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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 721 records · Page 40
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

miR-125b-5p inhibits breast cancer cell proliferation, migration and invasion by targeting KIAA1522

Highlights: • miR-125b-5p expression was found downregulated in breast cancer. • We explored the tumor suppressive role of miR-125b-5p in breast cancer. • KIAA1522 was reported to function as oncogene in breast cancer. • KIAA1522 was a direct target of miR-125b-5p in breast cancer. Abnormal gene expression due to the dysregulation of microRNAs (miRNAs) often occurred in the initiation or progression of cancers. The aim of this present study was to investigate the function role of miR-125b-5p in breast cancer (BC). Expression levels of miR-125b-5p were determined by quantitative Real-time PCR. Biological functions of miR-125b-5p in the progression of BC were investigated with a series of in vitro experiments including cell counting kit-8 assay, colony formation assay, wound-healing assay and transwell invasion assay. The target of miR-125b-5p in BC was validated by luciferase activity reporter assay and western blot assay. We found miR-125b-5p expression was significantly reduced in BC cell lines compared to the normal breast epithelial cell line. Functional assays showed that cell proliferation, colony formation ability, cell migration, and cell invasion can be suppressed by miR-125b-5p overexpression. Besides, KIAA1522 was validated as a direct target of miR-125b-5p in BC. Collectively, our study showed that miR-125b-5p functions as a tumor suppressor and regulates BC progression through targeting KIAA1522.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Platelet-derived growth factor receptor alpha (PDGFRα) is overexpressed in NK/T-cell lymphoma and mediates cell survival

Nasal-type natural killer/T-cell lymphoma (NKTCL) is a subtype of non-Hodgkin lymphoma (NHL) that is clinically aggressive and has a poor prognosis. Platelet-derived growth factor receptors (PDGFRs) and their ligands (PDGFs) play important roles in angiogenesis, cancer cell proliferation, survival, migration and poor prognosis in various tumours. However, the significance of PDGFRs in NKTCL remains unknown. Herein, the present study aimed to investigate the important role of PDGFRα in pathogenesis, progression and prognisis of NKTCL. Firstly, we performed immunohistochemical staining, qRT-PCR and western blotting to determine PDGFRα expression in formalin-fixed, paraffin-embedded tissue sections from 78 NKTCL cases and in cell lines. Secondly, correlations between PDGFRα expression and NKTCL clinical parameters and prognosis were analysed. Moreover, a biological assessment of PDGFRα blockade in two NKTCL cell lines was conducted through proliferation assay, cell-cycle evaluation and apoptosis detection by flow cytometry analyses. Furthermore, we detected in vivo activity of imatinib in mouse model of NKTCL. We found that the expression of PDGFRα was significantly higher in NKTCL tissues compared to the reactive lymphoid hyperplasia of the nasopharynx (P = 0.028). High PDGFRα expression was strongly associated with a high LDH level (P = 0.028) and III-IV stage (P = 0.013). NKTCL patients with high PDGFRα expression displayed a reduced median overall survival time and progression-free survival time when compared with those with low PDGFRα expression (P = 0.011, P = 0.005, respectively). Cox multivariate analysis showed that III-IV stage (P = 0.024) and high PDGFRα expression (P = 0.003) were independent prognostic factors in NKTCL patients. Biological assessment assays in two NKTCL cell lines revealed that a specific PDGFR antagonist, imatinib, inhibited cell viability, blocked cell cycle progression at G0/G1 stage and induced apoptosis. Similarly, the in vivo assay showed that imatinib delayed mouse model tumour growth. In conclusion, NKTCL tumour cells have prominent PDGFRα expression, which can serve as a candidate prognostic marker. PDGFR antagonists have significant biological effect on NKTCL and may be useful therapeutic agents for treatment of NKTCL.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Alleviation of A disintegrin and metalloprotease 10 (ADAM10) on thromboangiitis obliterans involves the HMGB1/RAGE/ NF-κB pathway

