Simulated Microgravity Inhibits the Proliferation of Chang Liver Cells by Attenuation of the Major Cell Cycle Regulators and Cytoskeletal Proteins

Simulated microgravity (SMG) induced the changes in cell proliferation and cytoskeleton organization, which plays an important factor in various cellular processes. The inhibition in cell cycle progression has been considered to be one of the main causes of proliferation inhibition in cells under SMG, but their mechanisms are still not fully understood. This study aimed to evaluate the effects of SMG on the proliferative ability and cytoskeleton changes of Chang Liver Cells (CCL-13). CCL-13 cells were induced SMG by 3D clinostat for 72 h, while the control group were treated in normal gravity at the same time. The results showed that SMG reduced CCL-13 cell proliferation by an increase in the number of CCL-13 cells in G0/G1 phase. This cell cycle phase arrest of CCL-13 cells was due to a downregulation of cell cycle-related proteins, such as cyclin A1 and A2, cyclin D1, and cyclin-dependent kinase 6 (Cdk6). SMG-exposed CCL-13 cells also exhibited a downregulation of α-tubulin 3 and β-actin which induced the cytoskeleton reorganization. These results suggested that the inhibited proliferation of SMG-exposed CCL-13 cells could be associate with the attenuation of major cell cycle regulators and main cytoskeletal proteins.     

Simulated Microgravity Increases the Permeability of HUVEC Monolayer through Up-Regulation of Rap1GAP and Decreased Rap2 Activation

Space microgravity condition has great physiological influence on astronauts’ health. The interaction of endothelial cells, which control vascular permeability and immune responses, is sensitive to mechanical stress. However, whether microgravity has significant effects on the physiological function of the endothelium has not been investigated. In order to address such a question, a clinostat-based culture model with a HUVEC monolayer being inside the culture vessel under the simulated microgravity (SMG) was established. The transmittance of FITC-tagged dextran was used to estimate the change of integrity of the adherens junction of the HUVEC monolayer. Firstly, we found that the permeability of the HUVEC monolayer was largely increased after SMG treatment. To elucidate the mechanism of the increased permeability of the HUVEC monolayer under SMG, the levels of total expression and activated protein levels of Rap1 and Rap2 in HUVEC cells, which regulate the adherens junction of endothelial cells, were detected by WB and GST pull-down after SMG. As the activation of both Rap1 and Rap2 was significantly decreased under SMG, the expression of Rap1GEF1 (C3G) and Rap1GAP in HUVECs, which regulate the activation of them, was further determined. The results indicate that both C3G and Rap1GAP showed a time-dependent increase with the expression of Rap1GAP being dominant at 48 h after SMG. The down-regulation of the expression of junctional proteins, VE-cadherin and β-catenin, in HUVEC cells was also confirmed by WB and immunofluorescence after SMG. To clarify whether up-regulation of Rap1GAP is necessary for the increased permeability of the HUVEC monolayer after SMG, the expression of Rap1GAP was knocked down by Rap1GAP-shRNA, and the change of permeability of the HUVEC monolayer was detected. The results indicate that knock-down of Rap1GAP reduced SMG-induced leaking of the HUVEC monolayer in a time-dependent manner. In total, our results indicate that the Rap1GAP-Rap signal axis was necessary for the increased permeability of the HUVEC monolayer along with the down-regulation of junctional molecules including VE-cadherin and β-catenin.     

Activation of Focal Adhesion Kinase Restores Simulated Microgravity-Induced Inhibition of Osteoblast Differentiation via Wnt/Β-Catenin Pathway

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/β-catenin pathway. However, the mechanism by which SMG alters the Wnt/β-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/β-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/β-catenin and Wnt/β-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/β-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients.     

