1.Wnt3a signaling pathway plays a role in neuropathic pain through epigenetic modification of JMJD6.
Chuchu PENG ; Zhigang CHENG ; Xiaoyan ZHU
Journal of Central South University(Medical Sciences) 2019;44(4):386-391
To explore whether Wnt3a exerts a role in neuropathic pain through Jumonji C domain 6 (JMJD6)-associated epigenetic modification.
Methods: SD rats were divided into 4 groups: A sham group, a chronic constriction injury (CCI) group, a CCI+negative lentiviral expression vector (LV-NC) group and a CCI+lentiviral overexpression vector (LV-JMJD6) group. The sciatic nerve CCI model of SD rat and JMJD6 lentiviral expression vector were constructed. On the third day after CCI, the intrathecal catheter was prepared, and 20 μL of normal saline and lentivirus-containing reagent (virus titer 1×108 TU/mL) were administered. The rats' paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were monitored, and Western blotting was used to detect the expression of Wnt3a and NR2B protein in the spinal cord. Co-immunoprecipitation was applied to detect the interaction between JMJD6 and Wnt3a.
Results: Compared with the sham group, the PWMT of the rats in each group after CCI was significantly decreased and the PWTL was significantly shortened (P<0.05). Compared with the CCI group and the CCI+LV-NC group, PWMT in the CCI+LV-JMJD6 group was increased significantly on the 10th day and the 14th day after CCI, and the PWTL was significantly prolonged on the 14th day after CCI (P<0.05). On the 14th day after CCI, the expression levels of Wnt3a and NR2B in the CCI group and the CCI+LV-NC group were significantly higher than those in the sham group. After intrathecal injection of lentiviral vector, Wnt3a and NR2B protein expression levels in the CCI+LV-JMJD6 group were lower compared with the CCI+LV-NC group (P<0.05). The results of co-immunoprecipitation showed no direct interaction between Wnt3a and JMJD6.
Conclusion: Wnt3a is involved in mediating neuropathic pain, and its effect may be related to the epigenetic modification of JMJD6, which is likely regulated through indirect interaction.
Animals
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Injections, Spinal
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Neuralgia
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Rats
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Rats, Sprague-Dawley
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Signal Transduction
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Spinal Cord
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Wnt3A Protein
2.Effects of orchinol on invasion, migration and Wnt3a/β-catenin signaling pathway of human gastric cancer SGC-7901 cells.
Tao LING ; Wei WANG ; Chen HU ; Xin YAN ; Yu-Ting XU ; Chun TANG ; Yu KOU ; Liang LIU
China Journal of Chinese Materia Medica 2018;43(23):4718-4723
The purpose of this study was to investigate the inhibitory effect of the main 9,10-dihydrophenanthrene orchinol isolated from Spiranthes sinensis Radix et Herba on the invasion and migration of human gastric cancer SGC-7901 cells and its preliminary molecular mechanism. SGC-7901 cells were cultured in vitro, after the cells were treated with different final concentrations(5, 10, 20, 40, 80 μmol·L⁻¹) of orchinol for 24, 48 or 72 hours, the effect of orchinol on cell viability was measured by MTT assay. Wound healing and Transwell assays were performed to determine the effects of different final concentrations(5, 10, 20, 40 μmol·L⁻¹) of orchinol for 48 hour on invasion and migration abilities of SGC-7901 cells, respectively. The protein expression levels of β-catenin, Wnt-3α, DvL2, cyclinD1 and GSK-3β were detected by Western blot. The results showed that 5-80 μmol·L⁻¹ orchinol inhibited the viability of SGC-7901 cells in a dose-dependent and time-dependent manner, and the IC₅₈ values of 24, 48 and 72 hours were 77.79, 42.96 and 7.85 μmol·L⁻¹, respectively. Compared with the control group, the ability of invasion and migration of SGC-7901 cells was significantly inhibited after treated with 5, 10 and 20 μmol·L⁻¹ orchinol for 48 hours (<0.05, <0.01), and the dose-effect relationship was observed. The results of Western blot showed that orchinol could significantly down-regulate the protein expression levels of β-catenin, Wnt3a, DvL2 and cyclinD1, and up-regulate the protein expression level of GSK-3β(<0.05, <0.01, <0.001). The above results suggest that orchinol can obviously inhibit the invasion and migration of SGC-7901 cells, which may be related to its inhibition of Wnt3a/β-catenin signaling pathway and the proteins expression of downstream genes.
Cell Line, Tumor
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Cell Movement
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Cell Proliferation
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Humans
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Phenanthrenes
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Stomach Neoplasms
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Wnt Signaling Pathway
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Wnt3A Protein
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beta Catenin
3.Effects of Wnt3a on proliferation, activation and the expression of TGFb/Smad in rat hepatic stellate cells.
