Improved urine DNA methylation panel for early bladder cancer detection.

BACKGROUND: Bladder cancer is one of the most common malignancies but the corresponding diagnostic methods are either invasive or limited in specificity and/or sensitivity. This study aimed to develop a urine-based methylation panel for bladder cancer detection by improving published panels and validate performance of the new panel with clinical samples. METHODS: Related researches were reviewed and 19 potential panels were selected. RRBS was performed on a cohort with 45 samples to reassess these panels and a new panel inherited best markers was developed. The new panel was applied with qMSP platform to 33 samples from the RRBS cohort and the results were compared to those of RRBS. Lastly, another larger cohort with 207 samples was used to validate new panel performance with qMSP. RESULTS: Three biomarkers (PCDH17, POU4F2 and PENK) were selected to construct a new panel P3. P3 panel achieved 100% specificity and 71% sensitivity with RRBS in corresponding cohort and then showed a better performance of 100% specificity and 84% sensitivity with qMSP platforms in a balanced cohort. When validated with 207-sample cohort, P3 with qMSP showed a performance of 97% specificity and 87% sensitivity which was modestly improved compared to the panels it derided from. CONCLUSIONS: Overall, the P3 panel achieved relatively high sensitivity and accuracy in bladder cancer detection.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension.

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

Transcription factor POU4F2 promotes colorectal cancer cell migration and invasion through hedgehog-mediated epithelial-mesenchymal transition.

As a POU homeodomain transcription factor, POU4F2 has been implicated in regulating tumorigenic processes in various cancers. However, the role of POU4F2 in colorectal cancer (CRC) remains unclear. Here, we revealed that POU4F2 functions as a tumor promotor in CRC. Bioinformatics analysis in specimens from CRC patients and expression analysis in CRC cell lines showed that POU4F2 was upregulated at the mRNA and protein levels in CRC. Depletion of POU4F2 suppressed the metastatic phenotypes of CRC cells, including cell migration, invasion, and the expression of epithelial-mesenchymal transition (EMT) markers. Moreover, depletion of POU4F2 decreased the number of lung metastatic nodes in nude mice. Mechanistically, POU4F2 positively regulated the Hedgehog signaling pathway, as inferred from the downregulation of the expression of sonic Hedgehog homolog, patched 1, Smoothened, and GLI family zinc finger 1 in vitro and vivo following silencing of POU4F2. Furthermore, the SMO agonist SAG reversed the effects of POU4F2 knockdown in CRC. Functionally, POU4F2 contributed to the Hedgehog signaling-regulated activation of the EMT process and promotion of CRC cell migration and invasion. Collectively, these findings elucidated the role of POU4F2 as a tumor promotor in CRC through the regulation of Hedgehog signaling-mediated EMT and suggested that POU4F2 suppression might be a promising therapeutic target in inhibiting CRC metastasis.

Neuroprotective effects of Acer palmatum thumb. leaf extract (KIOM-2015E) against ischemia/reperfusion-induced injury in the rat retina.

Purpose: The present study aimed to determine whether the administration of Acer palmatum thumb. leaf extract (KIOM-2015E) protects against the degeneration of rat retinal ganglion cells after ischemia/reperfusion (I/R) induced by midbrain cerebral artery occlusion (MCAO). Methods: Sprague-Dawley rats were subjected to 90 min of MCAO, which produces transient ischemia in both the retina and brain due to the use of an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. This was followed by reperfusion under anesthesia with isoflurane. The day after surgery, the eyes were treated three times (eye drop) or one time (oral administration) daily with KIOM-2015E for five days. Retinal histology was assessed in flat mounts and vertical sections to determine the effect of KIOM-2015E on I/R injury. Results: A significant loss of brain-specific homeobox/POU domain protein 3A (Brn3a) and neuron-specific class III beta-tubulin (Tuj-1) fluorescence and a marked increase in glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) expression were observed after five days in the PBS-treated MCAO group compared to the sham-operated control group. However, KIOM-2015E treatment reduced (1) MCAO-induced upregulation of GFAP and GS, (2) retinal ganglion cell loss, (3) nerve fiber degeneration, and (4) the number of TUNEL-positive cells. KIOM-2015E application also increased staining for parvalbumin (a marker of horizontal cell associated calcium-binding protein and amacrine cells) and recoverin (a marker of photoreceptor expression) in rats subjected to MCAO-induced retinal damage. Conclusions: Our findings indicated that KIOM-2015E treatment exerted protective effects against retinal damage following MCAO injury and that this extract may aid in the development of novel therapeutic strategies for retinal diseases, such as glaucoma and age-related macular disease.