Highlights: • TAO rats displayed higher whole blood viscosity and blood platelet count. • TAO rats had structural damages of vascular smooth muscle and endothelial cells. • ADAM treatment alleviated the symptoms of TAO rats in a dose-dependent pattern. • HMGB1, RAGE and NF-κB increased in TAO rats in both mRNA and protein level. • HMGB1, RAGE and NF-κB decreased by ADAM10 treatment in both mRNA and protein level. Thromboangiitis obliterans (TAO), also known as Buerger's disease, is a nonatherosclerotic inflammatory disease that influences medium- and small-sized blood vessels of extremities. However, mechanisms underlying TAO are still unclear. As a mediator associated with inflammation, A disintegrin and metalloprotease 10 (ADAM10) was hypothesized to play inhibitory roles in the development of TAO. Thus, the objective of this study is to investigate the effects of ADAM10 in a sodium laurate-induced TAO rat model and elucidate underlying mechanisms. Male Wistar rats were randomly divided into four groups (n = 6) for treatment: sham-operated (SHAM), TAO model (TAO), ADAM10 low dose injection (3 mg/kg; ADAM10-LD) and ADAM10 high dose injection (6 mg/kg; ADAM10-HD). After 14-day treatment, color Doppler ultrasound and hematology analysis indicated TAO rats displayed higher whole blood viscosity and blood platelet count compared with those in the SHAM group. Histologic evaluation and transmission electron microscopy revealed that the ultrastructural damages of vascular smooth muscle and endothelial cells were observed in TAO rats, such as fractured endoplasmic reticulum, decreased cell counts, and fibrillation. On the other hand, the typical signs and symptoms of TAO rats were significantly alleviated via ADAM10 treatment with a dose-dependent pattern. Real-time PCR and western blot results revealed that the expression of high-mobility-group box 1 (HMGB1), receptor for advanced glycation end-products (RAGE) and nuclear factor-kappa B (NF-κB) increased in TAO rats whereas decreased by ADAM10 treatment in both mRNA and protein levels. In conclusion, the results suggest ADAM10 alleviates symptoms of sodium laurate-induced TAO in rats via the RAGE/NF-κB signaling pathway and provides insight into the molecular basis and a potential therapeutic strategy for TAO.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Hydroxysafflor yellow A attenuates high glucose-induced pancreatic β-cells oxidative damage via inhibiting JNK/c-jun signaling pathway

Highlights: • High glucose-induced oxidative stress can lead to pancreatic β-cells damage. • HSYA can reduce pancreatic β-cells apoptosis by attenuating oxidative damage. • HSYA can scavenge oxidative stress and inhibit apoptosis through JNK/c-Jun signaling pathway. Pancreatic β-cells apoptosis and dysfunction induced by glucose toxicity were attributed to the formation of excess oxidative damage. Some studies have found that hydroxysafflor yellow A has strong effects to scavenge oxidative stress and inhibit apoptosis. In order to explore the influence of HSYA on oxidative stress induced by high glucose and the potential mechanisms, we set up a high glucose damage model and induced oxidative stress in INS-1 rat insulinoma cells. N-acetylcysteine was added as a group of oxidative stress scavenger. After 72 h of cultivation, the related indexes of oxidative stress (reactive oxygen species, catalase, glutathione peroxidase, lipid peroxidation, and superoxide dismutase), apoptosis (caspase3, parp) and the function of glucose stimulated insulin secretion were determined. In addition, the signaling pathway proteins of C-Jun NH2 -terminal kinases (JNK), phosphorylated JNK, C-jun, phosphorylated C-jun were evaluated. Fluorescence microscopy, qRT-PCR, western blotting were the main methods used in the experiment. Our results showed that hydroxysafflor yellow A reduced pancreatic β-cells apoptosis by attenuating oxidative damage, and JNK/c-Jun signaling pathway was involved. It indicated a significant mechanism for the positive impacts of HSYA on oxidative stress induced by high glucose, and provide important basis for using HSYA in diabetic prevention and therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

YY1-induced upregulation of lncRNA KCNQ1OT1 regulates angiotensin II-induced atrial fibrillation by modulating miR-384b/CACNA1C axis

Recent years, the role of long non-coding RNAs (lncRNAs) in atrial fibrillation (AF) has been gradually elucidated. In the current study, we measured the expression of ten AF-related lncRNAs to do qRT-PCR analysis. LncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) was found to be significantly upregulated in AF model and Ang-II-induced mice heart. CACNA1C has been reported as a biomarker in atrial fibrillation. Here, we found that the expression pattern of CACNA1C was consistent with that of KCNQ1OT1. Electrophysiological study was conducted to demonstrate the effect of KCNQ1OT1 and CACNA1C on the Effective refractory period (ERP), interatrial conduction time (IACT), incidence of AF and AF duration of Ang-II-induced mice heart. Mechanically, KCNQ1OT1 contributed to the upregulation of CACNA1C by binding with miR-384. Furthermore, YY1 could activate the transcription of KCNQ1OT1 and CACNA1C. In conclusion, the present study revealed that YY1-induced upregulation of lncRNA KCNQ1OT1 regulates angiotensin II-induced atrial fibrillation by regulating miR-384/CACNA1C axis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Administration of ubiquitin-activating enzyme UBA1 inhibitor PYR-41 attenuates angiotensin II-induced cardiac remodeling in mice