Yan-ping WANG ; Qi HE ; Fei WU ; Lan-lan ZHU ; Wei LIU ; Ya-nan ZHANG ; Yong-wen HE
Chinese Journal of Hepatology 2013;21(2):111-115
OBJECTIVETo observe the effects of Wnt3a on proliferation and, activation of hepatic stellate cells (HSCs) and their the expression of the transforming growth factor beta (TGFb) and /Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line.
METHODSSynchronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a (50, 100, 200, 250 and 300 ng/mL). Unstimulated cells served as controls. Edu Effects on proliferation were determined by EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a, and the Effects on the protein expression of TGFb/Smad signaling factors was assessed by western blot detection (gray-value analysis) of alpha-smooth muscle actin (a-SMA), a-SMA, TGFb1, Smad3, and and Smad7; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis .The correlation was also observed. The significance of inter-group differences was assessed by one-way ANOVA, and correlations were determined using bivariate statistical modeling.
RESULTSIn general, HSC The proliferation of hepatic stellate cells increased after the addition of in response to Wnt3a stimulation for 24 h, reaching its peak at the maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration (63.00+/-2.30%), and it increased dramatically compared with those in which was significantly higher than the proliferation rates of the unstimulated control cells, and the cells stimulated with 50, 100 and 150 ng/mLl group (P less than 0.05), but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mLl group,it had no obvious increase(P more than 0.05).; The Wnt3a stimulation also led to time-dependent increases in the protein expressions of a-SMA, TGFb1, and Smad3 increased with the addition of Wnt3a and the extension of time . For all three, The maximal amount of increased protein expression all reached to the was maximal produced by stimulation when hepatic stellate cells were treated by with 300 ng/mLl Wnt3a for 48 h hours,and the rations of(normalized gray- values:s of a-SMA, 1.0860+/-0.0101; TGFb1, 1.0346+/-0.0118; Smad3, to GAPDH were 1.0860+/-0.0101, 1.0346+/-0.0118, 1.0306+/-0.0122)respectively. However in contrast, the Wnt3a stimulation led to concentration- and time-dependent decreases in Smad7 expression varied inversely, with to them with the minimal ration of it to GAPDH the maximal decrease occurring with 300 ng/mL Wnt3a for 48 h (0.7736+/-0.0139) after being treated by 300 ng/ml Wnt3a for 48h. The comparison was remarkably discrepant, (P less than 0.05).There were positive correlations between a-SMA expression and was found to be positively correlated to TGFb1, Smad3 (r=0.968, P less than 0.05) and; Smad3 (r=0.997, P less than 0.01), but a-SMA and Smad7 had negatively correlated to Smad7 ion(r=0.960, P less than 0.05).
CONCLUSIONWnt3a can increase the stimulates proliferation as well as and activation of rat the hepatic stellate cells HSCs , and upregulate modifies the expression of TGFb/Smad signaling factors, of the hepatic stellate cells, and which may promote the hepatic fibrosis.
Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; metabolism ; Wnt3A Protein ; pharmacology
4.Wnt3a: functions and implications in cancer.
Sha HE ; Yi LU ; Xia LIU ; Xin HUANG ; Evan T KELLER ; Chao-Nan QIAN ; Jian ZHANG
Chinese Journal of Cancer 2015;34(12):554-562
Wnt3a, one of Wnt family members, plays key roles in regulating pleiotropic cellular functions, including self-renewal, proliferation, differentiation, and motility. Accumulating evidence has suggested that Wnt3a promotes or suppresses tumor progression via the canonical Wnt signaling pathway depending on cancer type. In addition, the roles of Wnt3a signaling can be inhibited by multiple proteins or chemicals. Herein, we summarize the latest findings on Wnt3a as an important therapeutic target in cancer.
Cell Division
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physiology
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Gene Expression Regulation, Neoplastic
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physiology
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Humans
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Neoplasm Proteins
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metabolism
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physiology
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Neoplasms
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genetics
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metabolism
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pathology
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Tumor Cells, Cultured
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Wnt Signaling Pathway
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physiology
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Wnt3A Protein
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metabolism
;
physiology
5.Low dose of triptolide ameliorates podocyte epithelial-mesenchymal transition induced by high dose of D-glucose via inhibiting Wnt3α/β-catenin signaling pathway activation.