HOXA9, PCDH17, POU4F2, and ONECUT2 as a Urinary Biomarker Combination for the Detection of Bladder Cancer in Chinese Patients with Hematuria.

BACKGROUND: DNA methylation biomarkers for bladder cancer (BCa) have not been evaluated extensively in the Chinese population. OBJECTIVE: To develop and validate a urinary biomarker combination of methylation assays in a group of Chinese patients with hematuria. DESIGN, SETTING, AND PARTICIPANTS: A total of 192 urine samples were collected and evaluated from patients with microscopic or gross hematuria, including 97 BCa patients and 95 controls with benign diseases. A two-stage study was conducted: the first stage being assay construction and the second stage being assay validation. Eighty-one urine samples were analyzed for the hypermethylation of eight selected genes in stage 1 and then a four-gene panel was constructed. An additional 111 urine samples were analyzed using the four-gene panel (including HOXA9, PCDH17, POU4F2, and ONECUT2) for independent validation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The positive predictive value (PPV) and negative predictive value (NPV) were calculated for the combination methylation assay. Uni- and multivariate binary logistic regression analyses (backward elimination, conditional) were performed to calculate the association between BCa and each predictor variable. RESULTS AND LIMITATIONS: The combination assay of HOXA9, PCDH17, POU4F2, and ONECUT2 was selected based on the results of multivariate logistic regression analysis in stage 1. Using a strategy of three-level risk stratification, the assay yielded a consistent PPV of 100%. With an estimated BCa prevalence of 10% in a general hematuria population, the assay would result in an overall NPV of 98%. This combined methylation biomarker would yield an overall area under the receiver operating characteristic curve of 0.871 (with a sensitivity of 90.5% and a specificity of 73.2%) if using the prediction model from multivariate regression analysis. In addition, over half of BCa cases would be predicted accurately and ∼60% of unnecessary cystoscopies could be spared. This study had several limitations. First, the sample size was relatively small. Second, it was performed in a case-control population rather than in a natural hematuria cohort. CONCLUSIONS: A combination methylation assay of HOXA9, PCDH17, POU4F2, and ONECUT2 resulted in high PPV and NPV in Chinese patients with hematuria. With accurate risk prediction, the urinary biomarker combination could spare a sizeable proportion of low-risk patients from extensive and invasive examination. PATIENT SUMMARY: In the present study, we looked at the predictive performance of a urinary biomarker combination of HOXA9, PCDH17, POU4F2, and ONECUT2. We found that this urinary biomarker combination may help discriminate bladder cancer from other benign diseases in patients with hematuria, resulting in a reduction of unnecessary invasive examination in patients at low risk.

Transplantation of Retinal Ganglion Cells Derived from Male Germline Stem Cell as a Potential Treatment to Glaucoma.

Glaucoma is characterized by retinal ganglion cell (RGC) degeneration and is the second leading cause of blindness worldwide. However, current treatments such as eye drop or surgery have limitations and do not target the loss of RGC. Regenerative therapy using embryonic stem cells (ESCs) holds a promising option, but ethical concern hinders clinical applications on human subjects. In this study, we employed spermatogonial stem cells (SSCs) as an alternative source of ESCs for cell-based regenerative therapy in mouse glaucoma model. We generated functional RGCs from SSCs with a two-step protocol without applying viral transfection or chemical induction. SSCs were first dedifferentiated to embryonic stem-like cells (SSC-ESCs) that resemble ESCs in morphology, gene expression signatures, and stem cell properties. The SSC-ESCs then differentiated toward retinal lineages. We showed SSC-ESC-derived retinal cells expressed RGC-specific marker Brn3b and functioned as bona fide RGCs. To allow in vivo RGC tracing, Brn3b-EGFP reporter SSC-ESCs were generated and the derived RGCs were subsequently transplanted into the retina of glaucoma mouse models by intravitreal injection. We demonstrated that the transplanted RGCs could survive in host retina for at least 10 days after transplantation. SSC-ESC-derived RGCs can thus potentially be a novel alternative to replace the damaged RGCs in glaucomatous retina.

