circPSD3 is a promising inhibitor of uPA system to inhibit vascular invasion and metastasis in hepatocellular carcinoma.

BACKGROUND: Vascular invasion is a major route for intrahepatic and distant metastasis in hepatocellular carcinoma (HCC) and is a strong negative prognostic factor. Circular RNAs (circRNAs) play important roles in tumorigenesis and metastasis. However, the regulatory functions and underlying mechanisms of circRNAs in the development of vascular invasion in HCC are largely unknown. METHODS: High throughput sequencing was used to screen dysregulated circRNAs in portal vein tumor thrombosis (PVTT) tissues. The biological functions of candidate circRNAs in the migration, vascular invasion, and metastasis of HCC cells were examined in vitro and in vivo. To explore the underlying mechanisms, RNA sequencing, MS2-tagged RNA affinity purification, mass spectrometry, and RNA immunoprecipitation assays were performed. RESULTS: circRNA sequencing followed by quantitative real-time PCR (qRT-PCR) revealed that circRNA pleckstrin and Sect. 7 domain containing 3 (circPSD3) was significantly downregulated in PVTT tissues. Decreased circPSD3 expression in HCC tissues was associated with unfavourable characteristics and predicted poor prognosis in HCC. TAR DNA-binding protein 43 (TDP43) inhibited the biogenesis of circPSD3 by interacting with the downstream intron of pre-PSD3. circPSD3 inhibited the intrahepatic vascular invasion and metastasis of HCC cells in vitro and in vivo. Serpin family B member 2 (SERPINB2), an endogenous bona fide inhibitor of the urokinase-type plasminogen activator (uPA) system, is the downstream target of circPSD3. Mechanistically, circPSD3 interacts with histone deacetylase 1 (HDAC1) to sequester it in the cytoplasm, attenuating the inhibitory effect of HDAC1 on the transcription of SERPINB2. In vitro and in vivo studies demonstrated that circPSD3 is a promising inhibitor of the uPA system. CONCLUSIONS: circPSD3 is an essential regulator of vascular invasion and metastasis in HCC and may serve as a prognostic biomarker and therapeutic target.

The role of plasminogen activator inhibitor-2 in pneumococcal meningitis.

Pneumococcal meningitis is associated with dysregulation of the coagulation cascade. Previously, we detected upregulation of cerebral plasminogen activator inhibitor-2 (PAI-2) mRNA expression during pneumococcal meningitis. Diverse functions have been ascribed to PAI-2, but its role remains unclear. We analyzed the function of SERPINB2 (coding for PAI-2) in patients with bacterial meningitis, in a well-established pneumococcal meningitis mouse model, using Serpinb2 knockout mice, and in vitro in wt and PAI-2-deficient bone marrow-derived macrophages (BMDMs). We measured PAI-2 in cerebrospinal fluid of patients, and performed functional, histopathological, protein and mRNA expression analyses in vivo and in vitro. We found a substantial increase of PAI-2 concentration in CSF of patients with pneumococcal meningitis, and up-regulation and increased release of PAI-2 in mice. PAI-2 deficiency was associated with increased mortality in murine pneumococcal meningitis and cerebral hemorrhages. Serpinb2-/- mice exhibited increased C5a levels, but decreased IL-10 levels in the brain during pneumococcal infection. Our in vitro experiments confirmed increased expression and release of PAI-2 by wt BMDM and decreased IL-10 liberation by PAI-2-deficient BMDM upon pneumococcal challenge. Our data show that PAI-2 is elevated during in pneumococcal meningitis in humans and mice. PAI-2 deficiency causes an inflammatory imbalance, resulting in increased brain pathology and mortality.

Fibrinogen and Antifibrinolytic Proteins: Interactions and Future Therapeutics.