SMG1 Acts as a Novel Potential Tumor Suppressor with Epigenetic Inactivation in Acute Myeloid Leukemia

Suppressor with morphogenetic effect on genitalia family member (SMG1) belongs to a family of phosphoinositide 3-kinase-related kinases and is the main kinase involved in nonsense-mediated mRNA decay. Recently, SMG1 was suggested as a novel potential tumor suppressor gene, particularly in hypoxic tumors. To investigate the function of SMG1 in acute myeloid leukemia (AML), we performed methylation-specific polymerase chain reaction and found that SMG1 was hypermethylated in the promoter region. SMG1 hypermethylation was found in 66% (33/50) of AML samples compared with none (0/14) of the normal controls. SMG1 mRNA was down-regulated in AML patients with hypermethylation status whereas it was readily expressed in patients without methylation. Moreover, treatment of AML cells with demethylating agent 5-aza-2''-deoxycytidine (decitabine) inhibited AML cell growth and induced apoptosis by reversing SMG1 methylation status and restoring SMG1 expression. On the other hand, knockdown of SMG1 by RNA interference inhibited apoptosis. We also found that mTOR expression level was negatively correlated to SMG1 expression in AML patients which indicated that SMG1 and mTOR maybe act antagonistically to regulate AML cell growth. In conclusion, our results indicate that SMG1 acts as a potential tumor suppressor with epigenetic regulation in AML.     

Physosmotic Induction of Chondrogenic Maturation Is TGF-β Dependent and Enhanced by Calcineurin Inhibitor FK506

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.     

The Influence of Angiotensin Peptides on Survival and Motility of Human High-Grade Serous Ovarian Cancer Cells in Serum Starvation Conditions

High-grade serous ovarian carcinoma (HGSOC) is the most frequent and malignant form of ovarian cancer. A local renin–angiotensin system (RAS) has been found in the ovary, and changes in selected components of this system were observed in pathological states and also in ovarian cancer. In the present study, we examined the effect of three peptides, Ang-(1-7), Ang-(1-9) and Ang-(3-7), on proliferation and motility of the OVPA8 cell line, a new well-defined and preclinical model of HGSOC. We confirmed the presence of mRNA for all angiotensin receptors in the tested cells. Furthermore, our findings indicate that all tested angiotensin peptides increased the metabolic serum in the medium by activation of cell defense mechanisms such as nuclear factor kappaB signaling pathway andapoptosis. Moreover, tested angiotensin peptides intensified serum starvation-induced cell cycle arrest at the G0/G1 phase. In the case of Ang-(3-7), a significant decrease in the number of Ki67 positive cells (Ki67+) and reduced percentage of activated ERK1/2 levels in ovarian cancer cells were additionally reported. The angiotensin-induced effect of the accumulation of cells in the G0/G1 phase was not observed in OVPA8 cells growing on the medium with 10% FBS. Moreover, in the case of Ang-(3-7), the tendency was quite the opposite. Ang-(1-7) but not Ang-(1-9) or Ang-(3-7) increased the mobility of reluctant-to-migrate OVAP8 cells cultured in the serum-free medium. In any cases, the changes in the expression of VIM and HIF1A gene, associated with epithelial–mesenchymal transition (EMT), were not observed. In conclusion, we speculate that the adaptation to starvation in nutrient-deprived tumors can be modulated by peptides from the renin–angiotensin system. The influence of angiotensin peptides on cancer cells is highly dependent on the availability of growth factors and nutrients.     

Simulated Microgravity Inhibits Rodent Dermal Fibroblastic Differentiation of Mesenchymal Stem Cells by Suppressing ERK/β-Catenin Signaling Pathway

Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into dermal fibroblasts to participate in skin-repairing. However, at present, little is known about how microgravity affects dermal fibroblastic differentiation of BMSCs in space. The aim of this study was to investigate the effect of simulated microgravity (SMG) on the differentiation of BMSCs into dermal fibroblasts and the related molecular mechanism. Here, using a 2D-clinostat device to simulate microgravity, we found that SMG inhibited the differentiation and suppressed the Wnt/β-catenin signaling and phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2). After upregulating the Wnt/β-catenin signaling with lithium chloride (LiCl) treatment, we found that the effect of the differentiation was restored. Moreover, the Wnt/β-catenin signaling was upregulated when phosphorylation of ERK1/2 was activated with tert-Butylhydroquinone (tBHQ) treatment. Taken together, our findings suggest that SMG inhibits dermal fibroblastic differentiation of BMSCs by suppressing ERK/β-catenin signaling pathway, inferring that ERK/β-catenin signaling pathway may act as a potential intervention target for repairing skin injury under microgravity conditions.     

