landrace sows (mean parity 4.04) were randomly assigned to two dietary treatments (NC diet, a basal diet with 0.5 mg/kg of VK3; VK diet, a basal diet with 5 mg/kg of VK3) with twelve replicates per treatment and one sow per replicate according to parity. The experiment started on day 107 of gestation and lasted until day 21 of lactation (weaning). RESULTS: We observed that there were no differences (p>0.05) in average daily feed intake, backfat loss of sows, live piglet number at birth and weaning, average birth weight, average weaning weight, and average daily gain of piglets between two treatments. The apparent total tract digestibility of phosphorus was increased (p<0.05) in VK sows compared with NC sows. The serum bone alkaline phosphatase, osteocalcin, type I procollagen aminoterminal peptide, and type I procollagen carboxyl-terminal peptide on day of farrowing were higher (p<0.05) in VK sows than in NC sows. The serum phosphorus, parathyroid hormone, tartrate-resistant acid phosphatase, and tumor necrosis factor-alpha on day of weaning were lower (p<0.05) in VK sows compared with NC sows. CONCLUSION: Therefore, the overall results suggested that increasing dietary VK3 (0.5 to 5 mg/kg) during lactation improved the apparent total tract digestibility of phosphorus and serum bone metabolism biochemical markers in sows.

Effects of a functional fatty acid blend on growth performance, intestinal morphology, and serum profiles in weaned piglets.

OBJECTIVE: The objective of this study was to determine whether dietary supplementation with a functional fatty acid blend (FA) that contains 31.4% butyric acid and 4.99% medium-chain FA improve growth performance, antioxidant capacity, immunity status, and anti-inflammatory ability in weaned piglets. METHODS: One hundred and forty-four healthy piglets (Duroc×Landrace×Yorkshire) with an average body weight (BW) of 7.98±3.43 kg were randomly divided into three groups with six replicate pens and eight piglets per pen: Normal control (NC): a corn-soybean basal diet; FA1: a basal diet supplemented with 1,000 mg/kg of a functional FA; FA2: a basal diet supplemented with 2,000 mg/kg of a functional FA. The experiment lasted for 28 d. On d 14 and 28, one piglet in each pen from NC and FA2 groups was randomly selected for antioxidative index and immunoglobulins. On d 28, one piglet in each pen from NC and FA2 groups was randomly selected for intestinal morphology and inflammatory factor. RESULTS: We observed that FA supplementation linearly increased (p<0.05) average daily gain and the final BW. There was higher (p<0.05) catalase on d 14, and immunoglobulin (Ig) A and IgM on d 28 in piglets supplemented with FA2 than in the NC group. Moreover, dietary FA2 reduced (p<0.05) crypt depth of ileum in piglets. The concentrations of tumor necrosis factor-α, interleukin (IL)-1ß, IL-8, and IL-10 in jejunum were lower (p<0.05) in the FA2 group compared with the NC group. CONCLUSION: Therefore, the overall results suggests that the FA may help to improve gut health, antioxidant status, and immune parameters resulting in the improvement of growth performance.

Effects of essential oil coated with glycerol monolaurate on growth performance, intestinal morphology, and serum profiles in weaned piglets.

OBJECTIVE: This study aimed to investigate the effects of essential oil coated with glycerol monolaurate (GML) on the growth performance, intestinal morphology, and serum profiles of weaned piglets. METHODS: A total of 144 weaned piglets (Duroc×[Landrace×Yorkshire], average weight 8.07±3.33 kg) were randomly assigned to three groups with six replicate pens and eight piglets per pen: i) CON: a corn-soybean basal diet; ii) LEG: with 1,000 mg/kg essential oil coated with GML; and iii) HEG: with 2,000 mg/kg essential oil coated with GML. RESULTS: Results showed that average daily gain was increased (p<0.05) linearly by essential oil coated with GML supplementation on day 14 to 28 and day 0 to 28 compared with the CON group. Dietary supplementation with HEG increased (p<0.05) total antioxidant capacity and catalase activity on day 14, and immunoglobulin A (IgA) and IgM concentration on day 28 and tended to increase IgG on day 28. In addition, the crypt depth in the jejunum was reduced (p<0.05), and villus height and villus height/crypt depth in the ileum were increased (p<0.05) in the HEG group compared with the CON group. Moreover, lower (p<0.05) concentrations of tumor necrosis factor-α, interferon-γ, interleukin-1ß (IL-1ß), IL-8, and IL-10 were observed in the jejunum of piglets supplemented with HEG compared with the CON group. In addition, dietary HEG tended to decrease IL-6 level in the jejunum of piglets compared with the CON group. CONCLUSION: Dietary essential oil coated with GML can improve growth performance of weaned piglets. Moreover, supplementing 2,000 mg/kg essential oil coated with GML was demonstrated to improve antioxidant ability, and intestinal morphology, and reduce jejunal inflammatory factor levels.

Vitamin K2 Improves Osteogenic Differentiation by Inhibiting STAT1 via the Bcl-6 and IL-6/JAK in C3H10 T1/2 Clone 8 Cells.

