BRD9 promotes the progression of gallbladder cancer via CST1 upregulation and interaction with FOXP1 through the PI3K/AKT pathway and represents a therapeutic target.

Gallbladder cancer (GBC) is highly aggressive and has poor prognosis, with most patients only diagnosed at an advanced stage. Furthermore, treatment options are limited, and their effect is unsatisfactory. Bromodomain-containing protein (BRD) is an epigenetic regulator that plays a carcinogenic role in several tumors, including squamous cell lung cancer, acute myeloid leukemia, synovial sarcoma, and malignant rhabdomyosarcoma. However, the expression, biological function, and molecular mechanisms of action of BRD9 in GBC are still unknown. Kaplan-Meier analysis, qRT-PCR, and analysis of clinical features were used to assess the clinical significance of BRD9 in GBC. Cell Counting Kit-8 and colony formation assays were performed to determine the effects of BRD9 on cell growth. The functional role of BRD9 in GBC was explored using qRT-PCR, western blotting, siRNA, and CHIP-qPCR. mRNA sequencing was performed to explore the underlying mechanisms of BRD9, and a nude mouse model of GBC was established to explore the anti-tumor effects of the BRD9 inhibitor I-BRD9 in vivo. BRD9 expression was elevated in GBC tissues compared with adjacent non-tumor tissues, and high BRD9 expression was associated with poor prognosis in patients with GBC. BRD9 knockdown by siRNA significantly decreased cell growth. Targeting BRD9 with I-BRD9 inhibited the proliferation of GBC cells without significant toxic effects. Additionally, I-BRD9 treatment suppressed CST1 expression in GBC cell lines, thereby inhibiting the PI3K-AKT pathway. The transcription factor FOXP1 was found to interact with BRD9 to regulate CST1 expression. Collectively, these results suggest that BRD9 may be a promising biomarker and therapeutic target for GBC.

PTBP3 Mediates IL-18 Exon Skipping to Promote Immune Escape in Gallbladder Cancer.

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system, with poor response to current treatments. Abnormal alternative splicing has been associated with the development of a variety of tumors. Combining the GEO database and GBC mRNA-seq analysis, it is found high expression of the splicing factor polypyrimidine region- binding protein 3 (PTBP3) in GBC. Multi-omics analysis revealed that PTBP3 promoted exon skipping of interleukin-18 (IL-18), resulting in the expression of ΔIL-18, an isoform specifically expressed in tumors. That ΔIL-18 promotes GBC immune escape by down-regulating FBXO38 transcription levels in CD8+T cells to reduce PD-1 ubiquitin-mediated degradation is revealed. Using a HuPBMC mouse model, the role of PTBP3 and ΔIL-18 in promoting GBC growth is confirmed, and showed that an antisense oligonucleotide that blocked ΔIL-18 production displayed anti-tumor activity. Furthermore, that the H3K36me3 promotes exon skipping of IL-18 by recruiting PTBP3 via MRG15 is demonstrated, thereby coupling the processes of IL-18 transcription and alternative splicing. Interestingly, it is also found that the H3K36 methyltransferase SETD2 binds to hnRNPL, thereby interfering with PTBP3 binding to IL-18 pre-mRNA. Overall, this study provides new insights into how aberrant alternative splicing mechanisms affect immune escape, and provides potential new perspectives for improving GBC immunotherapy.

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.

Degradation kinetics of vitamins in premixes for pig: effects of choline, high concentrations of copper and zinc, and storage time.

OBJECTIVE: The present work was undertaken to evaluate the effects of storage time, choline chloride, and high concentrations of Cu and Zn on the kinetic behavior of vitamin degradation during storage in two vitamin premixes and four vitamin-trace mineral (VTM) premixes. METHODS: Two vitamin premixes (with or without 160,000 mg/kg of choline) were stored at 25°C and 60% humidity. Besides, four VTM premixes were used to evaluate the effects of choline (0 vs 40,000 mg/kg) and trace minerals (low CuSO4+ZnO vs high CuSO4+ZnO) on vitamin stability in VTM premixes stored in room, and the VTM premixes were stored in room temperature at 22°C. Subsamples from each vitamin and VTM premix were collected at 0, 1, 2, 3, 6, and 12 months. The retention of vitamin A (VA), vitamin D3 (VD3), vitamin E (VE), vitamin K3 (VK3), vitamin B1 (VB1), vitamin B2 (VB2), vitamin B3 (VB3), vitamin B5 (VB5), and vitamin B6 (VB6) in vitamin premixes and VTM premixes during storage was determined. The stability of vitamins in vitamin premixes and VTM premixes was determined and reported as the residual vitamin activity (% of initial) at each sampling point. RESULTS: The effect of choline on VK3 retention was significant in vitamin premixes (p<0.05). The negative effect of storage time was significant for the retentions of VD3, VK3, VB1, VB2, VB5, and VB6 in vitamin premix (p<0.05). For VTM premixes, negative effect of storage time was significant (p<0.05) for the losses of vitamin in VTM premixes. Choline and high concentrations of Cu and Zn significantly increased VA, VK3, VB1, and VB2 loss during storage (p<0.05). The supplementation of high concentrations of Cu and Zn significantly decreased the concentrations of VD3 and VB6 (p<0.05) in VTM premixes at extended storage time. CONCLUSION: The maximum vitamin stability was detected in vitamin and VTM premixes containing no choline or excess Cu and Zn. The results indicated that extended storage time increased degradation of vitamin in vitamin or VTM premixes. These results may provide useful information for vitamin and VTM premixes to improve the knowledge of vitamin in terms of its stability.

The strategies for the supplementation of vitamins and trace minerals in pig production: surveying major producers in China.

OBJECTIVE: Adequate vitamin and trace mineral intake for pigs are important to achieve satisfactory growth performance. There are no data available on the vitamin and trace mineral intake across pig producers in China. The purpose of this study was to investigate and describe the amount of vitamin and trace minerals used in Chinese pig diets. METHODS: A 1-year survey of supplemented vitamin and trace minerals in pig diets was organized in China. A total of 69 producers were invited for the survey, which represents approximately 90% of the pig herd in China. Data were compiled by bodyweight stages to determine descriptive statistics. Nutrients were evaluated for vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, vitamin B6, vitamin B12, pantothenic acid, niacin, folic acid, biotin, choline, copper, iron, manganese, zinc, selenium, and iodine. Data were statistically analyzed by functions in Excel. RESULTS: The results indicated variation for supplemented vitamin (vitamin A, vitamin D, vitamin E, vitamin K, vitamin B12, pantothenic acid, niacin, and choline) and trace minerals (copper, manganese, zinc, and iodine) in pig diets, but most vitamins and trace minerals were included at concentrations far above the total dietary requirement estimates reported by the National Research Council and the China's Feeding Standard of Swine. CONCLUSION: The levels of vitamin and trace mineral used in China's pig industry vary widely. Adding a high concentration for vitamin and trace mineral appears to be common practice in pig diets. This investigation provides a reference for supplementation rates of the vitamins and trace minerals in the China's pig industry.