Human cytomegalovirus UL36 inhibits IRF3-dependent immune signaling to counterbalance its immunoenhancement as apoptotic inhibitor.

Apoptotic inhibition and immune evasion have particular importance to efficient viral infection, while a dilemma often faced by viruses is that inhibiting apoptosis can up-regulate antiviral immune signaling. Herein, we uncovered that in addition to inhibiting caspase-8/extrinsic apoptosis, human cytomegalovirus (HCMV)-encoded UL36 suppresses interferon regulatory factor 3 (IRF3)-dependent immune signaling by directly targeting IRF3 to abrogate IRF3 interaction with stimulator of interferon genes or TANK-binding kinase 1 and inhibit IRF3 phosphorylation/activation. Although UL36-mediated caspase-8/extrinsic apoptosis inhibition enhances immune signaling, the immunosuppressing activity of UL36 counterbalances this immunoenhancing "side effect" undesirable for virus. Furthermore, we used mutational analyses to show that only the wild-type, but not the UL36 mutant losing either inhibitory activity, is sufficient to support effective HCMV replication in cells, showing the functional importance of the dual inhibition by UL36 for the HCMV life cycle. Together, our findings demonstrate a sophisticated mechanism by which HCMV tightly controls innate immune signaling and extrinsic apoptosis for efficient infection.

Renin-angiotensin system inhibitors improve the survival of cholangiocarcinoma: a propensity score-matched cohort study.

BACKGROUND: Hypertension is a risk factor for cholangiocarcinoma (CCA). The effect of anti-hypertensive drugs on the prognosis of CCA is not clear. METHODS: This is a retrospective study of 102 patients (56.9% males, median age 66 years) diagnosed with CCA and hypertension concurrently and received radical surgery (R0), with a median follow-up of 36.7 months. Kaplan-Meier analysis, Cox regressions, and propensity score (PS) matching were applied for statistical analysis. RESULTS: Results of multivariable cox analysis showed that renin-angiotensin system inhibitors (RASis) usage was a protective factor for progression-free survival (PFS) (hazard ratio [HR] = 0.55, 95% confidence interval [95% CI]: 0.32-0.96) and overall survival (OS) (HR = 0.40, 95% CI: 0.20-0.79), respectively. Calcium channel blockers, diuretics, and ß-blockers didn't show significant associations. The association of RASis usage and PFS and OS was derived by PS matching, with a cohort of 28 RASis users and 56 RASis non-users. The median PFS and OS of RASis users (PFS, 17.6 months (9.2-34.4); OS, 24.8 months (16.5-42.3)) were longer than RASis non-users (PFS, 10.5 months (4.1-24.1); OS, 14.6 months (10.6-28.4)). The 1 year, 2 years, and 3 years' survival rates of RASis users (89.1%, 77.0%, and 65.5%) were higher than RASis non-users (70.9%, 54.0%, and 40.0%). CONCLUSIONS: RASis usage improves the survival of patients with CCA and hypertension concurrently.

SUMO specific peptidase 3 halts pancreatic ductal adenocarcinoma metastasis via deSUMOylating DKC1.

In the past few decades, advances in the outcomes of patients suffering from pancreatic ductal adenocarcinoma (PDAC) have lagged behind these gained in the treatment of many other malignancies. Although the pivotal role of the SUMO pathway in PDAC has been illustrated, the underlying molecule drivers have yet to be fully elucidated. In the present study, we identified SENP3 as a potential suppressor of PDAC progression through an in vivo metastatic model. Further studies revealed that SENP3 inhibited PDAC invasion in a SUMO system dependent fashion. Mechanistically, SENP3 interacted with DKC1 and, as such, catalyzed the deSUMOylation of DKC1, which accepted SUMO3 modifiers at three lysine residues. SENP3-mediated deSUMOylation caused DKC1 instability and disruption of the interaction between snoRNP proteins, which contributed to the impaired migration ability of PDAC. Indeed, overexpression of DKC1 abated the anti-metastasis effect of SENP3, and DKC1 was elevated in PDAC specimens and associated with a poor prognosis in PDAC patients. Collectively, our findings shed light on the essential role of SENP3/DKC1 axis in the progression of PDAC.

