Nucleolin lactylation contributes to intrahepatic cholangiocarcinoma pathogenesis via RNA splicing regulation of MADD.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a fatal malignancy of the biliary system. The lack of a detailed understanding of oncogenic signaling or global gene expression alterations has impeded clinical iCCA diagnosis and therapy. The role of protein lactylation, a newly unraveled post-translational modification that orchestrates gene expression, remains largely elusive in the pathogenesis of iCCA. METHODS: Proteomics analysis of clinical iCCA specimens and adjacent tissues was performed to screen for proteins aberrantly lactylated in iCCA. Mass spectrometry, macromolecule interaction and cell behavioral studies were employed to identify the specific lactylation sites on the candidate protein(s) and to decipher the downstream mechanisms responsible for iCCA development, which were subsequently validated using a xenograft tumor model and clinical samples. RESULTS: Nucleolin (NCL), the most abundant RNA-binding protein in the nucleolus, was identified as a functional lactylation target that correlates with iCCA occurrence and progression. NCL was lactylated predominantly at lysine 477 by the acyltransferase P300 in response to a hyperactivity of glycolysis, and promoted the proliferation and invasion of iCCA cells. Mechanistically, lactylated NCL bound to the primary transcript of MAP kinase-activating death domain protein (MADD) and led to efficient translation of MADD by circumventing alternative splicing that generates a premature termination codon. NCL lactylation, MADD translation and subsequent ERK activation promoted xenograft tumor growth and were associated with overall survival in patients with iCCA. CONCLUSION: NCL is lactylated to upregulate MADD through an RNA splicing-dependent mechanism, which potentiates iCCA pathogenesis via the MAPK pathway. Our findings reveal a novel link between metabolic reprogramming and canonical tumor-initiating events, and uncover biomarkers that can potentially be used for prognostic evaluation or targeted treatment of iCCA. IMPACT AND IMPLICATIONS: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive liver malignancy with largely uncharacterized pathogenetic mechanisms. Herein, we demonstrated that glycolysis promotes P300-catalyzed lactylation of nucleolin, which upregulates MAP kinase-activating death domain protein (MADD) through precise mRNA splicing and activates ERK signaling to drive iCCA development. These findings unravel a novel link between metabolic rewiring and canonical oncogenic pathways, and reveal new biomarkers for prognostic assessment and targeting of clinical iCCA.

A COMPARATIVE ANALYSIS TO DETERMINE THE OPTIMUM HISTONE DEACETYLASE INHIBITORS AND ADMINISTRATION ROUTE FOR IMPROVING SURVIVAL AND ORGAN INJURY IN RATS AFTER HEMORRHAGIC SHOCK.

ABSTRACT: Objective: Histone deacetylase inhibitors (HDACIs) have been reported to improve survival in rats with hemorrhagic shock (HS). However, no consensus exists on the most effective HDACIs and their administration routes. We herein aimed to determine the optimal HDACIs and administration route in rats with HS. Methods: Survival analysis: In experiment I, male Sprague-Dawley rats were subjected to HS (mean arterial pressure [MAP] was maintained at 30-40 mm Hg for 20 min), and intravenously injected with the following agents (n = 8 per group): (1) no treatment, (2) vehicle (VEH), (3) entinostat (MS-275), (4) [ N -((6-(Hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide] (LMK-235), (5) tubastatin A, (6) trichostatin A (TSA), and (7) sirtinol. In experiment II, rats were intraperitoneally injected with TSA. Mechanism research: In experiments I and II, rats were observed for 3 h, after which blood samples and liver, heart, and lung tissues were harvested. Results: In experiment I, 75% rats in the VEH group but only 25% rats in the LMK-235 and sirtinol groups died within ≤5 h of treatment, whereas the survival of rats in the MS-275, tubastatin A, and TSA groups was significantly prolonged. MS-275, LMK-235, tubastatin A, and TSA significantly reduced histopathological scores, apoptosis cell numbers, and inflammatory cytokine levels. In experiment II, the survival was longer after i.v. TSA treatment than after i.p. TSA treatment, and the IL-6 levels in the heart were significantly lower in rat who received i.p. TSA treatment than in those who received i.v. TSA treatment. Conclusions: The i.v. effect was superior to the i.p. effect, while nonselective and isoform-specific classes I and IIb HDACIs had similar effects.

Protective effect of HDACIs in improves survival and organ injury after CLP-induced sepsis.

Introduction: The effects of isoform-specific histone deacetylase inhibitors (HDACIs) and the non-selective HDACI on sepsis have been profoundly reported. However, the best HDAC classes have not been fully evaluated. Therefore, this study aimed to determine which HDACIs are responsible for survival and beneficial for organ injury. Methods: Experiment I, SD rats were subjected to cecal ligation and puncture and randomly assigned to the no treatment, dimethyl sulfoxide (DMSO) only, MS-275, LMK-235, tubastatinA (TubA), trichostatin-A (TSA), and sirtinol groups (n = 5). Survival was monitored for 48 h. Experiment II, the animals were monitored for 12 h, then, blood and tissues sample were collected. Interleukin (IL)-6, IL-1ß, tumour necrosis factor (TNF)-α, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) expressions were evaluated using ELISA. Liver, heart and lung tissues were analysed via hematoxylin and eosin staining. Liver and heart tissue lysates were analysed for acetylated histones H3, H4, a-tubulin and nuclear factor kappa B (NF-κB), IL-6, IL-1ß, and TNF-α using western blotting. Splenocytes were examined via flow cytometry to analyse the immune cell population. Results: MS-275, TubA and TSA treatments significantly prolonged survival. MS-275, LMK-235, TubA and TSA significantly reduced the histopathological scores and AST, ALT, CK, LDH, IL-6, IL-1ß and TNF-α levels, significantly increased acetylated of NF-κB and changed the immune cell proportion. Conclusion: Our results indicated that HDACI classes I and IIb and non-selective HDACI can significantly prolong survival. Moreover, non-selective and isoform-specific class I and IIa/IIb HDACIs can attenuate inflammation and organ injury.

BAP31 regulates the expression of ICAM-1/VCAM-1 via MyD88/NF-κB pathway in acute lung injury mice model.

AIMS: The cell adhesion molecules (CAMs) that mediate neutrophil-endothelium cell adhesion are deeply involved in the pathogenesis of acute lung injury (ALI). B-cell receptor associated protein 31 (BAP31) has been reported to engage in the expression of some CAMs. This study was undertaken to explore whether BAP31 in endotheliocyte affects the pathological process of ALI by regulating CAMs, and its possible mechanism. MAIN METHODS: Our study used the shBAP31 endothelium cell lines and endothelial-specific BAP31 conditional knockdown mice constructed via Cre/loxP system. Hematoxylin and eosin staining was used to observe the histopathological manifestations. The adhesion of neutrophils to vascular wall was examined by intravital microscopy. The nuclear translocation of NF-κB was observed by immunofluorescence staining assay. Flow cytometric, real-time polymerase chain reaction and Western blot assay were performed to determine the expression of CAMs and key proteins in MyD88/NF-κB-related signaling pathway. Luciferase reporter and chromatin immunoprecipitation assay were analyzed for transcriptional activity of ICAM-1 and VCAM-1. KEY FINDINGS: Mechanistic investigations indicated that endothelium-specific BAP31 depletion dramatically reduced the capacity of neutrophils adherence to endothelial cells (ECs), which was mainly attributed to the significant downregulation of ICAM-1 (p < 0.05) and VCAM-1 (p < 0.05) expression. Interestingly, BAP31 knockdown apparently deactivated MyD88/TRAF6-mediated TAK1/NF-κB and PI3K/Akt signaling cascades, resulting in the inhibition of NF-κB activation and nuclear translocation. SIGNIFICANCE: Our data furnished convincing evidence that BAP31 deficiency performs a mitigative effect on ALI by decreasing neutrophils-ECs adhesion. These findings identified BAP31 as a promising protein for regulating the pathogenesis process of ALI.

