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Objective@#To study the different expression of 4 microRNA (miRNA, miR) during the osteogenesis differentiation of bone marrow mesenchymal stem cell (BMSC) cultured in high-fat or normal environment and to explore the relationship of these miRNAs with disheveled 2 during osteogenesis differentiation.@*Methods@#BMSC were cultured with 2 ml normal osteogenic induction (control group) and high-fat osteogenic induction (high-fat group) respectively. On the 3rd, 5th, 7th,14th, 21st day, quantitative real-time PCR (qPCR) was used to analyze expression levels of four miRNAs (miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p), mRNA of disheveled 2, osteogenic related factors such as alkaline phosphatase (ALP), Runt-related transcription gene 2 (Runx2). And the protein was detected by Western blotting. After BMSC were transfected by 50 μl 50 nmol/L miRNA mimics/inhibitors/negative controls respectively, BMSC were put on osteogenic induction, on the 1st, 3rd, 5th, 7th day, ALP activity was detected. On the 7th day, ALP staining was to observe the degree of osteogenesis differentiation, and Western blotting was adopted to analyze the expression of dishevelled 2 and other osteogenic related factors, while qPCR was used to analyze the expression of disheveled 2 mRNA. After 293T cells were co-transfected with disheveled 2 wild-type/mutant firefly luciferase reporter plasmid with either negative control (NC) or a mimic of these four miRNAs respectively for 48 h, luciferase activities were measured.@*Results@#On the 21th day, the expressions of miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p in high-fat groups were higher by 20%, 60%, 340% and 4 420% respectively than those in control groups (P<0.05). The expression of ALP and Runx2 in BMSC decreased after BMSC transfected miR-21-5p and miR-29c-3p mimics, while increased after transfected miR-21-5p and miR-29c-3p inhibitors. The expression of disheveled 2 decreased by 35% after transfected by miR-29c-3p mimic, while it increased by 269% after transfected by miR-29c-3p inhibitor (P<0.05). Transfection of the miR-29c-3p mimics significantly decreased the luciferase activity of wild-type 3'-UTR compared with NC control (P<0.05). There were no statistical significances among other groups.@*Conclusions@#miRNAs had better expression during osteogenesis differentiation of BMSC in high-fat environment; miR-29c-3p could negatively regulate the osteogenesis differentiation of BMSC by targets on dishevelled 2.
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Objective To study the expression of dishevelled-2 (DVL2) in rheumatoid arthritis cartilage and its effect on cartilage destruction.Methods Cartilage DVL2 expression in rat models of rheumatoid arthritis (RA),osteoarthritis(OA) and collagen-induced arthritis(CIA) were tested by Western blotting.DVL2 overexpressed lentivirus was transfected into the knee of CIA rats.Primary chondrocytes were extracted from RA patients by knee arthroplasty and transfected with DVL2 overexpressed lentivirus.Gene expression of related inflammation related cytokines was detected by real-time polymerase chain reaction (PCR).Results Compared with knee articular cartilage in OA patients and normal rats,DVL2 protein was highly expressed in knee cartilage of RA patients and CIA rats (P values 0.041 and 0.032,respectively).DVL2 did not significantly affect the destruction of knee cartilage in CIA rats (P=0.885).DVL2 overexpression in chondrocytes enhanced gene expression of cyclo-oxygenase-2 (COX-2),inducible nitric oxide synthase (NOS),matrix metalloproteinase (MMP) 2,MMP-3,and MMP-9,which could be more pronounced when tumor necrosis factor alpha was added.Conclusions DVL2 is highly expressed in RA articular cartilage and promotes the expression of inflammatory cytokines and MMP gene in chondrocytes by activating Wnt/β-catenin pathway,which involves in the destruction of articular cartilage in RA.
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Objective To optimize the culture method for rheumatoid arthritis fibroblast-like synoviocytes in vitro,and observe the effect of Dishevelled (Dvl) 2 on vascular endothelial growth factor (VEGF) in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS).Methods Synovium from RA patients who underwent knee arthroplasties were cut into small piece,and RA-FLS were isolated and cultured in vitro using tissue block method.Dvl 2 lentivirus overexpressing plasmid was constructed and transfected into RAFLS.Q-polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect the mRNA and protein expression levels of VEGF.Then we used 10 ng/ml tumor necrosis factor (TNF)-α recombinant protein to stimulate the transfected RA-FLS.24 h after stimulation,mRNA and protein expression of VEGF were detected again.Student's t test was used for two group analyses.Results RA-FLS was successfully isolated and cultured in vitro.The multiplicity of infection was 30 and was in conjunction with appropriate concentration of polybrene to promote transfection.Transfection efficiency could meet the test requirements.The mRNA of Dvl 2 increased for 79-fold than the control group.Compared with the control group,Dvl 2 could mildly inhibit RA-FLS secretion of VEGF.After TNF-α stimulation,Dvl 2 could significantly inhibit the VEGF's mRNA (2.15±0.10,2.92±0.47 fold,t=-3.924,P=0.003) and protein [(285±100) pg/ml,(155±61) pg/ml,t=-2.714,P=0.022] expression compared with the control group.Conclusion Dvl 2 can inhibit the effect of TNF-α induced secretion of VEGF in RA-FLS.The specific mechanism needs further study.