The POU4F2/Brn-3b transcription factor is required for the hypertrophic response to angiotensin II in the heart.

Adult hearts respond to increased workload such as prolonged stress or injury, by undergoing hypertrophic growth. During this process, the early adaptive responses are important for maintaining cardiac output whereas at later stages, pathological responses such as cardiomyocyte apoptosis and fibrosis cause adverse remodelling, that can progress to heart failure. Yet the factors that control transition from adaptive responses to pathological remodelling in the heart are not well understood. Here we describe the POU4F2/Brn-3b transcription factor (TF) as a novel regulator of adaptive hypertrophic responses in adult hearts since Brn-3b mRNA and protein are increased in angiotensin-II (AngII) treated mouse hearts with concomitant hypertrophic changes [increased heart weight:body weight (HW:BW) ratio]. These effects occur specifically in cardiomyocytes because Brn-3b expression is increased in AngII-treated primary cultures of neonatal rat ventricular myocytes (NRVM) or foetal heart-derived H9c2 cells, which undergo characteristic sarcomeric re-organisation seen in hypertrophic myocytes and express hypertrophic markers, ANP/ßMHC. The Brn-3b promoter is activated by known hypertrophic signalling pathways e.g. p42/p44 mitogen-activated protein kinase (MAPK/ERK1/2) or calcineurin (via NFAT). Brn-3b target genes, e.g. cyclin D1, GLUT4 and Bax, are increased at different stages following AngII treatment, supporting distinct roles in cardiac responses to stress. Furthermore, hearts from male Brn-3b KO mutant mice display contractile dysfunction at baseline but also attenuated hypertrophic responses to AngII treatment. Hearts from AngII-treated male Brn-3b KO mice develop further contractile dysfunction linked to extensive fibrosis/remodelling. Moreover, known Brn-3b target genes, e.g. GLUT4, are reduced in AngII-treated Brn-3b KO hearts, suggesting that Brn-3b and its target genes are important in driving adaptive hypertrophic responses in stressed heart.

VEGF-A and neuropilin 1 (NRP1) shape axon projections in the developing CNS via dual roles in neurons and blood vessels.

Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.

Alteration of Neurotrophic Factors After Transplantation of Bone Marrow Derived Lin-ve Stem Cell in NMDA-Induced Mouse Model of Retinal Degeneration.

Retinal ganglion cell layer (RGCs) is one of the important layers of retina, depleted in Glaucoma. Loss of RGC neurons is a major cellular mechanism involved in its pathogenesis resulting in severe vision loss. Stem cell therapy has emerged as a potential strategy to arrest the apoptotic loss of RGCs and also replace the degenerative cells in damaged retina. Here, we have investigated the incorporation and survival of mouse bone marrow derived Lin-ve stem cells in N-methyl-d-aspartate (NMDA)-induced mouse model of retinal degeneration. Two days after intravitreal injection of NMDA (100 mM) showed significant decrease in ganglion cell number and increase in TUNEL positive apoptotic cells in retinal layers. The injury was further characterized by immunohistochemical expression of Brn3b, GFAP, Bcl2, pCREB, CNTF, GDNF, and BDNF in retinal layers. Lin-ve cells (100,000 dose) were intravitreally transplanted after 2 days of injury and evaluated after 7, 14, and 21 days of transplantation. Transplanted cells were found to have migrated from intravitreal space and incorporated into injured retina at 7, 14, and 21 days post-transplantation. At 21 days Brn3b, CNTF, and BDNF expression was found to be upregulated whereas GDNF was downregulated when compared to respective injury time points. Molecular data showed decrease in the expression of Brn3b, BDNF, CNTF, and GDNF post transplantation when compared with injury groups. This study reveals that Lin-ve stem cells may exert neuroprotective effect in damaged retina mediated by participation of neurotrophic factors induced by stem cell transplantation at the site of injury. J. Cell. Biochem. 118: 1699-1711, 2017. © 2016 Wiley Periodicals, Inc.

Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Brn3b is a class IV POU domain transcription factor that plays an important role in the development of retinal ganglion cells (RGCs), RGC survival, and particularly axon growth and pathfinding. Our previous study demonstrated that recombinant adenoassociated virus serotype 2 (rAAV-2)-mediated overexpression of Brn3b in RGCs promoted neuroprotection in a rodent model of glaucoma. However, the mechanisms underlying neuroprotection of RGCs in rats overexpressing Brn3b in animal models of glaucoma remain largely unknown. The goal of this study was to understand some of the mechanisms underlying the neuroprotection of RGCs overexpressing Brn3b during intraocular pressure (IOP) elevation in Brown Norway rats. METHODS: One eye of Brown Norway rats (Rattus norvegicus) was injected with an AAV construct encoding either green fluorescent protein (GFP; recombinant adenoassociated virus-green fluorescent protein, rAAV-hSyn-GFP) or Brn3b (rAAV-hSyn-Brn3b). Expression of antiapoptotic proteins, including B cell lymphoma/leukemia-2 (Bcl-2) family proteins (Bcl-2 and Bcl-xL), and p-AKT, was observed following immunostaining of rat retinas that overexpress Brn3b. In a different set of experiments, intraocular pressure was elevated in one eye of Brown Norway rats, which was followed by intravitreal injection with AAV constructs encoding either GFP (rAAV-CMV-GFP) or Brn3b (rAAV-CMV-Brn3b). Retinal sections were stained for prosurvival factors, including Bcl-2, Bcl-XL, and p-AKT. RESULTS: AAV-mediated expression of transcription factor Brn3b promoted statistically significant upregulation of the Bcl-2 protein and increased expression of p-AKT in RGCs of Brown Norway rats. In addition, following IOP elevation, AAV-mediated Brn3b expression also statistically significantly increased levels of Bcl-2 in the RGC layer in Brown Norway rats. CONCLUSIONS: Adenoassociated virus-mediated Brn3b protein overexpression may promote neuroprotection by upregulating key antiapoptotic proteins, including Bcl-2, Bcl-xL, and p-AKT, in animal models of glaucoma.

An epigenetic biomarker combination of PCDH17 and POU4F2 detects bladder cancer accurately by methylation analyses of urine sediment DNA in Han Chinese.

To develop a routine and effectual procedure of detecting bladder cancer (BlCa), an optimized combination of epigenetic biomarkers that work synergistically with high sensitivity and specificity is necessary. In this study, methylation levels of seven biomarkers (EOMES, GDF15, NID2, PCDH17, POU4F2, TCF21, and ZNF154) in 148 individuals-which including 58 urothelial cell carcinoma (UCC) patients, 20 infected urinary calculi (IUC) patients, 20 kidney cancer (KC) patients,20 prostate cancer (PC) patients, and 30 healthy volunteers (HV)-were quantified by qMSP using the urine sediment DNA. Receiver operating characteristic (ROC) curves were generated for each biomarker. The combining predictors of possible combinations were calculated through logistic regression model. Subsequently, ROC curves of the three best performing combinations were constructed. Then, we validated the three best performing combinations and POU4F2 in another 72 UCC, 21 IUC, 26 KC and 22 PC, and 23 HV urine samples. The combination of POU4F2/PCDH17 has yielded the highest sensitivity and specificity of 90.00% and 93.96% in all the 312 individuals, showing the capability of detecting BlCa effectively among pathologically varied sample groups.

Transcription Factor Brn-3b Overexpression Enhances Neurite Outgrowth in PC12 Cells Under Condition of Hypoxia.

Transcription factor Brn-3b plays a key role in retinal ganglion cell differentiation, survival, and axon outgrowth during development. However, the precise role of Brn-3b in the normal adult retina as well as during neurodegeneration is unclear. In the current study, the effect of overexpression of Brn-3b was assessed in vitro, in PC12 cells under conditions of normoxia and hypoxia. Immunoblot analysis showed that overexpression of Brn-3b in PC12 cells as well as 661W cells produced significant increase in the growth cone marker, growth-associated protein-43 (GAP-43), and acetylated-tubulin (ac-TUBA). In addition, an increased immunostaining for GAP-43 and ac-TUBA was observed in PC12 cells overexpressing Brn-3b, which was accompanied by a marked increase in neurite outgrowth, compared to PC12 cells overexpressing the empty vector. In separate experiments, one set of PC12 cells transfected either with a Brn-3b expression vector or an empty vector was subjected to conditions of hypoxia for 2 h, while another set of similarly transfected PC12 cells was maintained in normoxic conditions. It was found that the upregulation of GAP-43 and ac-TUBA in PC12 cells overexpressing Brn-3b under conditions of normoxia was sustained under conditions of hypoxia. Immunocytochemical analysis revealed not only an upregulation of GAP-43 and ac-TUBA, but also increased neurite outgrowth in PC12 cells transfected with Brn-3b as compared to PC12 cells transfected with empty vector in both normoxia and hypoxia. The findings have implications for a potential role of Brn-3b in neurodegenerative diseases in which hypoxia/ischemia contribute to pathophysiology of the disease.