B-cell receptor associated protein 31 deficiency decreases the expression of adhesion molecule CD11b/CD18 and PSGL-1 in neutrophils to ameliorate acute lung injury.

Acute lung injury (ALI) and its more severe condition acute respiratory distress syndrome (ARDS) are critical life-threatening disorders characterized by an excessive influx of neutrophils into the alveolar space. Neutrophil infiltration is a multi-step process involving the sequential engagement of adhesion molecules. The adhesion molecule CD11b/CD18 acts as an important role in the recruitment of neutrophils to lung tissues in the ALI model. B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum transmembrane protein, has been reported to regulate the cellular anterograde transport of CD11b/CD18 in human neutrophils. To explore how BAP31 regulates CD11b/CD18 in mouse neutrophils, we constructed myeloid-specific BAP31 knockdown mice in this study. Biological investigations indicated that BAP31 deficiency could significantly alleviated lung injury, as evidenced by the improved histopathological morphology, reduced pulmonary wet/dry weight ratio, inhibited myeloperoxidase level and decreased neutrophil counts in the bronchoalveolar lavage fluid. Further studies clarified that BAP31 deficiency obviously down-regulated the expression of CD11b/CD18 and P-selectin glycoprotein ligand-1 (PSGL-1) by deactivating the nuclear factor kappa B (NF-κB) signaling pathway. Collectively, our results revealed that BAP31 depletion exerted a protective effect on ALI, which was possibly dependent on the attenuation of neutrophil adhesion and infiltration by blocking the expression of adhesion molecules CD11b/CD18 and PSGL-1. These findings implied the potential of BAP31 as an appealing protein to mediate the occurrence of ALI.

BAP31 affects macrophage polarization through regulating helper T cells activation.

Previously, we reported that B cell receptor associated protein 31 (BAP31) is a positive regulator on T-cells activation. Helper T cells [cluster of differentiation 4+ (CD4+) T cells] can regulate macrophage activation in adaptive immune response against pathogens. In this study, we elucidate that M1 and M2 macrophages polarization is significantly suppressed in Lck Cre-BAP31flox/flox mice or the co-culture system of CD4+ T cells from Lck Cre-BAP31flox/flox mice and macrophages from WT mice. It means that BAP31 may affect the regulation of CD4+ T cells on macrophages. Further studies suggest that BAP31 deficiency significantly reduce the expressions of T helper 1 (Th1)/ Th2/ Th17/ Th9/ Th22/ Treg cells-related cytokines and transcription factors. The inhibition of macrophages activation caused by BAP31 knockdown is due to the reduction of IFN-γ and IL-4 secreted by Th1 and Th2 cells. BAP31 also affects the levels of early activation markers (CD69 and CD25) of CD4+ T cells. Moreover, BAP31 deficiency downregulates the expression of TCRαß-CD3 complex, and the adaptor proteins p-Zap70, p-Lck, and p-Lat in TCR signaling pathway. These results demonstrate that BAP31 deficiency inhibits TCR/CD3-mediated activation in CD4+ T cells and adversely affects macrophages polarization. These findings establish a theoretical foundation for the study of BAP31 in immunotherapy.