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Objective To explore the relationship of DVL2 expression and the development of (CCRCC) by comparing the changes of DVL2 mRNA and protein expression in CCRCC specimens and matched normal renal specimens and its clinical significance. Methods DVL2 mRNA expressions in 22 CCRCC tissues, the matched adjacent normal tissues, and 10 CCRCC tissues alone were examined by semiquantitative RT-PCR and fluorescence quantitative PCR (real-time RT-PCR). Meanwhile, the different expression of the CCRCC between TNM Stage Ⅲ + Ⅳ and Stage Ⅰ +Ⅱ was also examined. Furthermore,immunohistochemistry was employed to examine DVL2 protein expression in 22 CCRCC and the matched adjacent normal tissues, and the other 10 CCRCC tissuses without the matched tissues. Results The DVL2 mRNA expression levels in 17 CCRCC tissues were increased by semi-quantitative RT-PCR and by real time RT-PCR compared with that in corresponding adjacent normal tissues, with the difference being significantly different (t = 2.535, P =0.0197). The DVL2 expression of 8 in 13 Ⅲ + ⅣCCRCC was higher than Ⅰ +ⅡCCRCC. Immunohistochemical examination showed that the DVL2 protein was located in cytomembrane and cytoplasm. Moreover, the positive level of DVL2 protein in CCRCC tissues[81.8 % (18/22)]was significantly higher than those in the adjacent tissues. However the expression was not associated with patients' age, gender, TNM stages (Fisher exact frenquently, P >0.05). Conclusion The DVL2 expression in CCRCC is obviously higher than the corresponding normal tissues in the level of mRNA and protein. And the higher DVL2 expression might be closely associated with the development and progression of CCRCC in the level of mRNA, which may be a potential molecular marker of CCRCC development and metastasis mechanism.
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ObjectiveTo explore the relationship between the expression of Dishevelled2 and Vangl2 and the embryonic neural tube defects (NTDs) induced by all-trans retinoic acid (RA)in Kunming mouse. Methods Fifty pregnant mice were randomly divided into control and RA-treated groups.RA-treated mice were fed with 30mg/kg RA dissolved with peanut oil on embryo 7.75 days, while the mice of control group were administrated with an equal volume of peanut oil on the same time. Then all the embryos were sampled from pregnant mice at the 4th, 18th, 42nd, 66th and 90th hour after treatment. In situ hybridization and immunohistochemical staining technique were used to detect the expression of Dishevelled2 and Vangl2 in embryonic neural tube. Results The two proteins both existed in the epithelial tissue of the mouse embryonic neural tube and displayed different expression modes at various developmental stages.Compared with the control group, the RA treated group showed a significant decrease (P≤0.05) at the 18th and 42nd hour and a significant increase (P≤0.05) at the 66th hour in Dishevelled2 protein after maternal treatment, and no significant difference was found at the 90th hour. Compared with the control group, the Vangl2 mRNA expression in the RA treated group displayed a significant decrease (P≤0.05) at the 4th and 18th hour and a significant increase (P≤0.05) at the 66th hour after RA treatment, and no difference was found at the 42nd hour. Compared with the control group, the expression of Vangl2 protein in the RA treated group decreased (P≤0.05) at the 18th and 42nd hour, and increased (P≤0.05) at the 90th hour after RA treatment, no difference was found at the 66th hour. Conclusion Excessive RA may interfere with the normal embryonic neural tube closure by regulating the expression of Dishevelled2 and Vangl2.
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Objective To explore the relationship between the expression of Dishevelled2 and Vangl2 proteins and the development of mouse palate. Methods Twenty-four pregnant mice were randomly divided into eight groups, and the mouse embryos were obtained at eight clock of the pregnant day of thirteen(p13d8h), p13d14h,p13d22h,p14d8h,p14d14h,p14d22h,p15d8h and p15d22h respectively, then paraffin sections were made conventionally.The distrubution and dynamic changes of Dishevelled2 and Vangl2 proteins in the embryonic palatal shelves were detected by immunohistochemistry and image analysis. Results It was found that the two kinds of proteins expressed in the epithelium and mesenchyma of the mouse palatal shelves at different development stages. The expression levels of the Dishevelled2,in both of the epithelium and mesenchyme of the palatal shelves, increased first (p13d8h-p13d22h),then decreased rapidly(p13d22h-p14d14h), and then increased again(p14d14h-p15d22h). The expression of Vangl2 protein in the mesenchyma showed a similar trend to that of the Dishevelled2, but there was no obvious regularity in the epithelium. In addition, the expressive levels of both kinds of proteins in the epithelium were significantly higher than those in mesenchyma of the palatal shelves. Conclusion Dishevelled2 and Vangl2 proteins might directly or indirectly take part in the regulation process of mouse palate morphogenesis.