Impact of Musashi-1 and Musashi-2 Double Knockdown on Notch Signaling and the Pathogenesis of Endometriosis

The stem cell marker and RNA-binding protein Musashi-1 is overexpressed in endometriosis. Musashi-1-siRNA knockdown in Ishikawa cells altered the expression of stem cell related genes, such as OCT-4. To investigate the role of both human Musashi homologues (MSI-1 and MSI-2) in the pathogenesis of endometriosis, immortalized endometriotic 12-Z cells and primary endometriotic stroma cells were treated with Musashi-1- and Musashi-2-siRNA. Subsequently, the impact on cell proliferation, cell apoptosis, cell necrosis, spheroid formation, stem cell phenotype and the Notch signaling pathway was studied in vitro. Using the ENDOMET Turku Endometriosis database, the gene expression of stem cell markers and Notch signaling pathway constituents were analyzed according to localization of the endometriosis lesions. The database analysis demonstrated that expression of Musashi and Notch pathway-related genes are dysregulated in patients with endometriosis. Musashi-1/2-double-knockdown increased apoptosis and necrosis and reduced stem cell gene expression, cell proliferation, and the formation of spheroids. Musashi silencing increased the expression of the anti-proliferation mediator p21. Our findings suggest the therapeutic potential of targeting the Musashi–Notch axis. We conclude that the Musashi genes have an impact on Notch signaling and the pathogenesis of endometriosis through the downregulation of proliferation, stemness characteristics and the upregulation of apoptosis, necrosis and of the cell cycle regulator p21.     

Rac1/Wave2/Arp3 Pathway Mediates Rat Blood-Brain Barrier Dysfunction under Simulated Microgravity Based on Proteomics Strategy

The blood-brain barrier (BBB) is critical to maintaining central nervous system (CNS) homeostasis. However, the effects of microgravity (MG) on the BBB remain unclear. This study aimed to investigate the influence of simulated MG (SMG) on the BBB and explore its potential mechanism using a proteomic approach. Rats were tail-suspended to simulate MG for 21 days. SMG could disrupt the BBB, including increased oxidative stress levels, proinflammatory cytokine levels, and permeability, damaged BBB ultrastructure, and downregulated tight junctions (TJs) and adherens junctions (AJs) protein expression in the rat brain. A total of 554 differentially expressed proteins (DEPs) induced by SMG were determined based on the label-free quantitative proteomic strategy. The bioinformatics analysis suggested that DEPs were mainly enriched in regulating the cell–cell junction and cell–extracellular matrix biological pathways. The inhibited Ras-related C3 botulinum toxin substrate 1 (Rac1)/Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 (Wave2)/actin-related protein 3 (Arp3) pathway and the decreased ratio of filamentous actin (F-actin) to globular actin contributed to BBB dysfunction induced by SMG. In the human brain microvascular endothelial cell (HBMECs), SMG increased the oxidative stress levels and proinflammatory cytokine levels, promoted apoptosis, and arrested the cell cycle phase. Expression of TJs and AJs proteins were downregulated and the distribution of F-actin was altered in SMG-treated HBMECs. The key role of the Rac1/Wave2/Arp3 pathway in BBB dysfunction was confirmed in HBMECs with a specific Rac1 agonist. This study demonstrated that SMG induced BBB dysfunction and revealed that Rac1/Wave2/Arp3 could be a potential signaling pathway responsible for BBB disruption under SMG. These results might shed a novel light on maintaining astronaut CNS homeostasis during space travel.     