Highlights: • Ang II induces UBA1 expression. • PYR-41 inhibits cardiac remodeling. • PYR-41 stabilizes PTEN. Pathological cardiac hypertrophy is the main risk factor for heart diseases. The ubiquitin-proteasome system (UPS) is the major intracellular protein degradation system involved in the development of cardiac hypertrophic remodeling. Ubiquitin-activating enzyme E1, a key component of the UPS, catalyzes the first step in ubiquitin conjugation to mark cellular proteins for degradation via proteasome. However, the functional role of E1 (UBA1) in regulation of hypertrophic remodeling in angiotensin II (Ang II)-infused mice remains unknown. In this study, male wild-type mice were treated with UBA1 inhibitor PYR-41 at two doses of 5 and 10 mg and infused with Ang II (1000 ng/kg/min) for 14 days. Systolic blood pressure was detected by using tail-cuff system. Cardiac function was assessed by echocardiography. Hypertrophic remodeling was analyzed examined by histological examinations. The expressions of genes and proteins were detected by quantitative real-time PCR and immunoblotting analysis. After 14 days, Ang II infusion significantly increased UBA1 expression at both mRNA and protein levels in the hearts. Furthermore, Ang II-infused mice showed a significant increase in systolic blood pressure compensatory cardiac function, hypertrophy, interstitial fibrosis, inflammation and oxidative stress compared with saline-treated controls, whereas these effects were dose-dependently attenuated in PYR-41-treated mice. These beneficial actions were associated mainly with inhibition of PTEN degradation and multiple downstream mediators (AKT, ERK1/2, STAT3, TGF-β/Smad2/3 and NF-kB(p65)). In conclusion, these results indicate that inhibition of UBA1 suppresses Ang II-induced hypertrophic remodeling, and suggest that administration of low dose PYR-41 may be a new potential therapeutic approach for treating hypertensive heart diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica

Highlights: • A novel gene encoding a Y-box protein named DjY2 was cloned from Dugesia japonica. • DjY2 protein was structurally different from other Y-box proteins in planarians. • The expression patterns of DjY2 transcripts are different from DjY1 and DeY1. • DjY2 gene might participate in the regeneration process in planarian. Y-box binding protein (YB protein) is an ancient conserved multifunctional DNA/RNA-binding protein. A novel YB protein DjY2 gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 689 bp with a putative open reading frame of 197 amino acids. It has a predicted molecular mass of 22.14 kDa and an isoelectric point of 9.67. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjY2 in the process of planarian regeneration. Results showed that DjY2 was expressed in many parts of the body in intact planarian, but the expression level was low in head and pharynx. The transcripts of DjY2 was significantly increased both at the head parts and the tail parts after amputation, especially at the site of cutting. The spatial expression gradually recovered to the state of intact planarian with the time of regeneration. Our results indicated that DjY2 might participate in the process of regeneration in planarian.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Gene expression profiling in adipose tissue of Sprague Dawley rats identifies olfactory receptor 984 as a potential obesity treatment target

Highlights: • Olr984 is identified as candidate gene for obesity resistance in rats. • Olr984 is highly expressed in human adipose tissue. • Olr984mRNA expression in human adipose tissue correlates with obesity parameters. The aim of the study was to identify and functionally characterize novel candidate gene/s involved in the development of resistance to diet-induced obesity in rats. In a high-fat-diet (HFD) study of rats, we found subgroups which either developed resistance to HFD-induced obesity (DR) or showed an obesity-prone phenotype (DIO). Gene expression analysis in 10 samples (5 DIO vs 5 DR) was performed. The most promising gene, OR6C3 (orthologous with rat Olr984 and mouse Olfr788) was measured by qRT-PCR in paired samples of human visceral (Vis) and subcutaneous (SC) adipose tissue (AT) (n = 225) and in sub-fractions of adipocytes and cells of stromal vascular fraction. Gene expression analyses showed Olr984 with significantly reduced mRNA expression in DR rats. In the Vis AT of human samples we found an up-regulation of OR6C3 compared to SC AT, independent of gender, glucose tolerance or type 2 diabetes. We observed significantly lower levels of SC AT OR6C3 mRNA in subjects with obesity compared to those with normal-weight or overweight. OR6C3 is more expressed in SVF than in adipocytes. Olr984 could be a novel candidate gene related to diet-induced obesity in rats. Variation in human AT mRNA expression is related to obesity parameters and glucose homeostasis and linked to the regulatory role of insulin on the Olr984.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Evaluation of collagen mixture on promoting skin wound healing in zebrafish caused by acetic acid administration

The aim of this study is to use zebrafish embryos as a quick platform for wound healing studies. At beginning, we optimized a protocol to induce skin lesion by acetic acid injection. The acetic acid injection induced regional inflammation wound hyperpigmentation by recruiting pigment cells to the wound area. Later, we applied established platform to evaluate the effect of tilapia's collagen peptide mixtures, including demonstration on promoting skin wound healing and eliminating inflammatory response. Results showed that after treating TY001, one of the above fish collagen peptide mixtures, not only repair and proliferation were induced, but also death and apoptosis cells were cleared within cutaneous lesion. Moreover, inflammatory response was suppressed along with collagen mixture treatment. Finally, the TY001-associated signaling was validated by real time-PCR, and numbers of gene associated with tissue repair and vessel proliferation were induced. To sum up, our findings provided a permissive model that may apply to generate a platform for further screening on repair and restoration technology. In addition, the tilapia fish collagen peptide mixture we applied on our model has great potential on developing clinical application on wound healing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MicroRNA-182-5p attenuates cerebral ischemia-reperfusion injury by targeting Toll-like receptor 4

Highlights: • We investigated whether miRNA-mediated neuroprotection acts via TLR4. • We found that TLR4 is a direct target of miR-182-5p. • miR-182-5p is a candidate for cerebral ischemia-reperfusion injury treatment. Cerebral ischemia-reperfusion-induced microglial activation causes neuronal death through the release of inflammatory cytokines. Increasing evidence suggests that microRNAs (miRNAs) exert a neuroprotective effect by modulating the inflammatory process in cerebral ischemia-reperfusion injury. Furthermore, Toll-like receptor 4 (TLR4) is increasingly being considered to have a significant role in the regulation of inflammation. However, whether miRNAs mediate their neuroprotective effects by regulating TLR4-mediated inflammatory responses remains unknown. To explore this gap in the literature, we conducted both in vitro and in vivo experiments. In vitro: BV2 cells were activated by oxygen-glucose deprivation (OGD). TLR4 and inflammatory cytokine (TNF-a, IL-6, and IL-1β) transcription and translation expression levels were assessed using RT-PCR, ELISA, and western blot. BV2 cells were transfected with miR-182-5p mimics, inhibitors, siTLR4, or negative control (NC) using lipofectamine 2000 reagent. To confirm whether TLR4 is a direct target of miR-182-5p, we performed a luciferase reporter assay. In BV2 cells, we observed that OGD upregulated TLR4 expression, but downregulated miR-182-5p expression. We determined that miR-182-5p inhibited TLR4 by directly binding to its 3′-UTR. Furthermore, miR-182-5p suppressed the release of TNF-a, IL-6, and IL-1β. In vivo: A middle cerebral artery occlusion (MCAO) rat model was used to mimic cerebral ischemia-reperfusion. Iba1 and TLR4 double staining was used to demonstrate that the target of miR-182-5p in microglial cells, and the mediator of the anti-inflammatory effect, is TLR4. TTC staining was performed to evaluate the infarct volume. Compared to the animals treated with miR-182-5p NC and normal saline, rats treated with miR-182-5p mimics demonstrated significantly enhanced neurological functions. TTC staining results were consistent with neurological function test findings. In summary, our data suggested that miR-182-5p exhibits potential neuroprotective effects in the cerebral ischemia-reperfusion injury via the regulation of the TLR4-mediated inflammatory response.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Von Hippel-Lindau (VHL) protein antagonist, VH298, promotes functional activities of tendon-derived stem cells and accelerates healing of entheses in rats by inhibiting ubiquitination of hydroxy-HIF-1α

