Deoxyuridine Incorporation Research Articles

Inhibiting Wnt/beta-catenin in CTNNB1-mutated endometrial cancer.

The role of β-catenin/TCF transcriptional activity in endometrial cancer (EC) recurrence is not well understood. We assessed the impact of Wnt/β-catenin inhibition in EC models. In an analysis of the Cancer Genome Atlas, we confirmed that CTNNB1 mutations are enriched in recurrent low-risk EC and showed that aberrant Wnt/β-catenin pathway activation is associated with recurrence. We studied CTNNB1-wildtype (HEC1B, Ishikawa) and CTNNB1-mutant (HEC108, HEC265, HEC1B-S33Y, Ishikawa-S33Y) EC cell lines. Dose response curves were determined for 5 Wnt/β-catenin pathway inhibitors (Wnt-C59, XAV-939, PyrPam, PRI-724, SM04690). XAV939, Wnt-C59 and PyrPam inhibited function upstream of β-catenin transcriptional activity and were ineffective at inhibiting cell viability. In contrast, PRI724 and SM04690 indirectly inhibited β-catenin transcriptional activity and significantly reduced cell viability in CTNNB1-mutant cell lines. Treatment with SM04690 reduced cell viability (Licor Cell stain) in all EC cell lines, but viability was significantly lower in CTNNB1-mutant cell lines (p < 0.01). Mechanistically, SM04690 significantly inhibited proliferation measured via 5'-bromo-2'-deoxyuridine incorporation and reduced T cell factor (TCF) transcriptional activity. HEC1B, HEC1B-S33Y and HEC265 tumor-bearing mice were treated with vehicle or SM04690. Tumors treated with SM04690 had smaller mean volumes than those treated with vehicle (p < 0.001, p = 0.014, p = 0.06). In HEC1B-S33Y and HEC265 tumors, SM04690 treatment significantly reduced Ki67 H-scores compared to vehicle (p = 0.035, p = 0.024). Targeting the Wnt/β-catenin pathway in CTNNB1-mutant EC effectively inhibited proliferation and β-catenin/TCF transcriptional activity and blunted tumor progression in in vivo models. These studies suggest β-catenin transcriptional inhibitors are effective in EC and particularly in CTNNB1-mutant EC, highlighting a potential therapeutic vulnerability for treatment of CTNNB1-mutant EC.

HOXA13, Negatively Regulated by miR-139-5p, Decreases the Sensitivity of Gastric Cancer to 5-Fluorouracil Possibly by Targeting ABCC4.

PurposeChemoresistance remains a major challenge in the therapy of gastric cancer (GC). The homeobox (HOX) gene family has gained attention in carcinogenesis and chemoresistance. Here, this study aimed to explore the mechanism of HOXA13 in GC chemoresistance.MethodsQuantitative real-time PCR (qRT-PCR) and Western blot were used to evaluate the expression of HOXA13 in GC tissues. The Kaplan–Meier plotter database was mined for prognosis analysis of GC patients with different HOXA13 expression receiving 5-Fluorouracil (5-FU) therapy. The effects of HOXA13 on sensitivity of GC cells to 5-FU were investigated by Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2’-deoxyuridine (EdU) incorporation, flow cytometry and experiment in vivo. RNA-Sequencing analysis was performed to explore the underlying mechanism of HOXA13-mediated 5-FU resistance in GC. Chromatin immunoprecipitation (ChIP) and rescue experiments were applied to determine the relationship between HOXA13 and ABCC4. Luciferase reporter assay was performed to assess interaction of miR-139-5p and HOXA13.ResultsHOXA13 was upregulated in GC and its high expression was associated with poor prognosis of GC patients with 5-FU treatment. Overexpression of HOXA13 impaired the inhibitory effects of 5-FU on GC cells proliferation in vitro and vivo, and knockdown of HOXA13 exacerbated 5-FU-induced GC cells apoptosis. Mechanistically, HOXA13, directly targeted by miR-139-5p in GC, might upregulate ABCC4 expression, thereby accentuating 5-FU resistance of GC cells.ConclusionOur study suggests that HOXA13 attenuates 5-FU sensitivity of GC possibly by upregulating ABCC4. Thus, targeting HOXA13 would provide a novel prospective into the potential therapeutic strategy for reversing chemoresistance.