Subtype of Neuroblastoma Cells with High KIT Expression Are Dependent on KIT and Its Knockdown Induces Compensatory Activation of Pro-Survival Signaling

Neuroblastoma (NB) is a pediatric cancer with high clinical and molecular heterogeneity, and patients with high-risk tumors have limited treatment options. Receptor tyrosine kinase KIT has been identified as a potential marker of high-risk NB and a promising target for NB treatment. We investigated 19,145 tumor RNA expression and molecular pathway activation profiles for 20 cancer types and detected relatively high levels of KIT expression in NB. Increased KIT expression was associated with activation of cell survival pathways, downregulated apoptosis induction, and cell cycle checkpoint control pathways. KIT knockdown with shRNA encoded by lentiviral vectors in SH-SY5Y cells led to reduced cell proliferation and apoptosis induction up to 50%. Our data suggest that apoptosis induction was caused by mitotic catastrophe, and there was a 2-fold decrease in percentage of G2-M cell cycle phase after KIT knockdown. We found that KIT knockdown in NB cells leads to strong upregulation of other pro-survival growth factor signaling cascades such as EPO, NGF, IL-6, and IGF-1 pathways. NGF, IGF-1 and EPO were able to increase cell proliferation in KIT-depleted cells in an ERK1/2-dependent manner. Overall, we show that KIT is a promising therapeutic target in NB, although such therapy efficiency could be impeded by growth factor signaling activation.     

人参皂苷单体Rh2(S亚型)对人急性髓系白血病细胞株KG1α增殖、凋亡的影响及其机制

目的探讨人参皂苷单体Rh2(S亚型)对人急性髓系白血病细胞株KG1α增殖、凋亡的影响及其机制。方法取对数生长期的KG1α细胞,分为两组:空白对照组(常规培养)和人参皂苷单体Rh2(S)组,采用MTT法检测细胞的增殖活力,倒置显微镜观察细胞的形态及数量,透射电镜观察细胞的超微结构,流式细胞仪检测细胞周期的分布,Real-time PCR检测细胞中β-catenin基因mRNA水平,免疫细胞化学法检测细胞中β-catenin、TCF4、CyclinD1蛋白的定位及表达,Western blot检测细胞中β-catenin、TCF4、CyclinD1蛋白的表达水平。结果人参皂苷单体Rh2(S)对KG1α细胞的增殖具有明显的抑制作用,且呈浓度和时间依赖性。与空白对照组比较,Rh2(S)作用48 h后,可见KG1α细胞分散生长,数量明显减少;可见数量不等,程度不同的凋亡细胞;G0/G1期细胞比例明显上升(P<0.05),G2+M和S期细胞比例明显下降(P<0.05);细胞中β-catenin基因mRNA水平明显降低(P<0.01)。β-catenin、TCF4、CyclinD1蛋白的表达水平明显降低(P<0.05或P<0.01)。结论人参皂苷单体Rh2(S亚型)对KG1α细胞具有明显的增殖抑制作用,其机制可能抑制Wnt/β-catenin/TCF4/CyclinD1信号通路,使细胞周期阻滞,诱导细胞凋亡。     

Notch2胞内段基因过表达对K562细胞增殖的影响及其机制

目的探讨外源性Notch2胞内段基因过表达对慢性粒细胞白血病(Chronic myeloid leukemia,CML)细胞株K562增殖的影响及其机制。方法将携带Notch2胞内段(ICN2)基因的质粒转染K562细胞,MTT法检测细胞的增殖;流式细胞仪检测细胞的细胞周期分布;RT-PCR检测Notch2基因全长mRNA的转录,Western blot检测Notch2蛋白的表达;RT-PCR检测Notch通路下游靶基因Hes1、Hey1及细胞增殖、凋亡相关基因numb、Bcl-2、NF-κB和TGF-β1的mRNA转录水平。结果与未转染组相比,转染组K562细胞数量减少,增殖显著受抑(P<0.01);转染48 h后的K562细胞G1期细胞比例显著增多(P<0.01),S期细胞比例显著减少(P<0.01);Notch2基因mRNA及下游靶基因Hes1和Hey1 mRNA转录水平均明显增强(P<0.01),Notch2蛋白表达量增加(P<0.01);numb基因mRNA转录水平无变化;Bcl-2表达下调,NF-κB和TGF-β1表达上调。结论Notch2胞内段基因过表达可抑制K562细胞增殖,其机制可能是通过上调TGF-β1和NF-κB基因表达及下调Bcl-2基因表达,将细胞阻滞于G1期来实现的。     