Enthesis is the region where a tendon attaches to a bone. It is a relatively vulnerable position, and in most cases surgical treatment is required upon rupture. The reconstructed enthesis is usually weaker compared to the original, and is prone to rupture again. Hypoxia-inducible factor-1 α (HIF-1α) is known to be involved in extensive activities in cells. It is inhibited under normoxic conditions, and undergoes two essential processes, hydroxylation and ubiquitination, the latter of which has been largely unexplored. Herein, we measured the levels of HIF-1α and hydroxy-HIF-1α in VH298-treated rat tendon-derived stem cells (TDSCs) by immunoblotting. We also detected the proliferation of TDSCs using CCK-8 assay and the mRNA levels of related genes by quantitative RT-PCR. The TDSCs were observed to be induced and the chondrogenic differentiation related genes were found to be enhanced. We also simulated in-vitro wounding in a scratch test and reconstructed the enthesis in a rat model of Achilles tendon by classical surgery followed by administration of phosphate buffer saline (PBS) injection or VH298 injection. We observed that HIF-1α and hydroxy-HIF-1α levels were increased in VH298-treated TDSCs in a dose- and time-dependent manner. Thirty micromolar VH298 could significantly increase cell proliferation, migration, and expression of collagen-1α, collagen-3α, decorin, tenomodulin, tenascin C genes, and chondrogenic differentiation-related genes, collagen-2α, SRY-box9, aggrecan. VH298-treated enthesis could tolerate more load-to-failure, had a better healing pattern, and activation of HIF signaling pathway. VH298 can thus enhance the functional activities of TDSCs, enhance their chondrogenic differentiation potential, and accelerate enthesis healing by inhibiting the ubiquitination of hydroxy-HIF-1α.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Cyclic stretch increases mitochondrial biogenesis in a cardiac cell line

Unlike stable and immobile cell line conditions, animal hearts contract and relax to pump blood throughout the body. Mitochondria play an essential role by producing biological energy molecules to maintain heart function. In this study, we assessed the effect of heart mimetic cyclic stretch on mitochondria in a cardiac cell line. To mimic the geometric and biomechanical conditions surrounding cells in vivo, cyclic stretching was performed on HL-1 murine cardiomyocytes seeded onto an elastic micropatterned substrate (10% elongation, 0.5 Hz, 4 h/day). Cell viability, semi-quantitative Q-PCR, and western blot analyses were performed in non-stimulated control and cyclic stretch stimulated HL-1 cell lines. Cyclic stretch significantly increased the expression of mitochondria biogenesis-related genes (TUFM, TFAM, ERRα, and PGC1-α) and mitochondria oxidative phosphorylation-related genes (PHB1 and CYTB). Western blot analysis confirmed that cyclic stretch increased protein levels of mitochondria biogenesis-related proteins (TFAM, and ERRα) and oxidative phosphorylation-related proteins (NDUFS1, UQCRC, and PHB1). Consequently, cyclic stretch increased mitochondrial mass and ATP production in treated cells. Our results suggest that cyclic stretch transcriptionally enhanced mitochondria biogenesis and oxidative phosphorylation without detrimental effects in a cultured cardiac cell line.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Iron chelator-induced up-regulation of Ndrg1 inhibits proliferation and EMT process by targeting Wnt/β-catenin pathway in colon cancer cells

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), as the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells. However, whether Dp44mT has anticancer effects in colon cancer cells is still unknown. Here, we investigated the antitumor action of Dp44mT in colon cancer and its underlying mechanisms, and the connections between Dp44mT and N-myc downstream-regulated genes 1(Ndrg1). We used cell viability, migration and invasion assay, flow cytometry, western blot and qRT-PCR to examine the anticancer effects of Dp44mT and Ndrg1. We found that Dp44mT suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells and over-expression of Ndrg1 also suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells. Dp44mT attenuated the TGF-β1-induced EMT in colon cancer cells, and Dp44mT could up-regulate Ndrg1 expression level. Overexpression of Ndrg1 attenuates the TGF-β1-induced EMT, Dp44mT and Ndrg1 suppressed EMT through activation of Wnt/β catenin signaling pathway. In conclusion, our data demonstrated that Dp44mT/Ndrg1 have effective anticancer capability in colon cancer cells and that may represent a promising treatment strategy for human colon cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The proteomic study of serially passaged human skin fibroblast cells uncovers down-regulation of the chromosome condensin complex proteins involved in replicative senescence