Spread of X-chromosome inactivation into autosomal regions in patients with unbalanced X-autosome translocations and its phenotypic effects.

Patients with unbalanced X-autosome translocations are rare and usually present a skewed X-chromosome inactivation (XCI) pattern, with the derivative chromosome being preferentially inactivated, and with a possible spread of XCI into the autosomal regions attached to it, which can inactivate autosomal genes and affect the patients' phenotype. We describe three patients carrying different unbalanced X-autosome translocations, confirmed by G-banding karyotype and array techniques. We analyzed their XCI pattern and inactivation spread into autosomal regions, through HUMARA, ZDHHC15 gene assay and the novel 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and identified an extremely skewed XCI pattern toward the derivative chromosomes for all the patients, and a variable pattern of late-replication on the autosomal regions of the derivative chromosomes. All patients showed phenotypical overlap with patients presenting deletions of the autosomal late-replicating regions, suggesting that the inactivation of autosomal segments may be responsible for their phenotype. Our data highlight the importance of the XCI spread into autosomal regions for establishing the clinical picture in patients carrying unbalanced X-autosome translocations, and the incorporation of EdU as a novel and precise tool to evaluate the inactivation status in such patients.

An Optimized and Versatile Counter-Flow Centrifugal Elutriation Workflow to Obtain Synchronized Eukaryotic Cells.

Cell synchronization is a powerful tool to understand cell cycle events and its regulatory mechanisms. Counter-flow centrifugal elutriation (CCE) is a more generally desirable method to synchronize cells because it does not significantly alter cell behavior and/or cell cycle progression, however, adjusting specific parameters in a cell type/equipment-dependent manner can be challenging. In this paper, we used the unicellular eukaryotic model organism, Tetrahymena thermophila as a testing system for optimizing CCE workflow. Firstly, flow cytometry conditions were identified that reduced nuclei adhesion and improved the assessment of cell cycle stage. We then systematically examined how to achieve the optimal conditions for three critical factors affecting the outcome of CCE, including loading flow rate, collection flow rate and collection volume. Using our optimized workflow, we obtained a large population of highly synchronous G1-phase Tetrahymena as measured by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into nascent DNA strands, bulk DNA content changes by flow cytometry, and cell cycle progression by light microscopy. This detailed protocol can be easily adapted to synchronize other eukaryotic cells.

Bee Venom-Derived BBB Shuttle and its Correlation with Oligodendrocyte Proliferation Markers in Mice Model of Multiple Sclerosis.

Multiple sclerosis is a chronic demyelinating disease with a functional disturbance in the immune system and axonal damages. It was shown that Apamin as a blood-brain barrier shuttle acts as a Ca2+ activated K+ channels (SK channels) blocker. In this study, the effects of Apamin on oligodendrocyte differentiation markers were evaluated on an induced model of MS. Briefly, C57BL/6 male mice (22±5g) except the control group were fed with 0.2% (w/w) cuprizone pellets for 6 weeks. After cuprizone withdrawal, mice were divided randomly into six groups. Apamin (100µg/kg/BW) was administered intraperitoneally as a co-treatment during phase I (demyelination) or post-treatment phase II (remyelination) twice a week. Mice were anesthetized, perfused with phosphate-buffered saline, then fixed brains were coronally sectioned and the changes in oligodendrocytes markers such as Olig2, PDGFR-α, and BrdU incorporation were assessed by immunohistochemistry assay. Apamin administration increased Olig2+ cells in phase I as compared to the control group (p<0.0001). Also, a decreasing trend in PDGFRa+ cells observed after cuprizone withdrawal (p<0.001). 5-Bromo-2'-deoxyuridine (BrdU) incorporation test was confirmed stimulation of oligodendrocyte progenitor cell proliferation in phase I in the Apamin exposed group (p<0.0001), especially at the subventricular zone. This study highlights the potential therapeutic effects of Apamin as a bee venom-derived peptide on oligodendrocyte precursor proliferation and elevation in myelin content in an oxidative induced multiple sclerosis model due to cuprizone exposure.