Ge SHI ; Wei WU ; Yi-Gang WAN ; He Wei-Ming HEX ; Yue TU ; Wen-Bei HAN ; Bu-Hui LIU ; Ying-Lu LIU ; Zi-Yue WAN
China Journal of Chinese Materia Medica 2018;43(1):139-146
To explore the effects and molecular mechanisms of triptolide(TP)on improving podocyte epithelial-mesenchymal transition(EMT)induced by high dose of D-glucose(HG), the immortalized podocytes of mice were divided into the normal group(N), the high dose of D-glucose group(HG), the low dose of TP group(L-TP), the high dose of TP group(H-TP)and the mannitol group(MNT), and treated by the different measures respectively. More specifically, the podocytes in each group were separately treated by D-glucose(DG, 5 mmol·L⁻¹)or HG(25 mmol·L⁻¹)or HG(25 mmol·L⁻¹)+ TP(3 μg·L⁻¹)or HG(25 mmol·L⁻¹)+ TP(10 μg·L⁻¹)or DG(5 mmol·L⁻¹)+ MNT(24.5 mmol·L⁻¹). After the intervention for 24, 48 and 72 hours, firstly, the activation of podocyte proliferation was investigated. Secondly, the protein expression levels of the epithelial markers in podocytes such as nephrin and podocin, the mesenchymal markers such as desmin and collagen Ⅰ and the EMT-related mediators such as snail were detected respectively. Finally, the protein expression levels of Wnt3α and β-catenin as the key signaling molecules in Wnt3α/β-catenin pathway were examined severally. The results indicated that, HG could cause the low protein expression levels of nephrin and podocin and the high protein expression levels of desmin, collagen Ⅰ and snail in podocytes, and inducing podocyte EMT. On the other hand, HG could cause the high protein expression levels of Wnt3α and β-catenin in podocytes, and activating Wnt3α/β-catenin signaling pathway. In addition, L-TP had no effect on the activation of podocyte proliferation, the co-treatment of L-TP and HG could significantly recover the protein expression levels of nephrin and podocin, inhibit the protein expression levels of desmin, collagen I and snail in podocytes, thus, further improving podocyte EMT. And that, the co-treatment of L-TP and HG could obviously decrease the high protein expression levels of Wnt3α and β-catenin induced by HG in podocytes, and inhibit Wnt3α/β-catenin signaling pathway activation. On the whole, HG can induce podocyte EMT by activating Wnt3α/β-catenin signaling pathway; L-TP can ameliorate podocyte EMT through inhibiting Wnt3α/β-catenin signaling pathway activation, which may be one of the effects and molecular mechanisms .
Animals
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Cells, Cultured
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Diterpenes
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pharmacology
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Epithelial-Mesenchymal Transition
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Epoxy Compounds
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pharmacology
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Glucose
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Mice
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Phenanthrenes
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pharmacology
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Podocytes
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drug effects
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Wnt Signaling Pathway
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Wnt3A Protein
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metabolism
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beta Catenin
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metabolism
6.Curcumin inhibits proliferation,migration and invasion of gastric cancer cells via Wnt3a/β-catenin/EMT signaling pathway.
Wen-Hu LIU ; Jiang-Bei YUAN ; Fan ZHANG ; Jin-Xia CHANG
China Journal of Chinese Materia Medica 2019;44(14):3107-3115
The aim of this paper was to investigate the effects of curcumin on the proliferation,migration,invasion and apoptosis of human gastric cancer cells and to explore the potential mechanisms. SGC7901,MKN45 and NCI N87 cells lines were cultured under different concentrations of curcumin( 2. 5,5,10,20,40,80 and 160 μmol·L~(-1)) at different time points( 12,24,48 and 72 h),and the effect of curcumin on cell proliferation was detected by CCK-8 assay. The migration and invasiveness of cells were determined by wound healing and Transwell assays,the apoptosis rate was assessed by flow cytometry,the expression of N-cadherin,E-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6,Bcl-2 and Bax were detected by Western blot,and the enzymatic activity of caspase-3,caspase-8 and caspase-9 was evaluated via caspase kit. RESULTS:: indicated that the proliferation of MKN45 cells was significantly inhibited by curcumin in a dose-and time-dependent manner( IC50= 21. 93 μmol·L~(-1)). Moreover,curcumin could inhibit the migration and invasion of MKN45 cells,downregulate the expression of N-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6 and Bcl-2,and upregulate the expression of E-cadherin and Bax,it could increase the activity of caspase-3,caspase-8,caspase-9 and induce apoptosis as well. The potential mechanism is through inhibiting the Wnt3 a/β-catenin/EMT pathway,regulating Bcl-2 signaling and caspase pathway,which might provide new potential strategies for gastric cancer treatment.