Dermal fibroblast is one of the major constitutive cells of skin and plays a central role in skin senescence. The replicative senescence of fibroblasts may cause skin aging, bad wound healing, skin diseases and even cancer. In this study, a label-free quantitative proteomic approach was employed to analyzing the serial passaged human skin fibroblast (CCD-1079Sk) cells, resulting in 3371 proteins identified. Of which, 280 proteins were significantly changed in early passage (6 passages, P6), middle passage (12 passages, P12) and late passage (21 passages, P21), with a time-dependent decrease or increase tendency. Bioinformatic analysis demonstrated that the chromosome condensin complex, including structural maintenance of chromosomes protein 2 (SMC2) and structural maintenance of chromosomes protein 4 (SMC4), were down-regulated in the serially passaged fibroblast cells. The qRT-PCR and Western Blot experiments confirmed that the expression of these two proteins were significantly down-regulated in a time-dependent manner in the subculture of human skin fibroblasts (HSFb cells). In summary, we used serially passaged human skin fibroblast cells coupled with quantitative proteomic approach to profile the protein expression pattern in the temporal progress of replicative senescence in HSFb cells and revealed that the down-regulation of the chromosome condensin complex subunits, such as SMC2 and SMC4, may play an important role in the fibroblast senescence.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Roles of SDF-1/CXCR4 axis in cartilage endplate stem cells mediated promotion of nucleus pulposus cells proliferation

Highlights: • NPCs proliferation was promoted by co-cultured CESCs and CESCs-CM in vitro. • SDF-1 and CXCR4 were expressed in a complementary pattern in CESCs and NPCs. • CESCs promoted NPCs proliferation partially via SDF-1/CXCR4 axis. • SDF-1/CXCR4 axis mainly activated ERK1/2 pathway rather than Akt pathway in NPCs. • CESCs upregulated TIMP1 expression in NPCs in a SDF-1/CXCR4 axis independent manner. Stem cells transplantation has shown considerable promise in intervertebral disc repair and low-back pain release. Cartilage endplate stem cells (CESCs) also showed potential for nucleus pulposus (NP) regeneration in a rabbit disc degeneration model, the precise mechanism was unclear. Here we investigated the effects of CESCs on NP cells (NPCs) proliferation and the mechanism in vitro. CESCs and NPCs were isolated from surgical specimens of degenerative human lumbar disc. NPCs were co-cultured with CESCs at a 1:1 ratio or cultured in CESCs conditioned medium (CESCs-CM). NPCs proliferation was evaluated by Ki-67 staining, CCK-8 assay and cell cycle analysis. Gene expressions were detected by qRT-PCR and activation of Akt and ERK1/2 was detected by western blot. CXCR4 antagonist AMD3100 was used to block SDF-1/CXCR4 axis. ERK1/2 and Akt inhibitors were used to block Akt and ERK1/2 activation. Results showed that NPCs proliferation was promoted by direct-contact co-culturing with CESCs as well as culturing in CESCs-CM. SDF-1 expression level in CESCs was significantly higher than that in NPCs, while CXCR4 was the opposite. Promotion of NPCs proliferation mediated by CESCs-CM could be partially attenuated by AMD3100. CESCs-CM activated both Akt and ERK1/2 in NPCs, while rhSDF-1 scarcely activated Akt but obviously activated ERK1/2. Akt and ERK1/2 inhibitors could partially inhibited CESCs-CM mediated promotion of NPCs proliferation and showed cumulative effect, while ERK1/2 inhibitor and AMD3100 could significantly abrogate SDF-1 mediated promotion of NPCs proliferation. Our results suggested that CESCs might promote NPCs proliferation in a paracrine pathway, which was partially mediated by SDF-1/CXCR4 axis via ERK1/2 signaling transduction pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Introducing nano-scale quantitative polymerase chain reaction

Highlights: • qPCR remain the gold-standard-method for measuring and validating gene expression levels and alterations. • qPCR reaction volume can be significantly reduced. • 1 μL versus 10 μL total reaction volumes gave comparable results. • The reduction in reaction volume can greatly reduce sample use and reagent cost. We show that the quantitative PCR reaction volume can be reduced significantly compared to the standard procedures, without compromising data quality. By analyzing dilution series (10{sup 0}–10{sup −7}) we found that measurement in 1 μl reaction volume indeed gave valid results comparable to 10 μL, when using a routine pipetting robot. This may enable a significant cost reduction through cutback of both biological material as well as chemical reagents.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Transforming growth factor β1-induced collagen production in myofibroblasts is mediated by reactive oxygen species derived from NADPH oxidase 4