Thrombus formation remains a major cause of morbidity and mortality worldwide. Current antiplatelet and anticoagulant therapies have been effective at reducing vascular events, but at the expense of increased bleeding risk. Targeting proteins that interact with fibrinogen and which are involved in hypofibrinolysis represents a more specific approach for the development of effective and safe therapeutic agents. The antifibrinolytic proteins alpha-2 antiplasmin (α2AP), thrombin activatable fibrinolysis inhibitor (TAFI), complement C3 and plasminogen activator inhibitor-2 (PAI-2), can be incorporated into the fibrin clot by FXIIIa and affect fibrinolysis by different mechanisms. Therefore, these antifibrinolytic proteins are attractive targets for the development of novel therapeutics, both for the modulation of thrombosis risk, but also for potentially improving clot instability in bleeding disorders. This review summarises the main properties of fibrinogen-bound antifibrinolytic proteins, their effect on clot lysis and association with thrombotic or bleeding conditions. The role of these proteins in therapeutic strategies targeting the fibrinolytic system for thrombotic diseases or bleeding disorders is also discussed.

Zinc Favors Triple-Negative Breast Cancer's Microenvironment Modulation and Cell Plasticity.

Triple-negative breast cancer (TNBC) tends to metastasize to the brain, a step that worsens the patient's prognosis. The specific hallmarks that determine successful metastasis are motility and invasion, microenvironment modulation, plasticity, and colonization. Zinc, an essential trace element, has been shown to be involved in all of these processes. In this work, we focus our attention on the potential role of zinc during TNBC metastasis. We used MDA-MB-BrM2 (BrM2) cells, a brain metastasis model derived from the parental TNBC cell line MDA-MB-231. Our studies show that BrM2 cells had double the zinc content of MDA-MB-231 cells. Moreover, exploring different metastatic hallmarks, we found that the zinc concentration is especially important in the microenvironment modulation of brain metastatic cells, enhancing the expression of SerpinB2. Furthermore, we show that zinc promotes the tumorigenic capacity of breast cancer stem cells. In addition, by causing a disturbance in MDA-MB-231 zinc homeostasis by overexpressing the Zip4 transporter, we were able to increase tumorigenicity. Nevertheless, this strategy did not completely recapitulate the BrM2 metastatic phenotype. Altogether, our work suggests that zinc plays an important role in the transformative steps that tumoral cells take to acquire tumorigenic potential and niche specificity.

Deficiency of plasminogen activator inhibitor-2 results in accelerated tumor growth.

BACKGROUND: Upregulation of the plasminogen activation system, including urokinase plasminogen activator (uPA), has been observed in many malignancies, suggesting that co-opting the PA system is a common method by which tumor cells accomplish extracellular matrix proteolysis. PAI-2, a serine protease inhibitor, produced from the SERPINB2 gene, inhibits circulating and extracellular matrix-tethered uPA. Decreased SERPINB2 expression has been associated with increased tumor invasiveness and metastasis for several types of cancer. PAI-2 deficiency has not been reported in humans and PAI-2-deficient (SerpinB2-/- ) mice exhibit no apparent abnormalities. OBJECTIVES: We investigated the role of PAI-2 deficiency on tumor growth and metastasis. METHODS: To explore the long-term impact of PAI-2 deficiency, a cohort of SerpinB2-/- mice were aged to >18 months, with spontaneous malignancies observed in 4/9 animals, all of apparently vascular origin. To further investigate the role of PAI-2 deficiency in malignancy, SerpinB2-/- and wild-type control mice were injected with either B16 melanoma or Lewis lung carcinoma tumor cells, with markedly accelerated tumor growth observed in SerpinB2-/- mice for both cell lines. To determine the relative contributions of PAI-2 from hematopoietic or nonhematopoietically derived sources, bone marrow transplants between wild-type C57BL/6J and SerpinB2-/- mice were performed. RESULTS AND CONCLUSIONS: Our results suggest that PAI-2 deficiency increases susceptibility to spontaneous tumorigenesis in the mouse, and demonstrate that SerpinB2 expression derived from a nonhematopoietic compartment is a key host factor in the regulation of tumor growth in both the B16 melanoma and Lewis lung carcinoma models.

Plasminogen activator inhibitor-2 (PAI-2) overexpression supports bladder cancer development in PAI-1 knockout mice in N-butyl-N- (4-hydroxybutyl)-nitrosamine- induced bladder cancer mouse model.