Different Expression Levels of DLK2 Inhibit NOTCH Signaling and Inversely Modulate MDA-MB-231 Breast Cancer Tumor Growth In Vivo

NOTCH signaling is implicated in the development of breast cancer tumors. DLK2, a non-canonical inhibitor of NOTCH signaling, was previously shown to be involved in skin and breast cancer. In this work, we studied whether different levels of DLK2 expression influenced the breast cancer characteristics of MDA-MB-231 cells. We found that DLK2 overexpression inhibited NOTCH activation in a dose-dependent manner. Moreover, depending on the level of inhibition of NOTCH1 activation generated by different levels of DLK2 expression, cell proliferation, cell cycle dynamics, cell apoptosis, cell migration, and tumor growth in vivo were affected in opposite directions. Low levels of DLK2 expression produced a slight inhibition of NOTCH1 activation, and enhanced MDA-MB-231 cell invasion in vitro and cell proliferation both in vitro and in vivo. In contrast, MDA-MB-231 cells expressing elevated levels of DLK2 showed a strong inhibition of NOTCH1 activation, decreased cell proliferation, increased cell apoptosis, and were unable to generate tumors in vivo. In addition, DLK2 expression levels also affected some members of other cell signaling pathways implicated in cancer, such as ERK1/2 MAPK, AKT, and rpS6 kinases. Our data support an important role of DLK2 as a protein that can finely regulate NOTCH signaling and affect the tumor properties and growth dynamics of MDA-MB-231 breast cancer cells.     

Porcine G0/G1 Switch Gene 2 (G0S2) Expression is Regulated During Adipogenesis and Short-Term In-Vivo Nutritional Interventions

Adipose triglyceride lipase (ATGL), catalyzing the initial step of hydrolysis of triacylglycerol (TAG) in adipocytes, has been known to be inhibited by G0/G1 switch gene 2 (G0S2). In this study, we report the porcine G0S2 cDNA and amino acid sequences as well as the expression level of porcine G0S2. The porcine G0S2 mRNA was abundantly expressed in adipose tissue and liver among various tissues. In adipose tissue, porcine G0S2 expression was 16-fold higher in the fat cell fraction than the stromal vascular fraction. The G0S2 level increased significantly during adipogenesis in vitro and in vivo. These data indicate that G0S2 expression is closely associated with lipid accumulation and adipogenesis. Considering G0S2 as an inhibitor of cell proliferation, the relatively low levels of G0S2 in preadipocytes and adipose tissues of fetal and neonatal pigs compared to adipocytes and adipose tissues of adult pigs may allow the fast cell proliferation rates. Further studies showed that a short-term 24-h fast down-regulated G0S2 expression and increased ATGL expression in adipose tissue; however, a long-term calorie restriction for 8 days had no influence on the level of G0S2 but increased ATGL expression. Therefore, porcine G0S2, which is both a negative regulator of ATGL-mediated lipolysis and cell proliferation in adipose tissue, can be down-regulated in vivo by a short-term 24-h fast followed by increased ATGL-mediated lipolysis.     