Fabrication and bioactivity evaluation of curcumin and paclitaxel loaded lipid nanoparticles of pH-sensitive histidinylated cationic amphiphile

Drug resistance, inefficient cellular uptake and the subservient drug release to increase the intracellular drug concentration inside the tumor cells are the key reasons for low therapeutic efficacy of drug-loaded lipid nanoparticles in cancer therapy. Herein, we report on the design, synthesis and bioactivity evaluation of Curcumin &amp; Paclitaxel (PTX) encapsulated endosomal pH-Sensitive lipid nanoparticles of histidinylated cationic amphiphile (16-GH; 2 in 1 system) to overcome these challenges. Findings in fluorescence resonance energy transfer (FRET) assay and in vitro drug release studies showed a controlled pH dependent fusogenic and drug release properties of the lipid nanoparticles of cationic amphiphile 16-GH respectively. Further in vitro studies revealed that Curcumin &amp; PTX encapsulated nanoparticles of lipid 16-GH significantly inhibited proliferation of tumor cells than healthy cells. These lipid nanoparticles were further analyzed for their effect on 5-bromo-2'-deoxyuridine (BrdU) incorporation, Annexin V-FITC and cell cycle arrest (Sub-G1 phase). Further studies also confirmed that nanoparticles of lipid 16-GH containing Curcumin &amp; PTX displayed significantly enhanced the caspase3/9 activity. Remarkably, nanoparticles of lipid 16-GH containing Curcumin &amp; PTX are efficient in inducing apoptosis. The results in our initial mechanistic studies support the notion that the tumor cell selective cytotoxic capability of the lipid nanoparticles of the presently described endosomal pH-sensitive lipid probably instigates from depolarization of mitochondrial membrane potential and subsequent activation of caspases 3 and 9. The distinguishing feature of the currently described endosomal pH-sensitive system is that it not only efficiently delivers highly potent anti-cancer agents (Curcumin &amp; PTX) to tumor cells, but the lipid nanoparticle drug carrier itself also contributes to inhibiting tumor cell growth. In summary, the presently described lipid nanoparticles are expected to simultaneously delivering combination of drugs to various types of tumor models.

Dioscin inhibits SCC15 cell proliferation via the RASSF1A/MST2/YAP axis.

Dioscin, an extract from traditional Chinese herbal plants, displays various biological and pharmacological effects on tumors, including inhibition of cell proliferation and induction of DNA damage. However, the effects of dioscin on oral squamous cell carcinoma (OSCC) cells are not completely understood. The present study aimed to evaluate the impact of dioscin on OSCC cell proliferation. Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine incorporation assays were performed to assess cell proliferation. Flow cytometry was conducted to detect alterations in the cell cycle and cell apoptosis. Western blotting and coimmunoprecipitation were performed to determine protein expression levels. In SCC15 cells, dioscin treatment significantly induced cell cycle arrest, increased apoptosis and inhibited proliferation compared with the control group. Mechanistically, the tumor suppressor protein Ras association domain-containing protein 1A (RASSF1A) was activated and oncoprotein yes-associated protein (YAP) was phosphorylated by dioscin. Furthermore, YAP overexpression and knockdown reduced and enhanced the inhibitory effects of dioscin on SCC15 cells, respectively. In summary, the results demonstrated that, compared with the control group, dioscin upregulated RASSF1A expression in OSCC cells, which resulted in YAP phosphorylation, thus weakening its transcriptional coactivation function, enhancing cell cycle arrest and apoptosis, and inhibiting cell proliferation. The present study indicated that dioscin may serve as a therapeutic agent for OSCC.

OPA Interacting Protein 5 Antisense RNA 1 Expedites Cell Migration and Invasion Through FOXM1/ Wnt/β-Catenin Pathway in Pancreatic Cancer.

