OBJECTIVE: To investigate the role of NOV/CCN3 in regulating the proliferation of mesenchymal stem cells (MSCs) and its regulatory mechanism and assess the value of CCN3 as a proliferative factor in bone tissue engineering. METHODS: Mouse embryonic fibroblasts (MEFs) were used as the MSC model, in which CCN3 expression was up-regulated and downregulated by transfection with the recombinant adenovirus vectors Ad-CCN3 and Ad-siCCN3, respectively. Flow cytometry was used to analyze the changes in cell cycle and apoptosis of the transfected cells. Western blotting was used to detect the expression levels of the proliferation indicators (PCNA, cyclin E, and cyclin B1) and the apoptosis indicators (Bax and Bcl-2) to assess the effect of modulation of CCN3 expression on MEF proliferation and apoptosis. CCN3 protein secretion by the cells was detected using ELISA. RT-qPCR and Western blotting were employed to analyze the changes in the expressions of Notch1, ligand DLL1, the downstream key proteins or genes (Hey1, P300, H3K9) and MAPK pathway-related proteins ERK1+2 and p-ERK1+2. RESULTS: Flow cytometry showed that compared with the control cells, MEFs transfected with Ad-CCN3 exhibited significantly increased cell proliferation index ( CONCLUSIONS CCN3 over-expression promotes the proliferation and inhibits apoptosis of MEFs possibly by inhibiting the classical Notch signaling pathway and activating the MAPK pathway
Animals ; Apoptosis ; Cell Cycle ; Cell Proliferation ; Fibroblasts ; Mice ; Nephroblastoma Overexpressed Protein

This study was aimed to investigate the expression level of NOV and BNIP3 mRNA in mice myelomonocytic leukemia (AML-M(4)) and its significance. The mice were inoculated intravenously with myelomonocytic leukemia cells of WEHI-3, and divided randomly into chemotherapy group and control (untreated) group. Bone marrow samples were then collected from both groups at different times. The NOV and BNIP3 mRNA expression were detected by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and the relationship between these expression levels and clinical significance in leukemia incidence and progression were analyzed with β-actin as the housekeeping gene. The results showed that the mean values of NOV and BNIP3 increased gradually from 2 weeks after inoculation and achieved highest level at death in control group. Expression level of NOV increased from 1.85E-05 before inoculation to 3.57E-02 at death (p < 0.05), and BNIP3 from 3.44E-03 to 3.48E-02. While 2 gene expression in the chemotherapy group decreased quickly to 2.51E-05 and 1.58E-03 (p < 0.05) respectively after chemotherapy, which were close to the level before inoculation (p > 0.05). The 2 gene expressions again rose at relapse, and difference of expression level between 2 group at death were no statistically significant (p > 0.05). It is concluded that the expression of NOV and BNIP3 in leukemia AML-M(4) is significantly higher than that in normal controls, of which high level expression is an important factor in the development of leukemia. Close relation between the therapeutic effect and expression level of these two genes suggests the great value in prognostic evaluation and MRD detection.
Animals ; Cell Line, Tumor ; Female ; Gene Expression ; Leukemia, Myeloid ; genetics ; Membrane Proteins ; genetics ; Mice ; Mitochondrial Proteins ; genetics ; Nephroblastoma Overexpressed Protein ; genetics

OBJECTIVETo observe the effects of prenatal exposure to lead on nephroblastoma over-expressed gene (NOV) protein and mRNA expression in hippocampus of rats' offspring, and to explore the molecular mechanism of lead on learning and memory.

METHODSThe pregnant rats were divided into 1 control group and 3 lead expose groups randomly: low( 125 mg/L), middle (250 mg/L) and high (500 mg/L). 8 rats in each group. From pregnancy ld until birth, the rats were given double evaporated water or lead acetate water of different doses according to their groups. The samples of descendants were taken on embryo 18 th day, postnatal 1st day, 21st day, 60th day. The contents of lead in blood and hippocampus were determined by hydride generation atomic absorption spectrometry method. The expression of NOV protein and mRNA in hippocampus were observed by immunohistochemistry and in situ hybridization.

RESULTSThe lead contents of blood [(312.46 +/- 43.55), (419.35 +/- 62.25), (541.45 +/- 47.90) microg/L] and hippocampus[(2.10 +/- 0.18), (2.58 +/- 0.12), (3.41 +/- 0.23) microg/L] were significantly higher in lead exposed groups than that of control [(214.31 +/- 40.77), (0.76 +/- 0.13) microg/L] (P < 0.05) on the embryo 18th, 1st and 21 st day, while there was no significantly difference among them on 60 th day. The expression of NOV protein in all lead exposed groups were significantly decreased compared with control group (P < 0.05) on 1st and 21 st day, while there was no significantly difference among them on 60th day. The expression of NOV mRNA of all the lead exposed groups were significantly decreased compared with control group (P < 0.05) on the embryo 18th, 1st and 21st day, while there was significantly difference only in the high dose group (0.0355 +/- 0.0100) compared with control (0.0900 +/- 0.0200) (P < 0.01) on 60th day.

CONCLUSIONPregnancy low level lead exposure could decrease the NOV protein and mRNA expression in hippocampus of offspring, which might be one of the molecular mechanisms of effect of lead on learning and memory.

Animals ; Female ; Gene Expression ; Hippocampus ; metabolism ; Lead ; adverse effects ; blood ; Nephroblastoma Overexpressed Protein ; genetics ; metabolism ; Pregnancy ; Prenatal Exposure Delayed Effects ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar