We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
Arabidopsis/genetics* ; Arabidopsis Proteins/metabolism* ; Cloning, Molecular ; DNA, Complementary/metabolism* ; Flowers/physiology* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Homeodomain Proteins/metabolism* ; Lamiales/physiology* ; MicroRNAs/metabolism* ; Nuclear Proteins/metabolism* ; Plants, Genetically Modified/physiology* ; Plasmids/metabolism* ; Polymerase Chain Reaction ; Transgenes

Alpha-Ketoglutarate-Dependent Dioxygenase FTO ; genetics ; Body Mass Index ; Body Weight ; genetics ; physiology ; Female ; Genotype ; Homeodomain Proteins ; genetics ; Humans ; Male ; Obesity ; genetics ; metabolism ; Transcription Factors ; genetics

BACKGROUNDGastric cancer (GC) is one of the most prevalent malignancies in the world today, with a high mortality rate. CDX2 is a Drosophila caudal-related homeobox transcription factor that plays an important role in GC. Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) is an important tumor suppressor which is widely expressed in normal human tissues. The aim of the study was to determine the relationship and mechanism between CDX2 and PTEN in invasion and migration of GC cells.

METHODSpcDNA3-CDX2 plasmids were transfected into MGC-803 cells to up-regulate CDX2 protein, and small interfering RNA-CDX2 was transfected to down-regulate CDX2. The influence of CDX2 or PTEN on cell migration and invasion was measured by invasion, migration and wound healing assays. Western blotting assay and immunofluorescence were used to detect the expression of CDX2, PTEN, phosphorylation of Akt, E-cadherin and N-cadherin. Statistical significance was determined by one-way analysis of variance.

RESULTSThe results showed that CDX2 reduced the migration and invasion of GC cells (P < 0.05), and inhibited the activity of Akt through down-regulating PTEN expression (P < 0.05). CDX2 also restrained epithelial-mesenchymal transition of GC cells.

CONCLUSIONSCDX2 inhibited invasion and migration of GC cells by PTEN/Akt signaling pathway, and that may be used for potential therapeutic target.

CDX2 Transcription Factor ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Chromosomes, Human, Pair 10 ; genetics ; Epithelial-Mesenchymal Transition ; genetics ; physiology ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Microfilament Proteins ; genetics ; metabolism ; PTEN Phosphohydrolase ; genetics ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Tensins ; Wound Healing ; genetics ; physiology

OBJECTIVETo examine the transfection of Homeobox A13 (HOXA13) on epithelial-mesenchymal transition (EMT) and the expression of bone morphogenetic protein-7 (BMP-7) induced by albumin-overload in human kidney tubular epithelial cells (HKCs).

METHODSThe cultured HKCs were treated with 20 mg/mL human serum albumin (HSA) for 48 hours. Protein expression of cytokeratin (CK), vimentin and HOXA13 in the HKCs was assessed by Western blot. Protein expression of CK, vimentin, and BMP-7 was also detected in HKCs transfected with lipofectamine contained HOXA13 DNA.

RESULTSHSA induced EMT in HKCs, presented by decreased CK expression (P<0.01) and increased vimentin expression (P<0.01). The up-regulated expression of HOXA13 transfected by lipofectamine inhibited the level of EMT induced by HSA in HKCs (P<0.05). The decreased rate of BMP-7 protein expression induced by HSA was inhibited by over-expressed HOXA13 in HKCs (P<0.05).

CONCLUSIONSTransfection of HOXA13 in HKCs could inhibit the degree of EMT induced by albumin-overload, possibly by increasing BMP-7 expression.

Bone Morphogenetic Protein 7 ; genetics ; Cells, Cultured ; Epithelial Cells ; metabolism ; Epithelial-Mesenchymal Transition ; Homeodomain Proteins ; physiology ; Humans ; Keratins ; genetics ; Kidney Tubules ; metabolism ; Transfection ; Vimentin ; genetics

Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.
ARNTL Transcription Factors/physiology ; Animals ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism/physiology ; CLOCK Proteins/physiology ; Cholesterol/blood ; *Circadian Rhythm ; E-Box Elements ; Exons ; *Gene Expression Regulation ; Hep G2 Cells ; Homeodomain Proteins/*genetics/metabolism/physiology ; Homeostasis ; Humans ; Liver/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; *Promoter Regions, Genetic ; Receptors, LDL/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism/physiology ; Transcription, Genetic

OBJECTIVETo investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro.

METHODSThe hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS(1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups.

CONCLUSIONhAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.

Amnion ; cytology ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; Flow Cytometry ; Homeodomain Proteins ; metabolism ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; RNA, Messenger ; genetics ; Trans-Activators ; metabolism ; beta 2-Microglobulin ; metabolism

Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 μmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.
Animals ; Antioxidants ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Curcumin ; pharmacology ; Cyclic GMP ; metabolism ; Embryoid Bodies ; drug effects ; metabolism ; physiology ; Enzyme Activators ; pharmacology ; Gene Expression ; drug effects ; Guanylate Cyclase ; genetics ; metabolism ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Mice ; Myosin Heavy Chains ; genetics ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Donors ; pharmacology ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Nitroso Compounds ; pharmacology ; Pyrazoles ; pharmacology ; Pyridines ; pharmacology ; Second Messenger Systems ; Transcription Factors ; genetics ; metabolism ; Troponin ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

BACKGROUNDDelta-like 4 (DLL4) is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization (CNV) is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV.

METHODSWe utilized chemical hypoxia induced by 200 µmol/L CoCl2 to observe the expression of DLL4 in choroid-retinal endothelial cells (RF/6A cells), which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA (siRNA), mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used: one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated.

RESULTSThe results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplex1 targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplex1, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA (P < 0.05).

CONCLUSIONSDLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.

Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Blotting, Western ; Cell Cycle ; genetics ; physiology ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Line ; Cell Movement ; genetics ; physiology ; Cell Proliferation ; Choroidal Neovascularization ; Endothelial Cells ; cytology ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; RNA, Small Interfering ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factor HES-1

Cardiac lymphatic system in the remodeling after acute myocardial infarction (AMI) has been overlooked. We wanted to investigate the role of bone marrow-derived endothelial progenitor cells (EPCs) and their contribution to lymphatic distribution in myocardial remodeling after AMI. Mouse (C57bl/6J) MI models were created by ligation of the left anterior descending coronary artery and were treated with phosphate buffered saline (PBS) or EPCs. Real-time RT-PCR with 2- to 4-week myocardial tissue samples revealed that lymphangiogenetic factors such as vascular endothelial growth factor (VEGF)-C (8.5 fold, P < 0.05), VEGF-D (6.1 fold, P < 0.05), Lyve-1 (15 fold, P < 0.05), and Prox-1 (11 fold, P < 0.05) were expressed at significantly higher levels in the PBS group than the EPC group. The PBS group also showed a significantly higher density of lymphatic vessels in the peri-infarction area. Echocardiography showed that from 2 weeks after the treatment, left ventricle (LV) dimensions at both systole and diastole were significantly smaller in the EPC group than in the PBS group (P < 0.01) and LV fractional shortening was higher in the EPC group accordingly (P < 0.01). Lymphangiogenic markers increased in a mouse MI model. EPC transplantation decreased lymphangiogenesis and adverse ventricular remodeling after AMI. These novel findings suggest that new lymphatic vessels may be formed in severely damaged myocardium, and may be involved in adverse myocardial remodeling after AMI.
Animals ; Cell Transplantation ; Endothelial Cells/*cytology ; Homeodomain Proteins/genetics/metabolism ; Immunohistochemistry ; Lymphangiogenesis/genetics/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Myocardial Infarction/metabolism/physiopathology/*therapy ; Real-Time Polymerase Chain Reaction ; *Stem Cell Transplantation ; Tumor Suppressor Proteins/genetics/metabolism ; Vascular Endothelial Growth Factor A/genetics/metabolism ; Vascular Endothelial Growth Factor D/genetics/metabolism

OBJECTIVETo establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study.

METHODSAn endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry.

RESULTSDigital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01).

CONCLUSIONSThe endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

Animals ; Cell Line, Tumor ; Cells, Cultured ; Chorioallantoic Membrane ; blood supply ; Diagnostic Imaging ; methods ; Homeodomain Proteins ; genetics ; metabolism ; physiology ; Human Umbilical Vein Endothelial Cells ; Humans ; Neovascularization, Pathologic ; Neovascularization, Physiologic ; Software ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transcription Factors ; genetics ; metabolism ; physiology ; Transfection