Pancreatic cancer (PC) is a digestive tract malignancy with poor prognosis. Long noncoding RNA (lncRNA) OPA interacting protein 5 antisense RNA 1 (OIP5-AS1) was regarded to be correlated with human malignancy, working as tumor suppressor or promoter on the basis of tumor types. However, the function of OIP5-AS1 in PC remained unclear. The study focused on the function and regulatory mechanism of OIP5-AS1 in PC. OIP5-AS1 expression was assessed by the quantitative reverse transcription PCR (RT-qPCR) in tumor tissues and PC cell lines. 5-ethynyl-2'-deoxyuridine (EdU) incorporation and cell counting kit-8 (CCK-8) assays were applied to detect cell proliferation ability. Through wound healing and transwell assays, cell migration and invasion capacities were estimated. Flow cytometry analysis was performed to examine apoptosis capability of PC cells. OIP5-AS1 downregulating inhibited cell proliferation, migration, and invasion capacities, while promoting cell apoptosis rates. As a competing endogenous RNA (ceRNA), OIP5-AS1 competed with Forkhead Box M1 (FOXM1) for the binding sites on microRNA-320b (miR-320b). OIP5-AS1 was able to upregulate FOXM1 expression via silencing miR-320b. Furthermore, FOXM1 served as an activator of Wnt/β-catenin pathway and mediated the effect of OIP5-AS1 on Wnt/β-catenin pathway. OIP5-AS1 expedites the proliferative, migrated, and invasive capability of PC cells, while repressing cell apoptosis through regulating miRNA-320b/FOXM1 axis and FOXM1/Wnt/β-catenin pathway in PC. OIP5-AS1 regulation on FOXM1/Wnt/β-catenin pathway may offer novel efficient markers for PC treatments.

Involvement of circHIPK3 in the pathogenesis of diabetic cardiomyopathy in mice.

In a mouse model of diabetic cardiomyopathy (DCM) the expression of the circular RNA circHIPK3 was found to be significantly increased. This study aimed to discover the molecular mechanisms linking circHIPK3 to the pathogenesis of DCM. The diabetic mouse model was established by i.p. injection of streptozotocin, which led to the development of DCM. Echocardiographic measurements were used to evaluate cardiac structure and function, and histological staining was applied to detect myocardial fibrosis in mice. 5-Ethynyl-2'-deoxyuridine incorporation was performed to determine cell proliferation and RNA fluorescent in situ hybridisation was employed to examine circHIPK3 expression in cardiac fibroblasts. RNA immunoprecipitation and luciferase reporter assay were conducted to explore the pathological mechanism of circHIPK3 in myocardial fibrosis. Knockdown of circHIPK3 was found to attenuate myocardial fibrosis and enhance cardiac function in DCM mice. In addition, silencing of circHIPK3 could suppress proliferation of cardiac fibroblasts treated with angiotensin II. Furthermore, RNA immunoprecipitation and luciferase reporter assay revealed a circHIPK3-miR-29b-3p-Col1a1-Col3a1 regulatory network in the pathogenesis of myocardial fibrosis. circHIPK3 contributes to increased myocardial fibrosis during DCM by functioning as a competing endogenous RNA that upregulates Col1a1 and Col3a1 expression through suppressing miR-29b-3p.

VEGF-C/VEGFR-3/iNOS Signaling in Osteosarcoma MG63 Cells Mediates Stimulatory Effects on Human Umbilical Vein Endothelial Cell Proliferation

Objective To assess the effects of vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor-3 (VEGFR-3) signaling on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells (HUVECs). MethodsMG63 cells were treated with VEGF-C alone (VEGF-C group), VEGF-C + iNOS inhibitor aminoguanidine (AG; AG group), and VEGF-C + VEGFR-3 inhibitor MAZ51 (MAZ51 group); untreated MG63 cells were used as controls. NO production was evaluated by a colorimetric method involving nitrate reductase. Meanwhile, mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs, we set up six groups: HUVECs, HUVECs+MG63, HUVECs+VEGF-C, HUVECs+MG63+VEGF-C, HUVECs+MG63+VEGF-C+AG, and HUVECs+MG63+VEGF-C+MAZ51 groups. The proliferation of HUVEC cells was assessed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and proliferating cell nuclear antigen (PCNA) expression quantitation. ResultsVEGF-C treatment enhanced iNOS expression at both gene and protein levels (mRNA: LSD-t=4.152, P<0.01; protein: LSD-t=3.486, P<0.01) and increased NO release of MG63 cells (LSD-t=3.774, P<0.01); treatment with either AG or MAZ51 decreased these effects (mRNA: LSD-t=9.183, P<0.001; LSD-t=8.639, P<0.001; protein: LSD-t=5.170, P<0.001; LSD-t=7.255, P<0.001; NO production:LSD-t=10.326, P<0.001; LSD-t=10.540, P<0.001). Interestingly, co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation (EdU: LSD-t=5.374, P<0.001; LSD-t=2.984, P<0.05; LSD-t=8.526, P<0.001; PCNA: LSD-t=9.267, P<0.001; LSD-t=5.515, P<0.001; LSD-t=14.873, P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG (EdU: LSD-t=10.770, P<0.001; PCNA: LSD-t=19.940, P<0.001) or MAZ51 (EdU: LSD-t=6.950, P<0.001; PCNA: LSD-t=14.001, P<0.001). ConclusionIn human osteosarcoma MG63 cells, activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production, which subsequently induces HUVEC proliferation.