BACKGROUND: Accumulating evidence suggests that plasminogen activator inhibitor-1 (PAI-1) plays an important role in bladder tumorigenesis by regulating cell cycle. However, it remains unclear whether and how inhibition of PAI-1 suppresses bladder tumorigenesis. METHODS: To elucidate the therapeutic effect of PAI-1 inhibition, we tested its tumorigenicity in PAI-1 knockout (KO) mice exposed to a known bladder carcinogen. RESULTS: PAI-1 deficiency did not inhibit carcinogen-induced bladder cancer in mice although carcinogen-exposed wild type mice significantly increased PAI-1 levels in bladder tissue, plasma and urine. We found that PAI-1 KO mice exposed to carcinogen tended to upregulate protein C inhibitor (PAI-3), urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA), and significantly increased PAI-2, suggesting a potential compensatory function of these molecules when PAI-1 is abrogated. Subsequent studies employing gene expression microarray using mouse bladder tissues followed by post hoc bioinformatics analysis and validation experiments by qPCR and IHC demonstrated that SERPING1 is further downregulated in PAI-1 KO mice exposed to BBN, suggesting that SERPING1 as a potential missing factor that regulate PAI-2 overexpression (compensation pathway). CONCLUSIONS: These results indicate that serpin compensation pathway, specifically PAI-2 overexpression in this model, supports bladder cancer development when oncoprotein PAI-1 is deleted. Further investigations into PAI-1 are necessary in order to identify true potential targets for bladder cancer therapy.

miR-200c/PAI-2 promotes the progression of triple negative breast cancer via M1/M2 polarization induction of macrophage.

PURPOSE: To investigate the effect of miR-200c/PAI-2 on macrophage polarization into M2-type TAMs in TNBC. METHODS AND MATERIALS: PAI-2 expression in MDA-MB-231con, MDA-MB-231miR-200ab and MDA-MB-231miR-200c breast cancer cells was evaluated by RT-PCR and immunofluorescence (IF), while the expression of the TAM marker F4/80 and the M2-type TAM marker CD206 in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 mouse lung metastatic tumor tissues was examined with immunohistochemistry (IHC). The effects of RAW264.7 cells on MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 were examined by transwell co-culture. CD206 expression in RAW264.7 cells were confirmed by immunostaining. The level of PAI-2 and IL-10 in the co-culture supernatants were assessed using ELISA. RESULTS: 1. RT-PCR and IF analysis showed that PAI-2 was upregulated in MDA-MB-231miR-200c cells. 2. IHC assays analysis showed that the numbers of F4/80 and CD206 positive cells were increased in MDA-MB-231miR-200c tumor tissues, while in MDA-MB-231miR-200c/siPAI-2 tumor tissues were decreased. 3. Transwell co-culture assays analysis showed that MDA-MB-231miR-200c cells significantly promoted the cell migration ability compared with the control group, while knockdown PAI-2 significantly inhibited the cell migration ability (P < 0.05). 4. Transwell co-culture and immunostaining assays analysis showed that overexpression miR-200c in MDA-MB-231 cell line increased the CD206 expression in RAW264.7 cells, while knockdown PAI-2 decreased. 5. ELISA assays analysis showed that miR-200c-mediated MDA-MB-231 cells significantly increased the secretion of PAI-2 and IL-10, while decreased the secretion of PAI-2 and IL-10 in MDA-MB-231 miR-200c/siPAI-2 cells. CONCLUSIONS: miR-200c promotes the malignant progressions of TNBC by PAI-2 upregulation and M2 phenotype macrophages polarization.

SerpinB2 inhibits migration and promotes a resolution phase signature in large peritoneal macrophages.