Angiotensin receptor blockers retard the progression and fibrosis via inhibiting the viability of AGTR1+ CAFs in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy characterized by massive fibrosis and has ineffective adjuvant therapies. Here, we demonstrate the potential of angiotensin receptor blockers (ARBs) in targeting iCCA. METHODS: Masson's trichrome staining was used to assess the effect of ARBs in iCCA specimens, CCK8 and gel contraction assays in vitro and in xenograft models in vivo. RNA-seq and ATAC-seq were used for mechanistic investigations. RESULTS: Patients with iCCA who were administered ARBs had a better prognosis and a lower proportion of tumour stroma, indicating alleviated fibrosis. The presence of AGTR1, the ARBs receptor, is associated with a poor prognosis of iCCA and is highly expressed in tumour tissues and cancer-associated fibroblasts (CAFs). The ARBs strongly attenuated the viability of AGTR1+ CAFs in vitro and retarded tumour progression and fibrosis in xenograft models of co-cultured CAFs and iCCA cells. Still, they did not have a significant effect on AGTR1- CAFs. Moreover, ARBs decreased the secretion of AGTR1+ CAF-derived MFAP5 via the Hippo pathway, weakened the interaction between CAFs and iCCA cells, and impaired the aggressiveness of iCCA cells by attenuating the activation of the Notch1 pathway in iCCA cells. CONCLUSIONS: ARBs exhibit anti-fibrotic function by inhibiting the viability of AGTR1+ CAFs. These findings support using ARBs as a novel therapeutic option for targeting iCCA.

Pancreatic ductal adenocarcinoma cells regulated the gemcitabine-resistance function of CAFs by LINC00460.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy with extremely poor prognosis. Gemcitabine resistance is a major challenge in the treatment of PDAC. Here, we showed that LINC00460 was associated with the response to gemcitabine both in PDAC patients and PDAC-PDX. After knocking down LINC00460 in PDAC tumor cells, results of RNA sequencing followed by gene ontology analysis indicated that LINC00460 influenced the activity of growth factors and modified the extracellular matrix. FISH showed that LINC00460 is mostly located in the cytoplasm. Results of RNA pull-down, LC-MS/MS, RIP, and immunoblotting confirmed that LINC00460 could directly bind to PDAP1. Furthermore, we demonstrated that LINC00460 mediated the cellular communication of PDAC tumor cells and CAFs by PDAP1/PDGFA/PDGFR signaling pathway and regulated the gemcitabine-resistance function of CAFs, which could be reversed by treatment with a PDGFR inhibitor (crenolanib). PDAC-PDX tumors with lower expression of LINC00460 showed a better response to gemcitabine plus crenolanib treatment. Our finding supported the application of LINC00460 in precision medicine that uses gemcitabine plus crenolanib to treat PDAC with low expression of LINC00460.

Xiaoyaosan Exerts Antidepressant-Like Effect by Regulating Autophagy Involves the Expression of GLUT4 in the Mice Hypothalamic Neurons.

Many studies have proven that autophagy plays a pivotal role in the development of depression and it also affects the expression of GLUT4 in the hypothalamus. Xiaoyaosan has been shown to exert antidepressant effects in a variety of ways, but its underlying mechanism by which Xiaoyaosan regulates autophagy as well as GLUT4 in the hypothalamus remains unclear. Thus, in this study, we established a mouse model of depression induced by chronic unpredictable mild stress (CUMS), and set up autophagy blockade as a control to explore whether Xiaoyaosan exerts antidepressant effect by affecting autophagy. We examined the effects of Xiaoyaosan on behaviors exhibited during the open field test, tail suspension test and sucrose preference test, and the changes in autophagy in hypothalamic neurons as well as changes in GLUT4 and the related indicators of glucose metabolism in CUMS-induced depressive mouse model. We found that CUMS- and 3-MA-induced mice exhibited depressive-like behavioral changes, with decreased LC3 expression and increased p62 expression, suggesting decreased levels of autophagy in the mouse hypothalamus. The expression of GLUT4 was also decreased, and it was closely related to the level of autophagy through Rab8 and Rab10. Nevertheless, after the intervention of Xiaoyaosan, the above changes were effectively reversed. These results show that Xiaoyaosan can regulate the autophagy in hypothalamic neurons and the expression of GLUT4 in depressed mice.