S1P induces proliferation of pulmonary artery smooth muscle cells by promoting YAP-induced Notch3 expression and activation

Sphingosine-1-phosphate (S1P), a natural multifunctional phospholipid, is highly increased in plasma from patients with pulmonary arterial hypertension and mediates proliferation of pulmonary artery smooth muscle cells (PASMCs) by activating the Notch3 signaling pathway. However, the mechanisms underpinning S1P-mediated induction of PASMCs proliferation remain unclear. In this study, using biochemical and molecular biology approaches, RNA interference and gene expression analyses, 5′-ethynyl-2′-deoxyuridine incorporation assay, and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, we demonstrated that S1P promoted the activation of signal transducers and activators of transcription 3 (STAT3) through sphingosine-1-phosphate receptor 2 (S1PR2), and subsequently upregulated the expression of the microRNA miR-135b, which further reduced the expression of E3 ubiquitin ligase β-transduction repeat-containing protein and led to a reduction in yes-associated protein (YAP) ubiquitinated degradation in PASMCs. YAP is the core effector of the Hippo pathway and mediates the expression of particular genes. The accumulation of YAP further increased the expression and activation of Notch3 and ultimately promoted the proliferation of PASMCs. In addition, we showed that preblocking S1PR2, prior silencing of STAT3, miR-135b, or YAP, and prior inhibition of Notch3 all attenuated S1P-induced PASMCs proliferation. Taken together, our study indicates that S1P stimulates PASMCs proliferation by activation of the S1PR2/STAT3/miR-135b/β-transduction repeat-containing protein/YAP/Notch3 pathway, and our data suggest that targeting this cascade might have potential value in ameliorating PASMCs hyperproliferation and benefit pulmonary arterial hypertension.

LncRNA LIFR‑AS1 inhibits gastric carcinoma cell proliferation, migration and invasion by sponging miR‑4698.

The vital functions of long non-coding (lnc)RNAs have been verified in gastric carcinoma (GC). However, as a novel cancer-related lncRNA, the influence of leukemia inhibitory factor receptor antisense RNA 1 (LIFR-AS1) in GC cell biological behaviors remains unreported. The present study explored the biological effects of lncRNA LIFR-AS1 on GC progression. Reverse transcription-quantitative PCR was performed to examine lncRNA LIFR-AS1 expression in GC tissues and cells. Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine incorporation, cell wound healing and Transwell invasion assays were used to assess the functions of lncRNA LIFR-AS1 in GC cell proliferation, migration and invasion. Additionally, associations among lncRNA LIFR-AS1, microRNA (miR)-4698 and microtubule-associated tumor suppressor 1 (MTUS1) were investigated via bioinformatics software and a luciferase reporter system. In addition, western blotting was used to examine the expression of MEK and ERK. Decreased lncRNA LIFR-AS1 expression was observed in GC tissues and cells. Upregulated lncRNA LIFR-AS1 inhibited GC cell proliferation, migration and invasion. Upregulated miR-4698 and downregulated MTUS1 were identified in GC tissues and cells. The inhibitory interaction between lncRNA LIFR-AS1 and miR-4698 was confirmed. Additionally, MTUS1 was predicted as a target gene of miR-4698 positively regulated by lncRNA LIFR-AS1. The MEK/ERK pathway was inhibited by lncRNA LIFR-AS1 via regulating MTUS1. These findings revealed the inhibitory functions of lncRNA LIFR-AS1 in GC cell proliferation, migration and invasion. The process was mediated via miR-4698, MTUS1 and the MEK/ERK pathway.

