Mesenchymal stem cell (MSC)-based therapy has emerged as a promising treatment for spinal cord injury (SCI), but improving the neurogenic potential of MSCs remains a challenge. Mixed lineage leukemia 1 (MLL1), an H3K4me3 methyltransferases, plays a critical role in regulating lineage-specific gene expression and influences neurogenesis. In this study, we investigated the role and mechanism of MLL1 in the neurogenesis of stem cells from apical papilla (SCAPs). We examined the expression of neural markers, and the nerve repair and regeneration ability of SCAPs using dynamic changes in neuron-like cells, immunofluorescence staining, and a SCI model. We employed a coimmunoprecipitation (Co-IP) assay, real-time RT-PCR, microarray analysis, and chromatin immunoprecipitation (ChIP) assay to investigate the molecular mechanism. The results showed that MLL1 knock-down increased the expression of neural markers, including neurogenic differentiation factor (NeuroD), neural cell adhesion molecule (NCAM), tyrosine hydroxylase (TH), βIII-tubulin and Nestin, and promoted neuron-like cell formation in SCAPs. In vivo, a transplantation experiment showed that depletion of MLL 1 in SCAPs can restore motor function in a rat SCI model. MLL1 can combine with WD repeat domain 5 (WDR5) and WDR5 inhibit the expression of neural markers in SCAPs. MLL1 regulates Hairy and enhancer of split 1 (HES1) expression by directly binds to HES1 promoters via regulating H3K4me3 methylation by interacting with WDR5. Additionally, HES1 enhances the expression of neural markers in SCAPs. Our findings demonstrate that MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1. These results provide a potential therapeutic target for promoting the recovery of motor function in SCI patients.
Animals ; Humans ; Rats ; Cell Differentiation ; Intracellular Signaling Peptides and Proteins/therapeutic use* ; Leukemia/metabolism* ; Mesenchymal Stem Cells ; Neurogenesis ; Stem Cells ; Transcription Factor HES-1/metabolism*

OBJECTIVETo observe the effect of activation of Notch1 signaling pathway by Notch intracellular domain (NICD) plasmid transfection on pancreatic cancer cell proliferation and explore the underlying mechanism.

METHODSThe transfection rates were observed under microscope with fluorescence stimulation, and mRNA expression levels of Hes1 were detected by real-time PCR. Cell proliferation changes were evaluated by CCK-8 after NICD and control plasmid transfection in pancreatic cancer cells. Caspase 3 activity was examined using a caspase 3 detection kit.

RESULTSThe transfection rates of NICD plasmid were up to 80% by fluorescence stimulation observation. Hes1 expression was significantly increased after NICD plasmid transfection, suggesting the activation of Notch1 signaling pathway. NICD plasmid transfection significantly promoted cancer cell proliferation compared to control plasmid transfeciton. The activities of caspase 3 were obviously decreased after NICD plasmid transfection in 3 pancreatic cancer cell lines.

CONCLUSIONActivation of Notch1 signaling pathway by NICD plasmid transfection can promote the proliferation of pancreatic cancer cells by inhibiting the apoptosis pathway.

Apoptosis ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Homeodomain Proteins ; metabolism ; Humans ; Pancreatic Neoplasms ; metabolism ; pathology ; Plasmids ; Receptor, Notch1 ; genetics ; metabolism ; Signal Transduction ; Transcription Factor HES-1 ; Transfection

OBJECTIVETo detect the expression of Math1, Hes1 and Hes5 in greater epithelial ridge (GER) cells of rat cochlear and explore their influence on hair cell differentiation.

METHODSPostnatal day 0 (P0), day 1 (P1) , day 3 (P3) day 4 (P4) and day 5 (P5) rat cochlear were dissected respectively and then pure GER cells were separated by a combinatorial approach of attachment and mechanical separation. The total RNA of GER cells was extracted by Trizol one step method and the expression of Math1, Hes1 and Hes5 in GER cells was detected with reverse transcription polymerase chain reaction.

RESULTSMath1 was expressed in P0 - P5 rat GER cells and Hes1 was expressed only in PO - P3 rat GER cells, while there was no expression of Hes5 in P0 - P5 rat GER cells.