ACLP promotes activation of cancer-associated fibroblasts and tumor metastasis via ACLP-PPARγ-ACLP feedback loop in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis. Its fibrotic tumor microenvironment (TME) plays a crucial role in promoting tumor invasion and metastasis, which eventually leads to a dismal 5-year survival rate in PDAC patients. Aortic carboxypeptidase-like protein (ACLP) promotes tissue fibrosis in benign diseases. However, its role in cancer-associated fibrosis remains unelucidated. Here, we show that ACLP was mainly expressed in cancer-associated fibroblasts (CAFs) but not in cancer cells and highly expressed in PDAC tissues. High ACLP expression was correlated with poor overall survival. Moreover, ACLP expression in PDAC patients with liver metastases was higher than that in PDAC patients without liver metastases. By detecting activation marker expression and CAF contractility and motility, we found that ACLP promoted CAF activation in PDAC, leading to TME fibrosis. Furthermore, ACLP-activated CAFs could promote cancer cell invasion in vitro and tumor metastasis in vivo. Mechanistically, ACLP promotes the expressions of MMP1 and MMP3 in CAFs, thus promoting PDAC invasion and metastasis. Intriguingly, we identified an ACLP-PPARγ-ACLP feedback loop in PDAC CAFs. Abatement of this feedback loop might be a promising approach in CAF-targeting PDAC treatment.

Carabin Deficiency Aggravates Hepatic Ischemia-Reperfusion Injury Through Promoting Neutrophil Trafficking via Ras and Calcineurin Signaling.

Neutrophil infiltration plays an important role in the initial phase of hepatic ischemia and reperfusion injury (HIRI). Despite many different key molecules that have been reported to meditate neutrophil trafficking in HIRI, the mechanism of this process has not been fully elucidated. In this study, we found that Carabin deficiency in myeloid cells (LysMCre : Carabinfl/fl) aggravated IRI-induced hepatic injury and apoptosis through increasing the infiltration of CD11b+Ly6G+ neutrophils. ImmGen Datasets further revealed that Carabin was expressed in bone marrow neutrophils (GM.BM) but was significantly downregulated in thio-induced peripheral neutrophils (GN.Thio.PC), which was consistently verified by comparing GM.BM and liver-infiltrating neutrophils induced by IRI. Mechanistically, up-regulation of Carabin in GM.BM in vitro reduced the expression levels of P-selectin, E-selectin, and αvß3 integrin through inhibiting Ras-ERK and Calcineurin-NFAT signaling. Furthermore, blocking P-selectin, E-selectin, and αvß3 integrin in LysMCre : Carabinfl/fl mice decreased the frequency and number of CD11b+Ly6G+ neutrophils and reversed hepatic ischemia-reperfusion damage. In conclusion, our results provide a new understanding of Carabin, such that it is expressed and functions not only in adaptive immune cells (T and B cells) but also in innate immune cells (neutrophils), contributing to the migration of neutrophils. These findings provide novel and promising therapeutic targets for the prevention of HIRI during liver transplantation or hepatic surgery.

Nitric Oxide Synthase Regulates Gut Microbiota Homeostasis by ERK-NF-κB Pathway in Shrimp.

The gut microbiota is a complex group of microorganisms that is not only closely related to intestinal immunity but also affects the whole immune system of the body. Antimicrobial peptides and reactive oxygen species participate in the regulation of gut microbiota homeostasis in invertebrates. However, it is unclear whether nitric oxide, as a key mediator of immunity that plays important roles in antipathogen activity and immune regulation, participates in the regulation of gut microbiota homeostasis. In this study, we identified a nitric oxide synthase responsible for NO production in the shrimp Marsupenaeus japonicus. The expression of Nos and the NO concentration in the gastrointestinal tract were increased significantly in shrimp orally infected with Vibrio anguillarum. After RNA interference of Nos or treatment with an inhibitor of NOS, L-NMMA, NO production decreased and the gut bacterial load increased significantly in shrimp. Treatment with the NO donor, sodium nitroprusside, increased the NO level and reduced the bacterial load significantly in the shrimp gastrointestinal tract. Mechanistically, V. anguillarum infection increased NO level via upregulation of NOS and induced phosphorylation of ERK. The activated ERK phosphorylated the NF-κB-like transcription factor, dorsal, and caused nuclear translocation of dorsal to increase expression of antimicrobial peptides (AMPs) responsible for bacterial clearance. In summary, as a signaling molecule, NOS-produced NO regulates intestinal microbiota homeostasis by promoting AMP expression against infected pathogens via the ERK-dorsal pathway in shrimp.