FOXC1蛋白基因shRNA干扰质粒对胃癌SGC-7901细胞增殖的影响

目的探讨FOXC1蛋白基因RNA干扰质粒对胃癌SGC-7901细胞增殖的影响。方法采用RT-PCR及Westernblot法检测SGC-7901细胞和胃黏膜上皮细胞GES-1中FOXC1基因mRNA的转录水平和蛋白的表达水平。构建3个FOXC1基因shRNA真核表达质粒pshRNA-FOXC1a、pshRNA-FOXC1b、pshRNA-FOXC1c,分别转染SGC-7901细胞,并设pGenesil-1空质粒转染组和未转染的细胞对照组,RT-PCR及Western blot法分别检测转染细胞中FOXC1基因mRNA的转录水平和蛋白的表达水平;MTT法检测转染细胞的增殖活力;流式细胞术检测转染细胞的细胞周期。结果 SGC-7901细胞中FOXC1基因mRNA的转录水平及蛋白的表达水平均显著高于GES-1细胞(P<0.05)。pshRNA-FOXC1a组、pshRNA-FOXC1b组和pshRNA-FOXC1c组SGC-7901细胞中FOXC1基因mRNA的转录水平和蛋白的表达水平均显著低于pGenesil-1组和SGC-7901组(P<0.01);细胞增殖抑制率显著高于pGensil-1组(P<0.05)。处于G1、G2期的细胞比例显著高于SGC-7901组(P<0.01),S期细胞比例显著低于SGC-7901组(P<0.01)。结论成功构建了FOXC1蛋白基因shRNA真核表达质粒。FOXC1在胃癌细胞SGC-7901中呈高表达,抑制其表达后,细胞周期被阻滞在G1～G2期,细胞增殖受到抑制。     

A γ-Secretase Inhibitor Attenuates Cell Cycle Progression and Invasion in Human Oral Squamous Cell Carcinoma: An In Vitro Study

Notch signaling is associated with many human malignancies, including oral squamous cell carcinoma (OSCC). However, the exact function of Notch signaling in OSCC remains unclear. Here, we investigated the effect of Notch signaling inhibition using a γ-secretase inhibitor (DAPT) on OSCC behaviours in vitro. Bioinformatic analysis of public-available gene expression profiles revealed the dysregulation of the Notch signaling pathway in OSCC compared with normal tissues, indicating the role of Notch signaling in OSCC regulation. RNA sequencing analysis of DAPT-treated human OSCC cells revealed the dysregulation of genes related to cell cycle-related pathways. Blocking Notch signaling significantly inhibited cell proliferation. DAPT-induced G0/G1 cell cycle arrest induced cell apoptosis. Furthermore, cell migration and invasion were also reduced in DAPT-treated cells. These findings indicate that Notch signaling activation participates in OSCC regulation by promoting cell growth, cell cycle progression, cell migration, and invasion. These mechanisms could facilitate OSCC progression. These results imply the potential use of Notch signaling inhibitors as a candidate adjuvant treatment in OSCC patients.     

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.     

Apg-2基因沉默对BaF3-MIGR1及BaF3-P210细胞增殖的影响

目的探讨Apg-2基因沉默对pMIGR1空载体感染的BaF3-MIGR1细胞及稳定表达Bcr-Abl融合基因的BaF3-P210细胞增殖的影响,为进一步研究Apg-2在Bcr-Abl阳性的CML细胞中的作用奠定基础。方法针对Apg-2基因609～629、845～865和2 110～2 130核苷酸合成3条shRNA序列Hspa41、Hspa42、Hspa43,同时合成阴性对照序列HspaHK,电穿孔转染BaF3-MIGR1和BaF3-P210细胞,经G418筛选稳定抑制细胞株,RT-PCR和Western blot检测细胞中Apg-2基因mRNA的转录水平及蛋白的表达水平;Am-blue法检测细胞增殖;甲基纤维素法检测细胞克隆形成能力;流式细胞技术检测细胞周期的变化。结果转染的3条shRNA质粒中,shRNA-Hspa42的干扰效果最佳。Apg-2表达抑制后,BaF3-P210细胞的增殖与克隆形成能力均明显下降(P<0.05),处于G1期细胞的百分率明显升高(P<0.05),处于S期的细胞百分率明显下降(P<0.05);而BaF3-MIGR1细胞的增殖与克隆形成能力均明显增强(P<0.05),处于S期的细胞百分率明显升高(P<0.05)。结论干扰Apg-2基因的表达可抑制Bcr-Abl阳性细胞BaF3-P210的增殖,而对Bcr-Abl阴性细胞BaF3-MIGR1的增殖起促进作用。