CONCLUSIONSProbably only when the expression of Math1 reaches a certain level can it induce GER cells to differentiate into hair cells. Meanwhile this process might controlled by Hes1 to some extent.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Differentiation ; Cochlea ; cytology ; metabolism ; Epithelial Cells ; metabolism ; Gene Expression Regulation, Developmental ; Hair Cells, Auditory ; cytology ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transcription Factor HES-1

The aim of this study was to explore the effect of DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) on proliferation in vitro of human multiple myeloma cell line RPMI8226 and its underlying mechanism. The proliferation of RPMI8226 cells was detected by CCK-8 method; flow cytometry was employed to assay the cell apoptosis rate;the expressions of Notch1 and Hes1 proteins were detected by Western blot. The results indicated that the proliferation of human RPMI8226 cells significantly decreased after treatment with DAPT 0.5 - 5.0 µmol/L for 24 - 72 h (P < 0.05) in a concentration- and time-dependent manner. DAPT significantly induced apoptosis of RPMI8226 cells (P < 0.05). The expressions of Notch1 and Hes1 proteins were gradually downregulated with the increase of DAPT concentration. It is concluded that the DAPT can inhibit the proliferation of RPMI8226 cells, which may be related with the down-regulation of the protein expression of Notchl and Hes1.
Apoptosis ; drug effects ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dipeptides ; pharmacology ; Homeodomain Proteins ; metabolism ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Receptor, Notch1 ; metabolism ; Transcription Factor HES-1

OBJECTIVETo study the inhibitory effect of (-)-epigallocatechin-3-gallate (EGCG) on cancer cells line HCT-8 and HT29 and its influence on the expression of HES1 and JAG1.

METHODSColorectal cancer cells line HCT-8 and HT29 were cultured in vitro and treated with different concentrations of EGCG(10 mg/L, 20 mg/L, 35 mg/L). The inhibition of proliferation was tested by MTT analysis. Influence of EGCG on the cell apoptosis and cell cycle of HCT-8 and HT29 were detected with flow cytometry, and gene expression of HCT-8 and HT29 after EGCG treatment with real-time polymerase chain reaction.

RESULTSEGCG affected the proliferation and apoptosis of HCT-8 and HT29. The inhibition rates of the three different concentrations of EGCG were(28.894±5.076)%, (34.903±1.794)%, and (39.028±0.105)% on HCT-8, and (14.682±4.244)%, (22.429±3.847)%, and (29.840±5.076)% on HT29. EGCG caused G(2)/M phase arrest and M phase transition in HCT-8 cell line, and S phase arrest and G2 phase transition in HT29 cell line. EGCG down-regulated HES1 gene expression in both cell lines, however, the differences were not statistically significant(both P>0.05). EGCG upregulated JAG1 gene expression in both cell lines, however only the difference in HCT-8 was statistically significant(0.201±0.018 vs. 0.440±0.077, P=0.029).

CONCLUSIONSEGCG can significantly inhibit the proliferation of HT29 cells and HCT-8 cells by changing cell cycle and inducing cell apoptosis. The mechanism may be related to the upregulation of JAG1 gene expression.

Apoptosis ; drug effects ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Calcium-Binding Proteins ; metabolism ; Catechin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; pathology ; Flow Cytometry ; HT29 Cells ; Homeodomain Proteins ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; metabolism ; Serrate-Jagged Proteins ; Transcription Factor HES-1

OBJECTIVETo investigate the status of Notch signaling pathway in small cell lung cancer (SCLC).

METHODSExpression plasmids of pEFBOS-NIC-MYC and pEFBOS-neo were transfected into NCI-H446 cells. Stably transfected cell lines were selected and their growth rates were examined by MTT method. Expression of downstream genes along the Notch signaling pathway were studied by RT-PCR. Protein expression of euroendocrine markers of CgA and NSE were detected by Western blot analysis and immunocytochemistry.

RESULTSThe expression of HES1 was increased in the pEFBOS-NIC-MYC group, but the expression of hASH in the pEFBOS-NIC-MYC group was decreased significantly. The transfected cells with pEFBOS-NIC-MYC plasmid showed a significantly slower growth rate compared with that of two control groups (P < 0.05, Student's t-test). Immunocytochemistry of NSE showed that PUs in the NIC transfected group, sham group and negative control group were 7.21 ± 0.59, 28.25 ± 1.46, 30.57 ± 1.31 respectively, the former one was smaller than the values of the latter two significantly (P < 0.01). Western blot analysis showed the grave scales of CgA in NIC transfected group and sham group to be 0.54 ± 0.03 and 0.99 ± 0.05 respectively (grave scales of the negative control was set as 1.00), the former one significantly smaller than that of the other two groups (P < 0.01). The grave scales of NSE in the NIC transfected group and sham group were 0.43 ± 0.02 and 1.07 ± 0.09 respectively (grave scales of the negative control was set as 1.00) and the former one was significantly smaller than the other two groups (P < 0.01).