SerpinB2 (plasminogen activator inhibitor type 2) has been called the "undecided serpin" with no clear consensus on its physiological role, although it is well described as an inhibitor of urokinase plasminogen activator (uPA). In macrophages, pro-inflammatory stimuli usually induce SerpinB2; however, expression is constitutive in Gata6+ large peritoneal macrophages (LPM). Interrogation of expression data from human macrophages treated with a range of stimuli using a new bioinformatics tool, CEMiTool, suggested that SerpinB2 is most tightly co- and counter-regulated with genes associated with cell movement. Using LPM from SerpinB2-/- and SerpinB2R380A (active site mutant) mice, we show that migration on Matrigel was faster than for their wild-type controls. Confocal microscopy illustrated that SerpinB2 and F-actin staining overlapped in focal adhesions and lamellipodia. Genes associated with migration and extracellular matrix interactions were also identified by RNA-Seq analysis of migrating RPM from wild-type and SerpinB2R380A mice. Subsequent gene set enrichment analyses (GSEA) suggested SerpinB2 counter-regulates many Gata6-regulated genes associated with migration. These data argue that the role of SerpinB2 in macrophages is inhibition of uPA-mediated plasmin generation during cell migration. GSEA also suggested that SerpinB2 expression (likely via ensuing modulation of uPA-receptor/integrin signaling) promotes the adoption of a resolution phase signature.

An Endogenous Anti-aging Factor, Sonic Hedgehog, Suppresses Endometrial Stem Cell Aging through SERPINB2.

Endometrial stem cells are located in the basal layer of the endometrium, and they are responsible for the cyclic regeneration of the uterus during the menstrual cycle. Recent studies have revealed that recurrent pregnancy loss is associated with an age-related stem cell deficiency in the endometrium. Therefore, intensive study of endometrial stem cell aging may provide new insights for preventing recurrent pregnancy loss. Sonic hedgehog (SHH) signaling has been identified as a morphogen during the embryonic development processes. In addition to this canonical function, we found that the age-associated decline in regenerative potential in the endometrium may be due to decreased SHH-signaling integrity in local stem cells with aging. Importantly, the current study also showed that SHH activity clearly declines with aging both in vitro and in vivo, and exogenous SHH treatment significantly alleviates various aging-associated declines in multiple endometrial stem cell functions, suggesting that SHH may act as an endogenous anti-aging factor in human endometrial stem cells. Moreover, we found that stem cell senescence may enhance SERPINB2 expression, which in turn mediates the effect of SHH on alleviating senescence-induced endometrial stem cell dysfunctions, suggesting that SERPINB2 is a master regulator of SHH signaling during the aging process.

SerpinB2 is involved in cellular response upon UV irradiation.

Ultraviolet light induced pyrimidine dimer is a helix distortion DNA damage type, which recruits repair complexes. However, proteins of these complexes that take part in both DNA damage recognition and repair have been well-described, the regulation of the downstream steps of nucleotide excision repair (NER) have not been clearly clarified yet. In a high-throughput screen, we identified SerpinB2 (SPB2) as one of the most dramatically upregulated gene in keratinocytes following UV irradiation. We found that both the mRNA and the protein levels of SPB2 were increased upon UV irradiation in various cell lines. Additionally, UV damage induced translocation of SPB2 from the cytoplasm to the nucleus as well as the damage induced foci formation of it. Here we show that SPB2 co-localizes with XPB involved in the NER pathway at UV-induced repair foci. Finally, we demonstrated that UV irradiation promoted the association of SPB2 with ubiquitylated proteins. In basal cell carcinoma tumour cells, we identified changes in the subcellular localization of SPB2. Based on our results, we conclude that SPB2 protein has a novel role in UV-induced NER pathway, since it regulates the removal of the repair complex from the damaged site leading to cancerous malformation.

Plasminogen activator inhibitor 2 (PAI2) inhibits invasive potential of hepatocellular carcinoma cells in vitro via uPA- and RB/E2F1-related mechanisms.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues. METHODS: First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining. RESULTS: The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial-mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson-Steiner grade, overall as well as tumor-free survival. CONCLUSIONS: Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.

PAI-1 but Not PAI-2 Gene Deficiency Attenuates Ischemic Brain Injury After Experimental Stroke.