Toll-like receptor-4 knockout mice are more resistant to optic nerve crush damage than wild-type mice.

BACKGROUND: This study aims to investigate the role of the inflammatory response following optic nerve crush (ONC) in knockout mice for the toll-like receptor-4 gene (TLR4-/-) compared to wild-type (WT) mice. METHODS: ONC was induced in TLR4-/- and C57BL6 WT mice. Histological sections of the retina and optic nerve were analysed on days 1, 3 or 21 after injury. Molecular analysis with real-time quantitative polymerase chain reaction was used to study the expression of CD45, tumour necrosis-alpha (TNF-α) and glial fibrillary acidic protein, as well as retinal ganglion cell (RGC) markers THY-1 and Brn3b. RESULTS: There was a 25.5% and 38% loss in the RGC layer of the ONC-injured eyes of the TLR4-/- and the WT mice, respectively (with 27% and 9% of the remaining cells positive for Brn3a, respectively). Mean levels of Thy-1 and Brn3b were higher in the TLR4-/- mice. CD45 and Iba1 staining revealed infiltration of inflammatory cells into the injured nerve and retina in both groups. Molecular analysis of the optic nerve on day 1 showed increased TNF-α expression and reduced CD45 and GFAP expression; on day 3, CD45 reverted to baseline but GFAP remained low; on day 21, all 3 markers were at baseline in the TLR4-/- group and decreased in the WT group. CONCLUSION: Inflammation plays a major role in the response to ONC injury. Reduced levels of inflammation are associated with improved RGC preservation. The increase in TNF-α and reduction in CD45 in both TLR4-/- and WT mice may indicate the presence of an alternative pathway for induction of RGC death.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. METHODS: Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. RESULTS: Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. CONCLUSIONS: Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

The transcriptional regulator BCL6 participates in the secondary gene regulatory response to vitamin D.

The vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the transcription factor vitamin D receptor (VDR) and therefore a direct regulator of transcription. Transcriptome-wide analysis of THP-1 human monocytes had indicated more than 600 genes to be significantly (p<0.05) stimulated after 4h incubation with 1,25(OH)2D3, but only 67 of them where more than 1.5-fold up-regulated. These include the genes encoding for the transcription factors BCL6, NFE2, POU4F2 and ELF4, which are controlled by one or two VDR binding sites within their chromosomal domains. The latter are defined via DNA loop formation mediated by the transcription factor CTCF that is highly conserved in its genome-wide loci. We found BCL6 being most responsive to 1,25(OH)2D3 and selected it for further analysis. An incubation of THP-1 cells with 1,25(OH)2D3 for 24 h resulted in a significant (p<0.001) change in the mRNA expression of more than 1600 genes, of which 132 were at least 2-fold up-regulated. About half of the latter genes are secondary 1,25(OH)2D3 targets, since they do not carry any VDR binding site within their chromosomal domain. Chromatin immunoprecipitation sequencing datasets indicated that the majority of these domains contain a BCL6 binding site. We followed the secondary transcriptional response to 1,25(OH)2D3 for eight representative gene examples and confirmed the binding of CTCF and BCL6 to their respective chromosomal domains. In conclusion, our study indicated that in monocytes most of the physiological responses to 1,25(OH)2D3 involve the action of the transcription factor BCL6.

Co-expression of POU4F2/Brn-3b with p53 may be important for controlling expression of pro-apoptotic genes in cardiomyocytes following ischaemic/hypoxic insults.