Osteogenic activity of vitamin K2 (VK2), a small molecular nutrient, has been suggested. However, the underlying mechanisms have not been fully elucidated. Therefore, this study aimed to explore the mechanisms by which VK2 promotes osteogenic differentiation. The effects of VK2 on osteogenic differentiation indicators were determined in C3H10 T1/2 clone 8 cells. The RNA-seq analysis was used to explore the hypothesis that VK2 promotes osteogenic differentiation. Small interfering RNA (siRNA) assay and plasmid transfection assay were used to determine the potential role of VK2 in the modulation of Bcl-6/STAT axis and IL-6/JAK/STAT signaling pathway. VK2 significantly increased alkaline phosphatase (ALP) activity, ALP, osteocalcin (OCN), and RUNX2 abundance, and RUNX2 protein expression. RNA-seq analysis showed that there were 314 differentially expressed genes (DEGs) upregulated and 1348 DEGs downregulated by VK2. PPI analysis determined the top 10 hub genes upregulated or downregulated by VK2. Overexpression of Bcl-6 increased osteogenic differentiation and decreased expression of STAT1. Administration with VK2 restored the inhibition by siBcl-6 in osteogenic differentiation. Knockdown of IL-6 decreased the mRNA levels of genes associated with the JAK/STAT signaling pathway, and increased markers of osteoblast differentiation. Furthermore, treatment with VK2 improved inhibition in osteogenic differentiation and decreased enhancement of JAK/STAT signaling pathway related genes by overexpression of IL-6. Our study suggests that VK2 could improve osteogenic differentiation via the Bcl-6/STAT axis and IL-6/JAK/STAT signaling pathway.

Effects of the methionine hydroxyl analog chelated microminerals on growth performance, antioxidant status, and immune response of growing-finishing pigs.

This study aimed to investigate the effects of methionine hydroxyl analog chelated microminerals (MHA-M) replacing inorganic microminerals (ITMs) on the growth performance, fecal microminerals concentrations, immune function, and antioxidant status in growing-finishing pigs; 253 pigs (average 33.68 kg body weight) were randomly assigned to six treatments with six replicates each treatment: (1) ITM: a basal diet with Cu, Fe, Mn, and Zn from sulfates providing 20, 100, 40, and 60 mg/kg; (2-6): 1/5MHA-M, 2/5MHA-M, 3/5MHA-M, 4/5MHA-M, and MHA-M was replaced with 20%, 40%, 60%, 80%, and 100% MHA-M. Results showed that the average daily gain (ADG) in the 1/5MHA-M and 2/5MHA-M was greater than other groups in the whole period. Fecal Cu, Fe, Mn, and Zn concentrations had decreased as the intake of trace minerals decreases. The ITM group decreased the fecal Zn concentration on Days 35, 70, and 91, and Fe concentration on Day 70, and increased the Mn concentration on Day 70 compared with MHA-M group. Pigs fed 1/5MHA-M, 2/5MHA-M, and MHA-M had a higher immune function and antioxidant status in serum compared with ITM, 3/5MHA-M, and 4/5MHA-M on Day 35. In conclusion, treatment with 1/5MHA-M and 2/5MHA-M could reduce the excretion of fecal microminerals and improve the immune function and antioxidant capacity compared with the ITM group.

Effects of α-glycerol monolaurate on intestinal morphology, nutrient digestibility, serum profiles, and gut microbiota in weaned piglets.

This experiment was conducted to investigate the effects of dietary supplementation of α-glycerol monolaurate (α-GML) on the growth performance, nutrient digestibility, serum profiles, intestinal morphology, and gut microbiota of weaned piglets. A total of 96 healthy 28-d-old (Duroc × Landrace × Yorkshire) weaned piglets with body weight of 8.34 ± 0.05 kg were randomly divided into 2 treatment groups with 6 replicate pens and 8 piglets per pen. The control group was fed a basal diet and the experimental group was fed the basal diet supplemented with 1,000 mg/kg α-GML. The experiment lasted for 28 d. Dietary supplementation with α-GML had no effect on average daily gain, average daily feed intake, or gain to feed ratio in piglets (P > 0.05); however, it reduced (P < 0.05) diarrhea rate of piglets on days 15 to 28. The apparent total tract digestibility of dry matter (DM), crude protein (CP), ether extract (EE), and gross energy (GE) on day 14, and DM, organic matter, CP, EE, and GE on day 28 increased (P < 0.05) with α-GML supplementation. Moreover, higher (P < 0.05) glutathione peroxidase activity and interleukin-10 (IL-10) concentration, and lower (P < 0.05) malondialdehyde and tumor necrosis factor-α concentrations were observed in piglets supplemented with α-GML compared with the control group on day 14. Compared with the control group, the villus height/crypt depth in the duodenum and villus height in the jejunum and ileum were significantly greater (P < 0.05) in the α-GML group. Dietary α-GML supplementation significantly increased (P < 0.05) the relative abundance of Firmicutes, while decreasing (P < 0.05) Bacteroidota and Campilobacterota in the cecal contents; significantly increased (P < 0.05) the relative proportion of Lactobacillus and Blautia species, reduced (P < 0.05) Eubacterium_rectale_ATCC_33656, Campylobacter, and uncultured_bacterium_Alloprevotella species. Thus, dietary α-GML supplementation at 1,000 mg/kg reduces diarrhea rate, improves intestinal morphology, nutrient digestibility, antioxidant capacity, and immune status, and ameliorates gut microbiota in weaned piglets.