After stroke, secondary brain damage is influenced by the extent of fibrin clot formation. This is counteracted by the endogenous fibrinolysis. Of major interest are the key players of the fibrinolytic plasminogen activator system including the urokinase plasminogen activator (uPA), the tissue-type plasminogen activator (tPA), and their endogenous inhibitors plasminogen activator inhibitor 1 (PAI-1) and PAI-2. The role of PAI-1 in brain injury is well established, whereas the importance of PAI-2 is unknown at present. The objectives of the present were twofold: first, to characterize the time-dependent cerebral mRNA expression of the plasminogen activator system (PAS) after brain ischemia and second, to investigate the impact of PAI-1 and PAI-2 on brain infarct volume using gene-deficient mice. Adult C57Bl/6J mice were subjected to unilateral transient middle cerebral artery occlusion (MCAO) followed by reperfusion for 3, 24, 72, or 120 h. Quantitative PCR revealed that brain mRNA expression levels of the PAS components, and particularly of PAI-1 (237-fold) and PAI-2 (19-fold), peaked at 24 h after stroke. Accordingly, PAI-1 plasma activity was strongly increased. Brain infarct volume in TTC (2,3,5-triphenyltetrazolium chloride)-stained brain sections was significantly smaller 24 h after MCAO in PAI-1-deficient mice (- 31%), but not in PAI-2-deficient mice (- 6%). Thus, endogenous upregulation of PAI-1, but not of PAI-2, might contribute to increased brain damage after acute ischemic stroke. The present study therefore shows that PAI-2 is induced by brain ischemia, but does not play an important or relevant role for secondary brain damage after brain injury.

Post-injury Nose-to-Brain Delivery of Activin A and SerpinB2 Reduces Brain Damage in a Mouse Stroke Model.

Synaptic NMDA receptors activating nuclear calcium-driven adaptogenomics control a potent body-own neuroprotective mechanism, referred to as acquired neuroprotection. Viral vector-mediated gene transfer in conjunction with stereotactic surgery has previously demonstrated the proficiency of several nuclear calcium-regulated genes to protect in vivo against brain damage caused by toxic extrasynaptic NMDA receptor signaling following seizures or stroke. Here we used noninvasive nose-to-brain administration of Activin A and SerpinB2, two secreted nuclear calcium-regulated neuroprotectants, for post-injury treatment of brain damage following middle cerebral artery occlusion (MCAO) in C57BL/6N mice. The observed reduction of the infarct volume was comparable to the protection obtained by intracerebroventricular injection of recombinant Activin A or SerpinB2 or by stereotactic delivery 3 weeks prior to the injury of a recombinant adeno-associated virus containing an expression cassette for the potent neuroprotective transcription factor Npas4. These results establish post-injury, nose-to-brain delivery of Activin A and SerpinB2 as effective and possibly clinically applicable treatments of acute and chronic neurodegenerative conditions.

The role of SerpinB2 in human bronchial epithelial cells responses to particulate matter exposure.

Exposure to particulate matter (PM) has been related to the onset of adverse health effects including lung cancer, but the underlying molecular mechanisms are still under investigation. Epithelial-to-mesenchymal transition (EMT) is regarded as a crucial step in cancer progression. In a previous study, we reported EMT-related responses in the human bronchial epithelial cell line HBEC3-KT, exposed to Milan airborne winter PM2.5. We also found a strong modulation of SERPINB2, encoding for the PAI-2 protein and previously suggested to play an important role in cancer. Here we investigate the role of SERPINB2/PAI-2 in the regulation of EMT-related effects induced by PM exposure in HBEC3-KT. PM exposure (up to 10 µg/cm2) increased SERPINB2 expression, reduced cell migration and induced morphological alterations in HBEC3-KT. Changes in actin structure and cadherin-1 relocalization were observed in PM-exposed samples. Knockdown of SERPINB2 by siRNA down-regulated the CDH1 gene expression, as well as PAI-2 and cadherin-1 protein expression. SERPINB2 knockdown also increased cell migration rate, and counteracted the PM-induced reduction of cell migration and alteration of cell morphology. SERPINB2 was found to be greatly down-regulated in a HBEC2-KT transformed cell line, supporting the importance of this gene in the regulation of EMT. In conclusion, here we show that PAI-2 regulates CDH1 gene/cadherin-1 protein expression in bronchial HBEC3-KT cells, and this mechanism might be involved in the regulation of cell migration. SERPINB2 down-regulation should be considered part of EMT, and the over-expression of SERPINB2 in PM-exposed samples might be interpreted as an initial protective mechanism.