Garcinol inhibits the proliferation of endometrial cancer cells by inducing cell cycle arrest.

Endometrial cancer (EC) is the most common gynecological cancer, and one of the most important causes of cancer-related deaths in women worldwide. The long-term survival rate is lower in advanced-stage and recurrent EC, therefore it is important to identify new anticancer drugs. Garcinol, a polyisoprenylated benzophenone, is a promising anticancer drug for various cancer types but its effects on EC remain unclear. To investigate the anticancer effects of garcinol on EC, cell proliferation and cell cycle were assessed by real-time cell proliferation, cell counting, and colony formation assays, flow cytometric analysis, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, in EC Ishikawa (ISH) and HEC-1B cell lines. Western blotting was used to evaluate the expression of cell cycle-related protein cyclins, cyclin-dependent kinase and tumor suppression proteins. Garcinol inhibited ISH and HEC-1B cell proliferation in a dose-dependent manner, and induced ISH and HEC-1B cell cycle arrest at the G1 phase and G2/M phase, respectively, and decreased the S phase and DNA synthesis in these two cell lines. Following garcinol treatment the expression levels of p53 and p21 were increased, while the expression levels of CDK2, CDK4, cyclin D1 and cyclin B1 were gradually decreased in a dose-dependent manner in both ISH and HEC-1B cells. In addition, the expression levels of phosphorylated c-JUN N-terminal kinase (JNK) and p-c-JUN were significantly increased in both types of cells. Collectively, garcinol can induce EC cell cycle arrest and may be a promising candidate for EC chemotherapy.

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice.

Müller cells (MC) are considered dormant retinal progenitor cells in mammals. Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain. It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC. Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells. In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC. The level of ephrinAs/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC. Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2-/- A3-/- mice by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. We detected a significant increase of EdU+ cells in MC derived from A2-/- A3-/- mice. Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout (Rho-/-) mice. To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2-/- A3-/- , Rho-/- and Rho-/- A2-/- A3-/- mice and the numbers of EdU+ cells distributed among different layers of the retina. EphrinAs/EphA4 expression was upregulated in the retina of Rho-/- mice compared to the wild-type mice. In addition, cultured MC derived from ephrin-A2-/- A3-/- mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice. Interestingly, we detected a significant increase of EdU+ cells in the retinas of adult ephrin-A2-/- A3-/- mice mainly in the inner nuclear layer; and these EdU+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC. In Rhodopsin knockout mice (Rho-/- A2-/- A3-/- mice), a significantly greater amount of EdU+ cells were located in the ciliary body, retina and RPE than that of Rho-/- mice. Comparing between 6 and 12 weeks old Rho-/- A2-/- A3-/- mice, we recorded more EdU+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration. Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC. Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration. All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA (approval No. S-353-0715) on October 24, 2012.

Long non-coding RNA Peg13 attenuates the sevoflurane toxicity against neural stem cells by sponging microRNA-128-3p to preserve Sox13 expression.