Cell Line, Tumor
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Cell Movement
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Cell Proliferation
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Curcumin
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pharmacology
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Humans
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Stomach Neoplasms
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drug therapy
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pathology
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Wnt Signaling Pathway
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Wnt3A Protein
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metabolism
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beta Catenin
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metabolism
7.Functional electrical stimulation increases neural stem/progenitor cell proliferation and neurogenesis in the subventricular zone of rats with stroke.
Hui-Hua LIU ; Yun XIANG ; Tie-Bin YAN ; Zhi-Mei TAN ; Sheng-Huo LI ; Xiao-Kuo HE
Chinese Medical Journal 2013;126(12):2361-2367
BACKGROUNDFunctional electrical stimulation (FES) is known to promote the recovery of motor function in rats with ischemia and to upregulate the expression of growth factors which support brain neurogenesis. In this study, we investigated whether postischemic FES could improve functional outcomes and modulate neurogenesis in the subventricular zone (SVZ) after focal cerebral ischemia.
METHODSAdult male Sprague-Dawley rats with permanent middle cerebral artery occlusion (MCAO) were randomly assigned to the control group, the placebo stimulation group, and the FES group. The rats in each group were further assigned to one of four therapeutic periods (1, 3, 7, or 14 days). FES was delivered 48 hours after the MCAO procedure and divided into two 10-minute sessions on each day of treatment with a 10-minute rest between them. Two intraperitoneal injections of bromodeoxyuridine (BrdU) were given 4 hours apart every day beginning 48 hours after the MCAO. Neurogenesis was evaluated by immunofuorescence staining. Wnt-3 which is strongly implicated in the proliferation and differentiation of neural stem cells (NSCs) was investigated by Western blotting analysis. The data were subjected to one- way analysis of variance (ANOVA), followed by a Tukey/Kramer or Dunnett post hoc test.
RESULTSFES significantly increased the number of BrdU-positive cells and BrdU/glial fibrillary acidic protein double- positive neural progenitor cells in the SVZ on days 7 and 14 of the treatment (P < 0.05). The number of BrdU/doublecortin (DCX) double-positive migrating neuroblast cells in the ipsilateral SVZ on day 14 of the FES treatment group ((522.77 ± 33.32) cells/mm(2)) was significantly increased compared with the control group ((262.58 ± 35.11) cells/mm(2), P < 0.05) and the placebo group ((266.17 ± 47.98) cells/mm(2), P < 0.05). However, only a few BrdU/neuron-specific nuclear protein-positive cells were observed by day 14 of the treatment. At day 7, Wnt-3 was upregulated in the ipsilateral SVZs of the rats receiving FES ((0.44 ± 0.05)%) compared with those of the control group rats ((0.31 ± 0.02)%, P < 0.05) or the placebo group rats ((0.31 ± 0.04)%, P < 0.05). At day 14, the corresponding values were (0.56 ± 0.05)% in the FES group compared with those of the control group rats ((0.50 ± 0.06)%, P < 0.05) or the placebo group rats ((0.48 ± 0.06)%, P < 0.05).
CONCLUSIONFES augments the proliferation, differentiation, and migration of NSCs and thus promotes neurogenesis, which may be related to the improvement of neurological outcomes.
Animals ; Bromodeoxyuridine ; metabolism ; Cell Proliferation ; Cerebral Ventricles ; physiopathology ; Electric Stimulation Therapy ; Glial Fibrillary Acidic Protein ; analysis ; Male ; Neural Stem Cells ; physiology ; Neurogenesis ; Rats ; Rats, Sprague-Dawley ; Stroke ; physiopathology ; therapy ; Wnt3A Protein ; analysis
8.Mechanisms underlying the expression regulation of HOXB4 transcription in hematopoietic stem cells - review.
Journal of Experimental Hematology 2008;16(4):960-963
As a member of the hox gene family, hoxB4 gene encodes a class of DNA-dependent homeobox domain nucleoprotein, which is a specific transcription factor, playing an important role in regulating the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Therefore, it is important to understand the mechanisms involved in regulating expression of hoxB4 in the HSC. Previous studies have suggested that some hoxB4 upstream regulatory factors, such as USF-1 (upstream activating factor -1), USF-2 (upstream activating factor -2) and NF-Y complex, as well as hematopoietic cytokines, such as platelet growth factor (TPO) and Wnt3a protein, play important regulatory roles in the expression of hoxB4 in hematopoietic stem cells. In this review the structure and biological characteristics of hoxB4, mechanisms involved in regulating expression of hoxB4 in the HSC are summarized.