HNRNPC impedes m6A-dependent anti-metastatic alternative splicing events in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with poor prognosis due to early metastasis. The aberrant N6-methyladenosine (m6A) RNA modification has emerged as an important mechanism in cancer progression and metastasis, but its role in PDAC remained largely unknown. Here, we demonstrated that an m6A regulator, heterogeneous nuclear ribonucleoprotein C (HNRNPC), modulated alternative splicing events to promote PDAC metastasis. In clinical PDAC tissues, high expression of HNRNPC was correlated with metastasis, resulting in poor prognosis in PDAC patients. Knockdown of HNRNPC significantly reduced PDAC cell invasion in vitro and metastasis in vivo. In contrast, overexpression of HNRNPC provoked malignant phenotypes of PDAC cells. Mechanistically, HNRNPC antagonized the anti-metastatic isoform of TAF8 (TAF8L) but increased the pro-metastatic alternative splicing isoform of TAF8 (TAF8S). Mutation of the m6A-site of TAF8 attenuated the interaction between HNRNPC and TAF8 transcript, leading to the decrease of TAF8S. Furthermore, experimental manipulation of the anti-metastasis splicing isoform TAF8L revealed that splice isoform switching of TAF8 is crucial for PDAC metastasis. In conclusion, our findings demonstrate the essentiality of HNRNPC-mediated alternative splicing events that impinges on metastatic PDAC.

MFAP5 facilitates the aggressiveness of intrahepatic Cholangiocarcinoma by activating the Notch1 signaling pathway.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. The dismal outcome of ICC patients is due to lack of early diagnosis, the aggressive biological behavior of ICC and the lack of effective therapeutic options. Early diagnosis and prognosis of ICC by non-invasive methods would be helpful in providing valuable information and developing effective treatment strategies. METHODS: Expression of microfibrillar-associated protein 5 (MFAP5) in the serum of ICC patients was detected by ELISA. Human ICC specimens were immunostained by MFAP5 antibodies. The growth rate of human ICC cell lines treated with MFAP5 or MFAP5 shRNAs was examined by CCK8 and colony formation assays. Cell cycle analysis was performed with PI staining. The effect of MFAP5 inhibition was assessed by xenograft models in nude mice. RNA-seq and ATAC-seq analyses were used to dissect the molecular mechanism by which MFAP5 promoted ICC aggressiveness. RESULTS: We identified MFAP5 as a biomarker for the diagnosis and prognosis of ICC. Upregulated MFAP5 is a common feature in aggressive ICC patients' tissues. Importantly, MFAP5 level in the serum of ICC patients and healthy individuals showed significant differential expression profiles. Furthermore, we showed that MFAP5 promoted ICC cell growth and G1 to S-phase transition. Using RNA-seq expression and ATAC-seq chromatin accessibility profiling of ICC cells with suppressed MFAP5 secretion, we showed that MFAP5 regulated the expression of genes involved in the Notch1 signaling pathway. Furthermore, FLI-06, a Notch signaling inhibitor, completely abolished the MFAP5-dependent transcriptional programs. CONCLUSIONS: Raised MFAP5 serum level is useful for differentiating ICC patients from healthy individuals, and could be helpful in ICC diagnosis, prognosis and therapies.

EYA4 inhibits hepatocellular carcinoma by repressing MYCBP by dephosphorylating ß-catenin at Ser552.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the fourth leading cause of cancer-related death worldwide. Our previous study showed that EYA4 functioned by suppressing growth of HCC tumor cells, but its molecular mechanism is still not elucidated. Based on the results of gene microassay, EYA4 was inversely correlated with MYCBP and was verified in human HCC tissues by immunohistochemistry and western blot. Overexpressed and KO EYA4 in human HCC cell lines confirmed the negative correlation between EYA4 and MYCBP by qRT-PCR and western blot. Transfected siRNA of MYCBP in EYA4 overexpressed cells and overexpressed MYCBP in EYA4 KO cells could efficiently rescue the proliferation and G2/M arrest effects of EYA4 on HCC cells. Mechanistically, armed with serine/threonine-specific protein phosphatase activity, EYA4 reduced nuclear translocation of ß-catenin by dephosphorylating ß-catenin at Ser552, thereby suppressing the transcription of MYCBP which was induced by ß-catenin/LEF1 binding to the promoter of MYCBP. Clinically, HCC patients with highly expressed EYA4 and poorly expressed MYCBP had significantly longer disease-free survival and overall survival than HCC patients with poorly expressed EYA4 and highly expressed MYCBP. In conclusion, EYA4 suppressed HCC tumor cell growth by repressing MYCBP by dephosphorylating ß-catenin S552. EYA4 combined with MYCBP could be potential prognostic biomarkers in HCC.

The C/EBPß-LINC01133 axis promotes cell proliferation in pancreatic ductal adenocarcinoma through upregulation of CCNG1.