The microRNA-15a-PAI-2 axis in cholangiocarcinoma-associated fibroblasts promotes migration of cancer cells.

BACKGROUND: Cholangiocarcinoma (CCA) has an abundance of tumor stroma which plays an important role in cancer progression via tumor-promoting signals. This study aims to explore the microRNA (miRNA) profile of CCA-associated fibroblasts (CCFs) and the roles of any identified miRNAs in CCA progression. METHODS: miRNA expression profiles of CCFs and normal skin fibroblasts were compared by microarray. Identified downregulated miRNAs and their target genes were confirmed by real-time PCR. Their binding was confirmed by a luciferase reporter assay. The effects of conditioned-media (CM) of miRNA mimic- and antagonist-transfected CCFs were tested in CCA migration in wound healing assays. Finally, the levels of miRNA and their target genes were examined by real-time PCR and immunohistochemistry in clinical CCA samples. RESULTS: miR-15a was identified as a downregulated miRNA in CCFs. Moreover, PAI-2 was identified as a novel target gene of miR-15a. Recombinant PAI-2 promoted migration of CCA cells. Moreover, CM from miR-15a mimic-transfected CCFs suppressed migration of CCA cells. Lower expression of miR-15a and higher expression of PAI-2 were observed in human CCA samples compared with normal liver tissues. Importantly, PAI-2 expression correlated with poor prognosis in CCA patients. CONCLUSIONS: These findings highlight the miR-15a/PAI-2 axis as a potential therapeutic target in CCA patients.

Risk Factors and Outcomes of Thalidomide-induced Peripheral Neuropathy in a Pediatric Inflammatory Bowel Disease Cohort.

BACKGROUND: Thalidomide is an effective therapy in children with inflammatory bowel disease refractory to standard treatments, but thalidomide-induced peripheral neuropathy (TiPN) limits its long-term use. We aimed to investigate the risk factors and the outcome of TiPN in children with inflammatory bowel disease. METHODS: Within a retrospective multicenter cohort study, we evaluated prevalence and evolution of TiPN. Clinical data and candidate genetic profiles of patients with and without TiPN were compared with detect predisposing factors. RESULTS: One hundred forty-two patients were identified. TiPN was found in 72.5% of patients (38.7% clinical and instrumental alterations, 26.8% exclusive electrophysiological anomalies, and 7.0% exclusive neurological symptoms). Median TiPN-free period of treatment was 16.5 months; percentage of TiPN-free patients was 70.0% and 35.6% at 12 and 24 months of treatment, respectively. The risk of TiPN increased depending on the mean daily dose (50-99 mg/d adjusted hazard ratio 2.62; 95% confidence interval [CI], 1.31-5.21; 100-149 mg/d adjusted hazard ratio 6.16; 95% CI, 20.9-13.06; >150 mg/d adjusted hazard ratio 9.57; 95% CI, 2.6-35.2). Single nucleotide polymorphisms in ICAM1 (rs1799969) and SERPINB2 (rs6103) genes were found to be protective against TiPN (odds ratio 0.15; 95% CI, 0.03-0.82 and 0.36; 95% CI, 0.14-0.88, respectively). TiPN was the cause of drug suspension in 41.8% of patients. Clinical symptoms resolved in 89.2% of cases, whereas instrumental alteration persisted in more than half of the patients during a short follow-up. CONCLUSIONS: In children with inflammatory bowel disease, TiPN is common but mild and generally reversible. Cumulative dose seems to be the most relevant risk factor, whereas polymorphisms in genes involved in neuronal inflammation may be protective.

microRNA-200c/141 upregulates SerpinB2 to promote breast cancer cell metastasis and reduce patient survival.