LncRNA-URHC Functions as ceRNA to Regulate DNAJB9 Expression by Competitively Binding to miR-5007-3p in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is often diagnosed at a late stage, when the prognosis is poor. The regulation of long noncoding RNAs (lncRNAs) plays a crucial role in HCC. However, the precise regulatory mechanisms of lncRNA signaling in HCC remain largely unknown. Our study aims to investigate the underlying mechanisms of lncRNA (upregulated in hepatocellular carcinoma) URHC in HCC. OBJECTIVE: To study the in vivo and in vitro localization and biological effects of URHC on liver cancer cells. Through bioinformatics analysis, dual-luciferase reporter gene analysis and rescue experiments revealed the possible mechanism of URHC. METHODS: RT-qPCR, fluorescence in situ hybridization (FISH) staining, EdU, colony formation, and tumor xenograft experiments were used to identify localized and biological effects of URHC on HCC cells in vitro and in vivo. The bioinformatics analysis, dual-luciferase reporter assay, and rescue experiments revealed the potential mechanism of URHC. RESULTS: URHC silencing may inhibit the HCC cells' proliferation in vitro and in vivo. We found that URHC was mainly localized in the cytoplasm. The expression of miR-5007-3p was negatively regulated by URHC. And miR-5007-3p could reverse the effect of URHC in HCC cells. The expression of DNAJB9 was negatively regulated by miR-5007-3p but positively regulated by URHC. These suggestive of lncRNA-URHC positively regulated the level of DNAJB9 by sponging miR-5007-3p. CONCLUSION: Together, our study elucidated the role of URHC as a miRNA sponge in HCC and shed new light on lncRNA-directed diagnostics and therapeutics in HCC.

Wogonin alleviates liver injury in sepsis through Nrf2-mediated NF-κB signalling suppression.

Sepsis is a life-threatening organ dysfunction syndrome, and liver is a susceptible target organ in sepsis, because the activation of inflammatory pathways contributes to septic liver injury. Oxidative stress has been documented to participate in septic liver injury, because it not only directly induces oxidative genotoxicity, but also exacerbates inflammatory pathways to potentiate damage of liver. Therefore, to ameliorate oxidative stress is promising for protecting liver in sepsis. Wogonin is the compound extracted from the medicinal plant Scutellaria baicalensis Geogi and was found to exert therapeutic effects in multiple inflammatory diseases via alleviation of oxidative stress. However, whether wogonin is able to mitigate septic liver injury remains unknown. Herein, we firstly proved that wogonin treatment could improve survival of mice with lipopolysaccharide (LPS)- or caecal ligation and puncture (CLP)-induced sepsis, together with restoration of reduced body temperature and respiratory rate, and suppression of several pro-inflammatory cytokines in circulation. Then, we found that wogonin effectively alleviated liver injury via potentiation of the anti-oxidative capacity. To be specific, wogonin activated Nrf2 thereby promoting expressions of anti-oxidative enzymes including NQO-1, GST, HO-1, SOD1 and SOD2 in hepatocytes. Moreover, wogonin-induced Nrf2 activation could suppress NF-κB-regulated up-regulation of pro-inflammatory cytokines. Ultimately, we provided in vivo evidence that wogonin activated Nrf2 signalling, potentiated anti-oxidative enzymes and inhibited NF-κB-regulated pro-inflammatory signalling. Taken together, this study demonstrates that wogonin can be the potential therapeutic agent for alleviating liver injury in sepsis by simultaneously ameliorating oxidative stress and inflammatory response through the activation of Nrf2.

LINC00671 suppresses cell proliferation and metastasis in pancreatic cancer by inhibiting AKT and ERK signaling pathway.

Long noncoding RNAs (lncRNAs) represent an emerging field of tumor biology, playing essential roles in cancer cell proliferation, invasion, and metastasis. However, the overall functional and clinical significance of most lncRNAs in pancreatic cancer is not thoroughly understood. Here, we described most of the lncRNAs with aberrant expression patterns in pancreatic cancer as detected by microarray. Quantitative real-time polymerase chain reaction further verified that the expression of LINC00671 was decreased in pancreatic cancer cell lines and patient samples. Furthermore, lower LINC00671 expression was associated with reduced tumor differentiation, aggressiveness, and poor prognosis. Functionally, LINC00671 overexpression inhibited pancreatic cancer cell proliferation, invasion, and migration in vitro, and reduced tumor growth in vivo. LINC00671 is mainly located in the cytoplasm. RNA sequencing and bioinformatics analyses indicated that LINC00671 binds to multiple miRNAs and therefore could be involved in multiple tumor-associated pathways, such as the AMPK signaling pathway and PI3K-Akt signaling pathway. Western blotting and immunohistochemistry further confirmed that LINC00671 overexpression suppressed the AKT, ERK, and epithelial-mesenchymal transition pathways. Overall, these results indicated that LINC00671 acts as a novel tumor suppressor in pancreatic cancer. Our findings may provide a new potential target for the treatment of pancreatic cancer.