CCAAT-Binding Factor
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metabolism
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Gene Expression Regulation
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Genes, Homeobox
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genetics
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physiology
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Hematopoietic Stem Cells
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metabolism
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Homeodomain Proteins
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genetics
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metabolism
;
physiology
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Humans
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Transcription Factors
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genetics
;
metabolism
;
physiology
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Upstream Stimulatory Factors
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metabolism
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Wnt Proteins
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metabolism
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Wnt3 Protein
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Wnt3A Protein
9.Role of Wnt 2, Wnt 3a and β-catenin in skin lesions of patients with scleroderma.
Journal of Southern Medical University 2012;32(12):1781-1786
OBJECTIVETo study the role of abnormally activated Wnt/β-catenin signal pathway in the pathogenesis of scleroderma (SD) and its association with the clinical classification of SD.
METHODSThe expression and distribution of Wnt 2, Wnt 3a, and β-catenin in the skin lesions of 45 SD patients, including 25 with systemic sclerosis (SSc) and 20 with localized scleroderma (LSc), were detected with SP immunohistochemistry, using 20 samples from healthy skin tissues as normal control.
RESULTSIn the dermis and epidermis of the SD skin lesions, Wnt 2 and Wnt 3a were located in the cytoplasm and cell nuclei, respectively; β-catenin was distributed in the nuclei of dermal fibroblast-like cells, glandular epithelium cells and infiltrating lymphocytes, and on the cell membrane in normal and a part of the affected epidermis. The skin lesions of SD patients showed obviously increased staining intensity of cytoplasmic Wnt 2, nuclear Wnt 3a and β-catenin, but markedly lowered cell membrane staining of β-catenin than normal skins (P<0.01). Both Wnt 2 and Wnt 3a were positively correlated with nuclear β-catenin deposition (r=0.663 and 0.654, P<0.01) and negatively with cell membrane β-catenin staining (r=-0.532 and -0.529, P<0.01). No significant difference was found in the staining intensities of the 3 proteins between SSc and LSc (P>0.05).
CONCLUSIONAbnormal activation of Wnt/β-catenin pathway occurs in the skin lesions of SD patients, which may play an important role in the pathogenesis of SD. SSc and LSc represent the two opposite ends of the SD spectrum rather than two separate diseases.
Adolescent ; Adult ; Case-Control Studies ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Scleroderma, Systemic ; metabolism ; pathology ; Signal Transduction ; Skin ; pathology ; Wnt Signaling Pathway ; Wnt2 Protein ; metabolism ; Wnt3A Protein ; metabolism ; Young Adult ; beta Catenin ; metabolism
10.In vitro effects of Wnt3a gene modification on mitigating damage of mouse bone marrow mesenchymal stem cells induced by Ara-C.
Guang LU ; Zhen-Yu LI ; Wei-Wei MOU ; Xu-Peng HE ; Xiu-Ying PAN ; Kai-Lin XU
Journal of Experimental Hematology 2011;19(4):1033-1037
This study was aimed to investigate the protective effect of Wit3a gene modification on mouse bone marrow mesenchymal stem cells against the injury induced by Ara-C. The gene-modified MSC steadily expressing Wnt3a were established by adenovirus system. The acute direct damage effects of different concentrations of Ara-C on the unmodified MSC and the gene-modified MSC were assessed by using an in vitro culture system, and the corresponding controls were set. The proliferation and apoptosis of MSC exposed to Ara-C were detected by cell count kit-8 (CCK-8) and flow cytometry. The expression of BCL-2 protein related with cell apoptosis was assayed by Western blot. The results indicated that as compared with unmodified MSC, Ara-C exhibited a less inhibitory effect on the proliferation of gene-modified MSC. There was obvious difference between unmodified MSC and gene-modified MSC (p < 0.05). The proliferation of gene-modified MSC began to recover at 72 hours after removal of Ara-C. However, unmodified MSC showed sustained suppression of proliferation after withdrawal of Ara-C. In apoptosis, the apoptosis rate of gene-modified MSC induced by Ara-C was significantly lower than those of unmodified MSC (p < 0.05). In addition, the expression levels of BCL-2 protein in gene-modified MSC were up-regulated compared with unmodified MSC (p < 0.05). It is concluded that Wnt3a gene modification can significantly mitigate the damage of mouse bone marrow MSC induced by Ara-C.
Animals
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Bone Marrow Cells
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drug effects
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metabolism
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Cytarabine
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adverse effects
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Mesenchymal Stromal Cells
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drug effects
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metabolism
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Mice
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Organisms, Genetically Modified
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Proto-Oncogene Proteins
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metabolism
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Proto-Oncogene Proteins c-bcl-2
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Wnt3A Protein
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genetics