The microRNA-200 (miR-200) family is associated with tumor metastasis and poor patient prognosis. We found that miR-200c/141 cluster overexpression upregulated SerpinB2 in the MDA-MB-231 triple-negative (TN) breast cancer cell line. We observed transcription factor (c-Jun, c-Fos, and FosB) upregulation, nuclear localization of c-Jun, and increased SerpinB2 promoter-directed chloramphenicol acetyltransferase activity in miR-200c/141 cluster-overexpressing cells relative to controls. Additionally, miR-124a and miR-26b, which directly target SepinB2, were downregulated compared to controls. In mouse xenograft models, miR-200c/141 cluster overexpression promoted lymph node and lung metastasis, and siRNA-mediated SerpinB2 knockdown decreased lung metastasis, suggesting that SerpinB2 mediates miR-200c/141-induced lung metastasis. We also explored the clinical significance of SerpinB2 protein status through analysis of primary breast tumor samples and The Cancer Genome Atlas (TCGA) data. High SerpinB2 levels were associated with reduced survival and increased lymph node metastasis in breast cancer patients. SerpinB2 was overexpressed in the TN breast cancer subtype as compared to the luminal subtype. The present study demonstrates that SerpinB2 promotes miR-200c/141 cluster overexpression-induced breast cancer cell metastasis, and SerpinB2 overexpression correlates with increased metastatic potential and unfavorable outcomes in breast cancer patients. SerpinB2 may be a useful biomarker for assessing metastasis risk in breast cancer patients.

Leucocyte expression of genes implicated in the plasminogen activation cascade is modulated by yoghurt peptides.

The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3-4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50-60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils.

Down-regulation of SerpinB2 is associated with gefitinib resistance in non-small cell lung cancer and enhances invadopodia-like structure protrusions.

The failure of targeted therapy due to the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is considered a major problem in the treatment of non-small cell lung cancer (NSCLC) patients. SerpinB2, a component of the urokinase plasminogen activator (uPA) system, has been recognized as a biomarker for the progression and metastasis of lung cancer. Nevertheless, the relationship between SerpinB2 and EGFR-TKI resistance has not been elucidated. Here, we report that SerpinB2 is down-regulated in gefitinib-resistant (H292-Gef) cells compared to gefitinib-sensitive (H292) cells. The low SerpinB2 levels in H292-Gef cells were also associated with an enhancement in invasiveness and increase in the length of invadopodia-like structures in the cells. The effect on invasiveness and gefitinib sensitivity was confirmed by knockdown and overexpression of SerpinB2. In addition, the possibility to overcome the resistance through the up-regulation of SerpinB2 was supported by employing an antitumor agent yuanhuadine (YD). Treatment with YD effectively elevated SerpinB2 levels and suppressed invasive properties in H292-Gef cells. Collectively, these findings demonstrate the prospective role of SerpinB2 as a novel biomarker for acquired gefitinib resistance and a potential target for NSCLC treatment.

Azithromycin differentially affects the IL-13-induced expression profile in human bronchial epithelial cells.

The T helper 2 (Th2) cytokine interleukin(IL)-13 is a central regulator in goblet cell metaplasia and induces the recently described Th2 gene signature consisting of periostin (POSTN), chloride channel regulator 1 (CLCA1) and serpin B2 (SERPINB2) in airway epithelial cells. This Th2 gene signature has been proposed as a biomarker to classify asthma into Th2-high and Th2-low phenotypes. Clinical studies have shown that the macrolide antibiotic azithromycin reduced clinical symptoms in neutrophilic asthma, but not in the classical Th2-mediated asthma despite the ability of azithromycin to reduce IL-13-induced mucus production. We therefore hypothesize that azithromycin differentially affects the IL-13-induced expression profile. To investigate this, we focus on IL-13-induced mucin and Th2-signature expression in human bronchial epithelial cells and how this combined expression profile is affected by azithromycin treatment. Primary bronchial epithelial cells were differentiated at air liquid interface in presence of IL-13 with or without azithromycin. Azithromycin inhibited IL-13-induced MUC5AC, which was accompanied by inhibition of IL-13-induced CLCA1 and SERPINB2 expression. In contrast, IL-13-induced expression of POSTN was further increased in cells treated with azithromycin. This indicates that azithromycin has a differential effect on the IL-13-induced Th2 gene signature. Furthermore, the ability of azithromycin to decrease IL-13-induced MUC5AC expression may be mediated by a reduction in CLCA1.