Highlights: • Intestinal fibrosis with stricture is severe complication of Crohn's disease. • TGF-β1-induced collagen production in intestinal myofibroblast is dependent on NOX4. • NOX inhibitor significantly diminished TGF-β1-induced ROS and collagen production. • NOX4 may constitute a therapeutic target in fibrotic disorders among Crohn's disease. Intestinal fibrosis with stricture formation is a severe complication of Crohn's disease (CD). Though new therapeutic targets to enable the prevention or treatment of intestinal fibrosis are needed, markers of this condition and the basic mechanisms responsible have not been established. NADPH oxidase (NOX) 4 has already been reported to play a key role in models of fibrogenesis, including that of the lung. However, its importance in intestinal fibrogenesis remains unclear. In this study, we examined the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with transforming growth factor (TGF)-β1. Using LmcMF cells, an intestinal subepithelial myofibroblast (ISEMF) line, we first examined the induction of collagen production by TGF-β1. Subsequently, we investigated the role of NOX4 in TGF-β1-induced collagen I production in these cells using SB525334 (an SMAD2/3 inhibitor), diphenyleneiodonium (an NOX inhibitor), and Nox4 small interfering RNA (siRNA). Production of collagen was assessed with Sirius red staining, and Nox4 expression was measured by quantitative real-time PCR. Reactive oxygen species (ROS) production was determined using DCFDA and fluorescent microscopy. We observed that TGF-β1 induced collagen production via NOX4 activation and ROS generation in LmcMF cells. Nox4 siRNA and inhibitors of TGF-β1 receptor and NOX significantly reduced TGF-β1-induced ROS and collagen production. Thus, in the present study, we revealed that collagen production in ISEMFs is induced via an NOX4-dependent pathway. This work supports a function for NOX4 in intestinal fibrogenesis and identifies it as a potential therapeutic target in recalcitrant fibrotic disorders of CD patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MicroRNA-503 regulates hypoxia-induced cardiomyocytes apoptosis through PI3K/Akt pathway by targeting IGF-1R

Highlights: • Hypoxia induces miR-503 expression. • Mir-503 promoted cell apoptosis and mitochondrial impairment. • MiR-503 directly target IGF-1R in H9c2 cells. • The biological activities of MiR-503 were abolished by siIGF-1R or LY294002. Coronary heart disease is the second highest specific cause of death. H9c2 cardiomyocytes were subjected to hypoxia (1% O{sub 2}) for 0, 6, 12, 24 and 48 h. Cell apoptosis and the activity of caspase3/7 was detected using ELISA; western blot was applied to determine the cleaved-caspase3 (c-caspase3), cleaved-PARP (c-PARP) and cytochrome C (Cyto C) expression after the inhibitor negative control (in-NC), miR-503 inhibitor, mimic negative control (mi-NC) and miR-503 mimic were transfected into cells for 48 h. Moreover, flow cytometry was applied to evaluate mitochondrial membrane potential. In addition, luciferase reporter gene assay was used for detection the relationship between miR-503 and insulin-like growth-factor-1 receptor (IGF-1R). Real-time PCR showed microRNA-503 (miR-503) was elevated in a time-dependent manner under hypoxia. MiR-503 inhibition prevented cell apoptosis and reduced caspase3/7 activity and the expression of c-caspase3 and c-PARP, prevented mitochondrial membrane potential collapse and reduced the cyto C level in cytosol. While, miR-503 overexpression showed a pro-apoptotic role and resulted in mitochondrial membrane potential loss. MiR-503 directly targets IGF-1R in H9c2 cardiomyocytes. The depletion of IGF-1R using a specific IGF-1R siRNA (siIGF-1R) abolished anti-apoptotic function of miR-503 inhibitor, and LY294002 showed a similar trend. In summary, miR-503 promoted cell apoptosis, caused mitochondrial membrane potential collapse and the emancipation of cyto C from mitochondrial through PI3K/Akt pathway via targeting IGF-1R in H9c2 cardiomyocytes.

60 APPLIED LIFE SCIENCES↗