Cardiomyocyte death following ischaemic/hypoxic injury causes irreversible damage to cardiac function and contributes to chronic diseases such as heart failure. Understanding the mechanisms associated with myocyte loss under these conditions can help to identify strategies to minimise/abrogate such detrimental effects. The p53 protein can induce apoptosis or cell cycle arrest, but effects on cell fate depend on interactions with other regulators such as POU4F2/Brn-3b (Brn-3b), which co-operates with p53 to increase the expression of pro-apoptotic genes. In contrast, the related POU4F1/Brn-3a (Brn-3a) blocks p53-mediated apoptosis but co-operates with p53 to enhance cell cycle arrest. In this study, we showed that permanent coronary artery ligation in mouse hearts, which induced apoptotic markers, activated caspase-3 and -8 and necroptosis markers; RIP-1 and -3 also increased Brn-3b and Brn-3a expression. However, Brn-3a was only detected in uninjured myocardium but not at the site of injury, whereas Brn-3b showed generalised increase, including within the infarct zone. Conversely, p53 was detected in the infarct zone and in some cells adjacent to the site of injury but not in uninjured myocardium. Co-localisation studies showed Brn-3a co-expression with p53 in cardiomyocytes adjacent to the infarct zone, whereas Brn-3b was co-localised with p53 in the infarct zone only. Increased Brn-3b and p53 correlated with elevated expression of pro-apoptotic target genes, Bax, Noxa and PUMA, whereas cleaved caspase-3 confirmed the presence of apoptotic cells within this region of the injured heart. Similarly, simulated ischaemia/reoxygenation (sI/R) injury in neonatal rat ventricular cardiomyocytes (NRVM) and heart derived H9c2 myoblasts increased Brn-3b, p53 as well as apoptotic genes, and this was associated with enhanced apoptosis. Furthermore, targeted reduction of Brn-3b using shRNA caused reduction in pro-apoptotic Bax and Noxa proteins, even though p53 expression remained intact, suggesting that Brn-3b is important for controlling the fate of the myocardium in the injured heart.

Genome-wide profiling in melatonin-exposed human breast cancer cell lines identifies differentially methylated genes involved in the anticancer effect of melatonin.

Epigenetic alterations have emerged as an important mechanism involved in tumorigenesis. The epigenetic impact of DNA methylation in various types of human cancer is not completely understood. Previously, we observed melatonin-induced differential expression of miRNA and miRNA-related genes in human breast cancer cell lines that indicated an anticancer effect of melatonin. In this report, we further characterized epigenetic changes in melatonin-exposed MCF-7 cells through the analysis of DNA methylation profiles in breast cancer cells to provide new insights into the potential mechanisms of the anticancer effect of melatonin. Microarray-based DNA methylation and gene expression profiling were carried out using human breast cancer cell lines. We further identified a number of mRNAs whose expression levels show an inverse correlation with DNA methylation levels. The mRNA expression levels and methylation status of candidate genes in melatonin-exposed cells were confirmed by real-time quantitative PCR and bisulfite PCR. This approach led to the detection of cancer-related genes, which were oncogenic genes, including EGR3 and POU4F2/Brn-3b were down-regulated, while the tumor suppressor gene, GPC3, was up-regulated by 1 nm melatonin-treated MCF-7 cells. Our results provide detailed insights into the DNA methylation patterns induced by melatonin and suggest a potential mechanism of the anticancer effect of aberrant DNA methylation in melatonin-treated breast cancer cells.

Human Müller glia with stem cell characteristics differentiate into retinal ganglion cell (RGC) precursors in vitro and partially restore RGC function in vivo following transplantation.

Müller glia with stem cell characteristics have been identified in the adult human eye, and although there is no evidence that they regenerate retina in vivo, they can be induced to grow and differentiate into retinal neurons in vitro. We differentiated human Müller stem cells into retinal ganglion cell (RGC) precursors by stimulation with fibroblast growth factor 2 together with NOTCH inhibition using the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). Differentiation into RGC precursors was confirmed by gene and protein expression analysis, changes in cytosolic [Ca(2+)] in response to neurotransmitters, and green fluorescent protein (GFP) expression by cells transduced with a transcriptional BRN3b-GFP reporter vector. RGC precursors transplanted onto the inner retinal surface of Lister hooded rats depleted of RGCs by N-methyl-d-aspartate aligned onto the host RGC layer at the site of transplantation but did not extend long processes toward the optic nerve. Cells were observed extending processes into the RGC layer and expressing RGC markers in vivo. This migration was observed only when adjuvant anti-inflammatory and matrix degradation therapy was used for transplantation. RGC precursors induced a significant recovery of RGC function in the transplanted eyes as determined by improvement of the negative scotopic threshold response of the electroretinogram (indicative of RGC function). The results suggest that transplanted RGC precursors may be capable of establishing local interneuron synapses and possibly release neurotrophic factors that facilitate recovery of RGC function. These cells constitute a promising source of cells for cell-based therapies to treat retinal degenerative disease caused by RGC dysfunction.