BackgroundExposure to anesthetics during brain development may impair neurological function, however, the mechanisms underlying anesthetic neurotoxicity are unclear. Recent studies indicate that long non-coding RNAs (lncRNAs) are crucial for regulating the functional brain development during neurogenesis. This study aimed to determine the regulatory effects and potential mechanisms of lncRNA Peg13 (Peg13) on sevoflurane exposure-related neurotoxicity against neural stem cells (NSCs).MethodsMouse embryotic NSCs were isolated and their self-renewal and differentiation were characterized by immunofluorescence. NSCs were exposed to 4.1% sevoflurane 2 h daily for three consecutive days. The potential toxicities of sevoflurane against NSCs were evaluated by neurosphere formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation and flow cytometry assays. The Peg13, miR-128-3p and Sox13 expression in NSCs were quantified. The potential interactions among Peg13, miR-128-3p and Sox13 were analyzed by luciferase reporter assay. The effects of Peg13 and/or miR-128-3p over-expression on the sevoflurane-related neurotoxicity and Sox13 expression were determined in NSCs.ResultsThe isolated mouse embryotic NSCs displayed potent self-renewal ability and differentiated into neurons, astrocytes and oligodendrocytes in vitro, which were significantly inhibited by sevoflurane exposure. Sevoflurane exposure significantly down-regulated Peg13 and Sox13, but enhanced miR-128-3p expression in NSCs. Transfection with miR-128-3p mimics, but not the control, significantly mitigated the Peg13 or Sox13-regulated luciferase expression in 293T cells. Peg13 over-expression significantly reduced the sevoflurane-related neurotoxicity and increased Sox13 expression in NSCs, which were mitigated by miR-128-3p transfection.ConclusionSuch data indicated that Peg13 mitigated the sevoflurane-related neurotoxicity by sponging miR-128-3p to preserve Sox13 expression in NSCs.

Trypanosoma cruzi amastigotes that persist in the colon during chronic stage murine infections have a reduced replication rate

Chronic Trypanosoma cruzi infections are typically lifelong, with small numbers of parasites surviving in restricted tissue sites, which include the gastrointestinal tract. There is considerable debate about the replicative status of these persistent parasites and whether there is a role for dormancy in long-term infection. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2′deoxyuridine incorporation into DNA to provide ‘snapshots’ of parasite replication status. Highly sensitive imaging of the extremely rare infection foci, at single-cell resolution, revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections of the colon are associated with a reduced rate of parasite replication. Despite this, very few host cells survived infection for more than 14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. We could find no evidence for wide-spread dormancy in parasites that persist in this tissue reservoir.

Xanthohumol Inhibits TGF-β1-Induced Cardiac Fibroblasts Activation via Mediating PTEN/Akt/mTOR Signaling Pathway.

BackgroundXanthohumol (Xn) is the most abundant prenylated flavonoid in Hops (Humulus lupulus L.), and exhibits a range of pharmacological activities. This study aimed to investigate the effect of Xn on TGF-β1-induced cardiac fibroblasts activation and elucidate the underlying mechanism.Materials and MethodsThe cellTiter 96® AQueous one solution cell proliferation assay kit was adopted to determine the cell viability of cardiac fibroblasts, and the proliferation was detected through 5-ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay. The α-SMA protein expression was measured by using immunofluorescence and Western blotting. Western blotting was conducted to test the protein expressions of collagen I and III, PTEN, p-Akt, Akt, p-mTOR, mTOR, p-Smad3, Smad3 and GAPDH. The mRNA levels of α-SMA, collagen I and III were determined by quantitative real-time polymerase chain reaction (PCR).ResultsXn inhibited the TGF-β1-induced proliferation, differentiation and collagen overproduction of cardiac fibroblasts. TGF-β1 induced the down-regulated PTEN expression, Akt and mTOR phosphorylation. These effects of TGF-β1 were suppressed by Xn, while blocking of PTEN reduced Xn-mediated inhibitory effect on cardiac fibroblasts activation induced by TGF-β1.ConclusionXn inhibits TGF-β1-induced cardiac fibroblasts activation via mediating PTEN/Akt/mTOR signaling pathway.

Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma.