Circular RNA circRHOT1 is upregulated and promotes cell proliferation and invasion in pancreatic cancer.

AIM: We aimed to identify the roles of circRHOT1 in pancreatic cancer. MATERIALS & METHODS: The circRHOT1 was acquired from our previous study followed by quantitative real-time PCR and fluorescence in situ hybridization validation in pancreatic cancer. We used siRNA and shRNA to explore the function of circRHOT1 in pancreatic cancer cells. Bioinformatic analyses were applied to study the potential mechanism of circRHOT1. RESULTS: The circRHOT1 was upregulated in pancreatic cancer and predominantly located in the cytoplasm. Reducing the circRHOT1 expression may inhibit the pancreatic cancer cell proliferation, invasion and migration. The circRHOT1 may play a role in pancreatic cancer through binding miR-26b, miR-125a, miR-330 and miR-382 to regulate multiple tumor-associated pathways. CONCLUSION: This study demonstrated that circRHOT1 may serve as an oncogenic circRNA that promotes tumor progression.

Sestrin 2 confers primary resistance to sorafenib by simultaneously activating AKT and AMPK in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the malignancy derived from normal hepatocytes with increasing incidence and extremely poor prognosis worldwide. The only approved first-line systematic treatment agent for HCC, sorafenib, is capable to effectively improve advanced HCC patients' survival. However, it is gradually recognized that the therapeutic response to sorafenib could be drastically diminished after short-term treatment, defined as primary resistance. The present study is aimed to explore the role of stress-inducible protein Sestrin2 (SESN2), one of the most important sestrins family members, in sorafenib primary resistance. Herein, we initially found that SESN2 expression was significantly up-regulated in both HCC cell lines and tissues compared to normal human hepatocytes and corresponding adjacent liver tissues, respectively. In addition, SESN2 expression was highly correlated with sorafenib IC50 of HCC cell lines. Thereafter, we showed that sorafenib treatment resulted in an increase of SESN2 expression and the knockdown of SESN2 exacerbated sorafenib-induced proliferation inhibition and cell apoptosis. Further mechanistic study uncovered that SESN2 deficiency impaired both AKT and AMPK phosphorylation and activation after sorafenib treatment. Moreover, the correlations between SESN2 expression and both phosphor-AKT and phosphor-AMPK expression were illustrated in HCC tissues. Taken together, our study demonstrates that SESN2 activates AKT and AMPK signaling as a novel mechanism to induce sorafenib primary resistance in HCC.

[Sestrin 2(SESN2) promotes primary resistance to sorafenib by activating AKT in hepatocellular carcinoma cells].

Objective To explore the role of sestrin2 (SESN2) in sorafenib primary resistance and the underlying mechanism in hepatocellular carcinoma (HCC) cells. Methods Real-time quantitative PCR (qRT-PCR) and Western blot analysis were performed to examine SESN2 mRNA and protein levels in Bel-7404, SNU-398, HLE, HLF and Hep3B cell lines. Immunohistochemical staining was used to detect SESN2 expression in HCC tissues. After the treatment of 0, 2, 5, 10, 15, 20, 25 µmol/L sorafenib for 24 hours, CCK-8 assay was performed to detect the cell viability and subsequent IC50 of sorafenib in the above HCC cell lines. After the treatment of 0, 2, 4, 6, 8 µmol/L sorafenib for 24 hours, qRT-PCR and Western blot analysis were conducted to measure the alterations of SESN2 mRNA and protein expressions in Bel-7404 and SNU-398 cells. CCK-8 assay and flow cytometry were performed to examine the viability and apoptosis of Bel-7404 and SNU-398 cells after sorafenib treatment with or without SESN2 knockdown by siRNA transfection. Western blot analysis was used to test the expressions of AKT and phosphorylated-AKT. Results Compared with the control, SESN2 expression markedly increased in both HCC cell lines and tissues and there was a positive correlation between SESN2 expression and IC50 of sorafenib in different HCC cell lines. Subsequently, the mRNA and protein levels of SESN2 were significantly elevated after sorafenib treatment in Bel-7404 and SNU-398 cells, and SESN2 knockdown led to decreased cell viability and increased cell apoptosis after sorafenib treatment. More importantly, SESN2 knockdown impaired sorafenib-induced AKT activation in HCC cells. Conclusion SESN2 up-regulation conferred primary resistance to sorafenib by activating AKT in HCC cells.