Diagnosis of bladder cancer recurrence based on urinary levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 hypermethylation.

BACKGROUND: Non muscle invasive bladder cancer (NMIBC) has the highest recurrence rate of any malignancy and as many as 70% of patients experience relapse. Aberrant DNA methylation is present in all bladder tumors and can be detected in urine specimens. Previous studies have identified DNA methylation markers that showed significant diagnostic value. We evaluated the significance of the biomarkers for early detection of tumor recurrence in urine. METHODOLOGY/PRINCIPAL FINDINGS: The methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens were measured by real-time PCR (MethyLight). We analyzed 390 urine sediments from 184 patients diagnosed with NMIBC. Urine from 35 age-matched control individuals was used to determine the methylation baseline levels. Recurrence was diagnosed by cystoscopy and verified by histology. Initially, we compared urine from bladder cancer patients and healthy individuals and detected significant hypermethylation of all six markers (P<0.0001) achieving sensitivity in the range 82%-89% and specificity in the range 94%-100%. Following, we validated the urinary hypermethylation for use in recurrence surveillance and found sensitivities of 88-94% and specificities of 43-67%. EOMES, POU4F2, VIM and ZNF154 were more frequently methylated in urine from patients with higher grade tumors (P ≤ 0.08). Univariate Cox regression analysis showed that five markers were significantly associated with disease recurrence; HOXA9 (HR=7.8, P=0.006), POU4F2 (HR=8.5, P=0.001), TWIST1 (HR=12.0, P=0.015), VIM (HR=8.0, P=0.001), and ZNF154 (HR=13.9, P<0.001). Interestingly, for one group of patients (n=15) we found that hypermethylation was consistently present in the urine samples despite the lack of tumor recurrences, indicating the presence of a field defect. CONCLUSION/SIGNIFICANCE: Methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.

Brn3a/Pou4f1 regulates dorsal root ganglion sensory neuron specification and axonal projection into the spinal cord.

The sensory neurons of the dorsal root ganglia (DRG) must project accurately to their central targets to convey proprioceptive, nociceptive and mechanoreceptive information to the spinal cord. How these different sensory modalities and central connectivities are specified and coordinated still remains unclear. Given the expression of the POU homeodomain transcription factors Brn3a/Pou4f1 and Brn3b/Pou4f2 in DRG and spinal cord sensory neurons, we determined the subtype specification of DRG and spinal cord sensory neurons as well as DRG central projections in Brn3a and Brn3b single and double mutant mice. Inactivation of either or both genes causes no gross abnormalities in early spinal cord neurogenesis; however, in Brn3a single and Brn3a;Brn3b double mutant mice, sensory afferent axons from the DRG fail to form normal trajectories in the spinal cord. The TrkA(+) afferents remain outside the dorsal horn and fail to extend into the spinal cord, while the projections of TrkC(+) proprioceptive afferents into the ventral horn are also impaired. Moreover, Brn3a mutant DRGs are defective in sensory neuron specification, as marked by the excessive generation of TrkB(+) and TrkC(+) neurons as well as TrkA(+)/TrkB(+) and TrkA(+)/TrkC(+) double positive cells at early embryonic stages. At later stages in the mutant, TrkB(+), TrkC(+) and parvalbumin(+) neurons diminish while there is a significant increase of CGRP(+) and c-ret(+) neurons. In addition, Brn3a mutant DRGs display a dramatic down-regulation of Runx1 expression, suggesting that the regulation of DRG sensory neuron specification by Brn3a is mediated in part by Runx1. Our results together demonstrate a critical role for Brn3a in generating DRG sensory neuron diversity and regulating sensory afferent projections to the central targets.