Long non-coding RNAs (lncRNAs) are emerging as important regulators and prognostic markers of multiple cancers. Our aim was to determine functional involvement of lncRNAs in pancreatic ductal adenocarcinoma (PDAC). In this study, we report that LINC01133 expression is higher in PDAC tissues compared to adjacent non-cancerous tissues, and this overexpression is associated with poorer prognosis among the patients. In vitro, a knockdown of LINC01133 substantially decreased PDAC cell proliferation. Tumorigenicity of PDAC cells with the LINC01133 knockdown was significantly impaired in a xenograft model assay. Moreover, we determined that CCAAT/enhancer-binding protein ß (C/EBPß) positively regulates LINC01133 expression by binding to the response elements within the LINC01133 promoter. Higher expression of C/EBPß was observed in PDAC tissues, and this overexpression was also associated with the poorer prognosis. Furthermore, the LINC01133 knockdown decreased cyclin G1 (CCNG1) expression. Overexpression of CCNG1 attenuated the LINC01133 silencing-induced impairment of proliferation in PDAC cells. In summary, our findings revealed that the C/EBPß-LINC01133 axis performs an oncogenic function in PDAC by activating CCNG1, which may serve as a prognostic biomarker or a therapeutic target in PDAC.

BTG2 Is Down-Regulated and Inhibits Cancer Stem Cell-Like Features of Side Population Cells in Hepatocellular Carcinoma.

BACKGROUND: Our previous study found that B cell translocation gene 2 (BTG2) was hyper-methylated and down-regulated in side population (SP) cells of hepatocellular carcinoma (HCC) cell line. However, its clinical significances and biological impacts on HCC SP cells remained unclear. AIMS: To investigate the prognostic value of BTG2 gene in HCC and its influences on cancer stem cells (CSCs)-like traits of HCC cell line SP cells. METHODS: BTG2 expression in human HCC and adjacent non-cancerous tissues was detected by immunohistochemical staining and quantitative real-time PCR, and also obtained from GEO and TCGA data. Its prognostic values were assessed. Its biological influences on HCC cell line SP cells were evaluated using cell viability, cell cycle, plate clone-forming assay, and chemoresistance in vitro and tumorigenicity in vivo. RESULTS: BTG2 expression was significantly suppressed in human HCC compared to adjacent non-cancerous tissues. BTG2 expression was correlated with TNM stage, tumor size and vascular invasion. Lower expression of BTG2 was associated with poorer overall survival and disease-free survival. In vitro, overexpression of BTG2 substantially suppressed cell proliferation and accumulation of HCC cell line SP cells in G0/G1 phase. Colony formation ability was markedly suppressed by BTG2 overexpression. Moreover, sensitivity of HCC cell line SP cells to 5-fluorouracil was substantially increased by overexpression of BTG2. Furthermore, tumorigenicity of HCC cell line SP cells transfected with BTG2 plasmids was significantly reduced in vivo. CONCLUSIONS: BTG2 gene could regulate the CSC-like traits of HCC cell line SP cells, and it represented as a molecular prognostic marker for HCC.

Hepatoprotective effect of ulinastatin in a rat model of major hepatectomy after obstructive jaundice.

BACKGROUND: To date, major hepatectomy with obstructive jaundice is still a highly risky and difficult surgery because of the high rate of complications. An excessive inflammatory response may be the primary hindrance to postoperative recovery of liver function. AIMS: Recent research has demonstrated that ulinastatin blocks the release of inflammatory factors and prevents the cytokine cascade reaction. This study was conducted to investigate the effect of ulinastatin on major hepatectomy after obstructive jaundice and to explore the potential mechanisms of this effect. METHODS: Male Sprague-Dawley rats were divided into three groups: sham, control and treated groups. In the control and treated groups, obstructive jaundice was induced, and a 70 % major hepatectomy was performed with implementation of ulinastatin treatment in the treated group but not the control group. The rats were sacrificed after hepatectomy on day 1, day 3, day 5 and day 7. The survival time, liver function, inflammatory cytokine expression and the indices of proliferation activities were examined. Kupffer cells were isolated, and the mRNA and protein levels of CD14 and NF-κB P65 in the Kupffer cells were determined. RESULTS: Compared to the control group, the survival rates, postoperative liver function, and the indices of proliferation activities were better in the treated group; in the treated group serum TNF-α and IL-6 levels were lower whereas serum IL-10 levels were higher. The expression of CD14 and NF-κB P65 in Kupffer cells at both the mRNA and protein levels was significantly higher in the control group than in the treated group. CONCLUSIONS: Ulinastatin has a protective effect in major hepatectomy with obstructive jaundice by inhibiting Kupffer cell activation and modulating the hepatic cytokine response.