Glycerol monolaurate (GML) is naturally present in breast milk as well as other natural sources such as coconut oil and is widely used as a food additive. Dietary α-GML is used in animal production due to its safe-guarding health and growth-promoting effects. In the present study, α-GML was evaluated for growth performance, blood parameters, and intestinal health in piglets. Dietary α-GML helped piglets digest dry matter, crude protein, ether extract, and gross energy in feed. The blood parameters and intestinal structure of piglets fed the diet containing 1,000 mg/kg α-GML were improved. In addition, α-GML supplementation promoted the colonization of beneficial bacteria and inhibited the number of harmful bacteria. In the current study, dietary α-GML was responsible for improving the health status, intestinal morphology, and digestion and absorption of nutrients of weaned piglets with less diarrhea.

Long noncoding RNA Linc00337 functions as an E2F1 co-activator and promotes cell proliferation in pancreatic ductal adenocarcinoma.

BACKGROUND: Long noncoding RNA (lncRNA) Linc00337 has been implicated in lung, gastric, colorectal and esophageal squamous cell carcinoma progression via various mechanisms; however, its clinicopathological significance and role in pancreatic ductal adenocarcinoma (PDAC) progression remains largely unknown. METHODS: Multiple approaches such as bioinformatic analysis, Transfection, quantitative real-time-PCR, Western blotting, animal studies, RNA-immunoprecipitation (RIP), RNA-pulldown and RNA-Fluorescence in situ hybridization (RNA-FISH) and were utilized to explore the role of Linc00337 in PDAC. RESULTS: Here we identified Linc00337 is an oncogenic lncRNA during PDAC progression. We found that the expression of Linc00337 is elevated in PDAC tissues and the higher Linc00337 predicts dismal prognosis. Functionally, Linc00337 promotes PDAC cell proliferation and cell cycle transition both in vitro and in vivo. Mechanistically, Linc00337 binds to E2F1 and functions as an E2F1 coactivator to trigger the targets expression during PDAC progression. CONCLUSION: Our results demonstrate a reciprocal regulation mechanism between Linc00337 and E2F1 in PDAC progression and report the clinical value of Linc00337 for PDAC prognosis and treatment.

Rapamycin ameliorates inflammation and fibrosis in the early phase of cirrhotic portal hypertension in rats through inhibition of mTORC1 but not mTORC2.

OBJECTIVE: Hepatic stellate cells (HSCs) transdifferentiation and subsequent inflammation are important pathological processes involved in the formation of cirrhotic portal hypertension. This study characterizes the pathogenetic mechanisms leading to cholestatic liver fibrosis and portal hypertension, and focuses on mammalian target of rapamycin (mTOR) pathway as a potential modulator in the early phase of cirrhotic portal hypertension. METHODS: Early cirrhotic portal hypertension was induced by bile duct ligation (BDL) for three weeks. One week after operation, sham-operated (SHAM) and BDL rats received rapamycin (2 mg/kg/day) by intraperitoneal injection for fourteen days. Vehicle-treated SHAM and BDL rats served as controls. Fibrosis, inflammation, and portal pressure were evaluated by histology, morphometry, and hemodynamics. Expressions of pro-fibrogenic and pro-inflammatory genes in liver were measured by RT-PCR; alpha smooth muscle actin (α-SMA) and antigen Ki67 were detected by immunohistochemistry; expressions of AKT/mTOR signaling molecules, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and interleukin-1 beta (IL-1ß) were assessed by western blot. RESULTS: The AKT/mTOR signaling pathway was markedly activated in the early phase of cirrhotic portal hypertension induced by BDL in rats. mTOR blockade by rapamycin profoundly improved liver function by limiting inflammation, fibrosis and portal pressure. Rapamycin significantly inhibited the expressions of phosphorylated 70KD ribosomal protein S6 kinase (p-P70S6K) and phosphorylated ribosomal protein S6 (p-S6) but not p-AKT Ser473 relative to their total proteins in BDL-Ra rats. Those results suggested that mTOR Complex 1 (mTORC1) rather than mTORC2 was inhibited by rapamycin. Interestingly, we also found that the level of p-ERK1/2 to ERK1/2 was significantly increased in BDL rats, which was little affected by rapamycin. CONCLUSIONS: The AKT/mTOR signaling pathway played an important role in the early phase of cirrhotic portal hypertension in rats, which could be a potential target for therapeutic intervention in the early phase of such pathophysiological progress.