BAP31 is involved in T cell activation through TCR signal pathways.

BAP31 is a ubiquitously expressed endoplasmic reticulum (ER) membrane protein. The functions of BAP31 in the immune system have not been investigated due to the lack of animal models. Therefore we created a BAP31 conditional knockdown mouse by performing a knockdown of BAP31 in the thymus. In doing so, we demonstrate that the maturation of T cells is normal but the number of T cells is less in the thymus of the knockout mouse. In addition, the spleen and lymph nodes of peripheral immune organs contained a lesser proportion of the mature T cells in the thymus specific BAP31 knockout mice. The BAP31 knockout T cells decreased the proliferation activated by TCR signal pathways. Further studies clarified that BAP31 affects the phosphorylation levels of both Zap70/Lck/Lat of the upstream members and Akt/GSK/Jnk/Erk of the downstream members of TCR signal pathways. Furthermore, BAP31 can regulate the expression of some markers such as CD3/TCRα/TCRß and some cytokines like IL-2/IFN-γ/IL-6/TNF-α which are important for T cell activation. Taken together, these results demonstrate that BAP31 may play an important role in T cell activation by regulating TCR signaling.

Autoimmune Regulator Expression in DC2.4 Cells Regulates the NF-κB Signaling and Cytokine Expression of the Toll-Like Receptor 3 Pathway.

Autoimmune regulator (Aire) mutations result in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which manifests as multi-organ autoimmunity and chronic mucocutaneous candidiasis (CMC). Indendritic cells (DCs), pattern recognition receptors (PRR), such as Toll-like receptors (TLRs), are closely involved in the recognition of various pathogens, activating the intercellular signaling pathway, followed by the activation of transcription factors and the expression of downstream genes, which take part in mediating the immune response and maintaining immune tolerance. In this study, we found that Aire up-regulated TLR3 expression and modulated the downstream cytokine expression and nuclear factor-κB (NF-κB) of the TLR3 signaling pathway.

Aire-Overexpressing Dendritic Cells Induce Peripheral CD4⁺ T Cell Tolerance.

Autoimmune regulator (Aire) can promote the ectopic expression of peripheral tissue-restricted antigens (TRAs) in thymic medullary epithelial cells (mTECs), which leads to the deletion of autoreactive T cells and consequently prevents autoimmune diseases. However, the functions of Aire in the periphery, such as in dendritic cells (DCs), remain unclear. This study's aim was to investigate the effect of Aire-overexpressing DCs (Aire cells) on the functions of CD4⁺ T cells and the treatment of type 1 diabetes (T1D). We demonstrated that Aire cells upregulated the mRNA levels of the tolerance-related molecules CD73, Lag3, and FR4 and the apoptosis of CD4⁺ T cells in STZ-T1D mouse-derived splenocytes. Furthermore, following insulin stimulation, Aire cells decreased the number of CD4⁺ IFN-γ⁺ T cells in both STZ-T1D and WT mouse-derived splenocytes and reduced the expression levels of TCR signaling molecules (Ca(2+) and p-ERK) in CD4⁺ T cells. We observed that Aire cells-induced CD4⁺ T cells could delay the development of T1D. In summary, Aire-expressing DCs inhibited TCR signaling pathways and decreased the quantity of CD4⁺IFN-γ⁺ autoreactive T cells. These data suggest a mechanism for Aire in the maintenance of peripheral immune tolerance and provide a potential method to control autoimmunity by targeting Aire.