CONCLUSIONNotch signaling pathway regulates SCLC cells through its inhibitory effect on hASH1 transcription via HES1 along with an expression inhibition of neuroendocrine markers in SCLC.

Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Chromogranin A ; metabolism ; Homeodomain Proteins ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Phosphopyruvate Hydratase ; metabolism ; Plasmids ; Receptor, Notch1 ; metabolism ; physiology ; Recombinant Proteins ; metabolism ; Signal Transduction ; Small Cell Lung Carcinoma ; metabolism ; pathology ; Transcription Factor HES-1 ; Transfection

OBJECTIVETo investigate the effect of both fermented Cordyceps powder (CS) and prednisone on the Notch2/hes-1 signaling activation in the kidney tubules of rats with acute aristolochic acid nephropathy (AAAN).

METHODSTotally 50 SD rats were randomly divided into 4 groups, i.e., the normal group, the model group, the CS group, the prednisone group, and the CS plus prednisone group, 10 in each group. The AAAN rat model was induced by intragastric administration of pure aristolochic acid A at the daily dose of 100 mg/kg for 3 days. Rats in the CS group were administered with CS at the daily dose of 5.0 g/kg by gastrogavage, while those in the prednisone group were administered with prednisone at the daily dose of 0.5 mg/kg. Rats in the CS plus prednisone group were treated by CS and prednisone. All treatment lasted for 3 successive weeks. Kidney functions [urea nitrogen (BUN) and serum creatinine (SCr)] were detected. The pathological changes of kidneys were observed by Hematoxylin-Eosin staining. The apoptosis of the renal tubular epithelial cells was detected by TUNEL. The protein expressions of Notch2 and Hes-1 in the renal tissue were detected by immunohistochemical assay and Western blot.

RESULTSResults of HE staining showed the structure in the nephridial tissue was regular in rats of the normal group. The renal tubular necrosis occurred in the rats of the model group. The pathological changes of kidneys were obviously improved in the CS group, the prednisone group, and the CS plus prednisone group. Compared with the normal group, levels of BUN and SCr, semi-quantitative score of the tubular interstitial tissue, ratio of apoptotic cells, and expressions of Notch2 and Hes-1 proteins significantly increased in the model group (P < 0.01). Compared with the model group, the aforesaid indices significantly decreased in the 3 treatment groups (P < 0.01). All indices decreased most obviously in the CS plus prednisone group (P < 0.05, P < 0. 01).

CONCLUSIONSNotch2/hes-1 signaling activation might be associated with apoptosis of renal tubular epithelial cells. Both CS and prednisone could play a nephroprotective role for AAAN. But CS plus prednisone could achieve the best effect. Inhabiting the Notch2/hes-1 signaling activation could be its nephroprotective mechanism.

Animals ; Apoptosis ; drug effects ; Aristolochic Acids ; toxicity ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cordyceps ; Female ; Homeodomain Proteins ; metabolism ; Kidney ; metabolism ; Kidney Diseases ; chemically induced ; metabolism ; Kidney Function Tests ; Kidney Tubules ; metabolism ; Male ; Prednisone ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch2 ; metabolism ; Signal Transduction ; drug effects ; Transcription Factor HES-1

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

OBJECTIVE: To investigate the role of Notch signaling in differentiation of Sprague-Dawley (SD) rat retinal progenitor cells (RPCs). METHODS: RPCs were isolated from 16-day embryonic SD rats and cultured in suspension. RPCs were cultured respectively in media with (treatment group) or without (control group) gamma-secretase inhibitor X which was used to block Notch signaling. Morphological observation and immunocytochemistry were applied at day 14 to determine the cell types and analyze the expression of Notch pathway genes in both groups. RESULTS: Most RPCs expressed Notch1 intracellular domains or its downstream transcriptional factor Hes1. A few expressed bHLH transcriptional factors NeuroD and Mash1. Most auto-differentiated RPCs expressed NeuroD or Mash1, while a few of them expressed Notch1 intracellular domains or Hes1. In the group treated with gamma-secretase inhibitor X, the positive rate of Nestin or GFAP was much lower than that in the control group while the positive rate of beta-tubulin was much higher than that in the control group. The difference in the positive rate of recovering between the two groups was not significant. CONCLUSION In vitro Notch signaling may inhibit retinal stem cells differentiation. Inhibiting Notch signaling in vitro may promote differentiation to neurons and partially inhibit glial differentiation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fetus ; Homeodomain Proteins ; metabolism ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; metabolism ; Retina ; cytology ; Signal Transduction ; drug effects ; physiology ; Stem Cells ; cytology ; Transcription Factor HES-1