The abnormal upregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) expression levels were reported to be involved in the progression of various types of cancer. Therefore, it is hypothesized that hnRNP K may serve as a useful diagnostic marker and antitumor target; however, only a few studies to date have investigated the exact role of hnRNP K in head and neck squamous cell carcinoma (HNSCC) and the potential downstream signaling pathway involved. The present study aimed to identify the roles of hnRNP K in the proliferation and migration of HNSCC, and the possible signaling pathways hnRNP K may be associated with in HNSCC. hnRNP K expression levels in clinical HNSCC samples were analyzed using the Oncomine and UALCAN databases, and its association with the survival of patients with HNSCC was analyzed using the tumor-immune system interactions database. Short hairpin RNA targeting hnRNP K was transfected into the CAL-27 cell line to establish HNSCC cells with stable hnRNP K-knockdown. Cell viability was analyzed using a Cell Counting Kit-8 assay and an absolute count assay, and cell proliferation was measured using 5-ethynyl-2′-deoxyuridine incorporation and colony formation assays. Migratory ability of cells was analyzed using wound healing assay and transwell assay. The growth of xenografts derived from hnRNP K-knockdown cells was also evaluated, and bioinformatics analyses were performed using the Gene Ontology and Kyoto Encyclopedia for Genes and Genomes databases to determine the possible downstream signaling pathways of hnRNP K. Furthermore, the status of the Wnt/β-Catenin signaling pathway in hnRNP K-knockdown cells mediated by small interfering RNA was determined using reverse transcription-quantitative PCR and western blotting. The results revealed that the expression levels of hnRNP K were upregulated in HNSCC cell lines and tissues. Moreover, the upregulation of hnRNP K expression levels was associated with poor survival of patients with HNSCC. The knockdown of hnRNP K also decreased HNSCC cell proliferation and migration, and inhibited tumor growth in nude mice. Bioinformatics analyses identified the Wnt/β-Catenin signaling pathway as a possible downstream signaling pathway of hnRNP K. Knockdown of hnRNP K significantly downregulated the expression levels of Wnt/β-Catenin signaling pathway-related proteins; while with knockdown of hnRNP K and overexpression of β-Catenin, the expression levels of Wnt/β-Catenin signaling pathway-related proteins were partially rescued. In conclusion, the present findings indicated that hnRNP K may serve as a candidate diagnostic biomarker and a promising therapeutic target for HNSCC.

Ferulic acid (FA) protects human retinal pigment epithelial cells from H2 O2 -induced oxidative injuries.

The aim of present study is to investigate whether Ferulic acid (FA), a natural polyphenol antioxidant, was able to protect ARPE‑19 cells from hydrogen peroxide (H2O2)‑induced damage, and elucidate the underlying mechanisms. Our results revealed that FA pre‐treatment for 24 hours can reverse cell loss of H2O2‐induced ARPE‐19 cells via the promotion of cell proliferation and prevention of apoptosis, as evidenced by 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) assay, respectively. Moreover, the addition of FA (5 mM) can decrease Bax and cleaved caspase‐3 protein expression, but increase Bcl‐2 protein expression in ARPE‐19 cells. Furthermore, H2O2‐induced oxidative stress in ARPE‐19 cells was significantly alleviated by FA, illustrated by reduced levels of ROS and MDA. In addition, the attenuated antioxidant enzymes activities of (SOD, CAT and GPX) and GSH level were reversed almost to the normal base level by the pre‐addition of FA for 24 hours. In all assays, FA itself did not exert any effect on the change of the above parameters. These novel findings indicated that FA effectively protected human ARPE‐19 cells from H2O2‐induced oxidative damage through its pro‐proliferation, anti‐apoptosis and antioxidant activity, suggesting that FA has a therapeutic potential in the prevention and treatment of AMD.

USP21 upregulation in cholangiocarcinoma promotes cell proliferation and migration in a deubiquitinase-dependent manner.

Ubiquitin-specific protease 21 (USP21) has been implicated in several types of cancer. It promotes or suppresses tumor growth in a cell-context dependent manner. Cholangiocarcinoma is a malignant tumor with a high mortality rate. However, the role of USP21 in cholangiocarcinoma remains unknown. Here, we identify that the level of USP21 is upregulated in cholangiocarcinoma using bioinformatics analysis and confirm this elevation in RBE cell lines. Cell counting and 5-ethynyl-2'-deoxyuridine incorporation assays reveal that USP21 promotes the proliferation of cholangiocarcinoma. Wound healing and transwell assays demonstrate that USP21 accelerates RBE cell migration. In addition, rescue assays reveal that reintroduction of USP21 wildtype other than the deubiquitinase-deficient C221A mutant restores USP21 depletion-induced attenuation in cell proliferation and migration, indicative of the requirement of the deubiquitinase activity. Collectively, these data indicate that USP21 is critically involved in cholangiocarcinoma tumorigenesis and may be an effective target for the treatment of cholangiocarcinoma.