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Objective:To investigate the inhibitory effect of miR-497 on the corneal epithelial healing in diabetic mice and its possible mechanism.Methods:Forty healthy clean-grade wild-type C57BL/J6 mice were randomly divided into a blank control group and a model control group, with 20 mice in each group.Another 20 CRISPR/Cas9-mediated miR-497 knockout mice and miR-497 overexpression mice were taken as miR-497 knockout and miR-497 overexpression groups, respectively.The diabetes model was constructed by continuous intraperitoneal injection of streptozotocin (STZ) to the mice in model control, miR-497 knockout and miR-497 overexpression groups, and the mice in blank control group were injected with an equal amount of citrate buffer, followed by 8-week normal feeding.After the establishment of diabetes model, the corneal epithelial injury model was further constructed by scraping off part of the corneal epithelium with a central diameter of 2 mm.The corneal epithelial defect area of mice in 0, 12, 24 and 36 hours after corneal epithelial injury was observed by corneal fluorescein sodium staining.The expression of Wnt3a and β-catenin proteins in mice corneal tissues was detected by Western blot.The expression of miR-497 as well as the mRNA expression levels of cell proliferation-associated factor genes CyclinD1, c-Myc, and Ki-67 mRNA was detected by real-time quantitative fluorescence PCR.The targeting relationship between miR-497 and wnt3a was detected by a dual luciferase reporter gene assay.Human corneal epithelial cells (HCEC) were cultured in vitro and transfected with miR-497 mimics, miR-497 mimics negative control, miR-497 inhibitor, and miR-497 inhibitor negative control by Lipo8000 as miR-497 mimics group, mimics negative control group, miR-497 inhibitor group, andmiR-497 inhibitor negative control group, respectively, all of which were cultured in high glucose medium containing 25% glucose.Another two groups of HCEC were taken and cultured in medium containing 5% and 25% glucose as control and high glucose groups, respectively.The cell proliferation viability was determined by CCK8 method.The use and care of animals complied ith the ARVO statement.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (2019K-K010). Results:Eight weeks after STZ injection, the blood glucose of mice was significantly higher and the weight was significantly lower in each diabetic model group than those of blank control group (all at P<0.05). At 12, 24 and 36 hours after the corneal epithelial injury, the percentages of corneal epithelial defect area observed by slit-lamp microscopy in model control group were significantly higher than those in blank control group and miR-497 knockout group and lower than those in miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expressions of wnt3a and β-catenin proteins in the corneal tissues of model control group were significantly lower than those of blank control group and miR-497 knockout group, but higher than those of miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expressions of CyclinD1, c-Myc and Ki-67 mRNA in model control group were lower than those in miR-497 knockout group, but higher than those in miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expression of miR-497 in model control group, miR-497 knockout group and miR-497 overexpression group was 1.00±0.02, 0.63±0.06 and 1.48±0.03, respectively, with a statistically significant difference ( F=19.62, P<0.01). The luciferase activity of miR-497-5p mimics group in wild-type wnt3a transfected cells was lower than that of miR-497-5p negative control group and empty vector group, and the differences were statistically significant (all at P<0.05). In the mutant wnt3a transfected cells, there was no significant difference in the luciferase activity among various groups ( F=0.73, P=0.59). The cell proliferation A value of high glucose group was 0.59±0.03, which was significantly lower than 0.59±0.03 of normal control group and 0.88±0.08 of miR-497 inhibitor group, but significantly higher than 0.48±0.11 of miR-497 mimics group (all at P<0.05). Conclusions:The silencing of miR-497 may promote the repair of diabetic corneal epithelial defects by targeting wnt/β-catenin pathway.
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Abstract Objective To investigate the effect of peroxynitrite on the cultured cochlear hair cells of C57BL/6 P3 mice in vitro as well as the role of Wnt3a, as an activator of the canonical Wnt signaling pathway, underlying the action of such an oxidative stress. Methods The in vitro primary cultured cochlear hair cells were subjected to l00 μM peroxynitrite and l00 μM peroxynitrite +25 ng/mL Wnt3a for 24 h, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy. Results The number of surviving hair cells was significantly reduced in the 100 μM peroxynitrite group, while it was significantly higher in the Wnt3a + peroxynitrite treated group compared with the peroxynitrite treated group. The transmission electron microscopy showed that exposure to peroxynitrite induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, while Wnt3a clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. Conclusion These results indicated that peroxynitrite could cause oxidative damage to the cochlear hair cells, and low concentrations of Wnt3a has a protective effect against oxidative damage. Level of evidence: Level 2.
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SUMMARY OBJECTIVE: Thyroid neoplasm incidence has increased worldwide, mostly due to the advancements in medical imaging and screening rates. The aberrant Wnt/β-catenin pathway has been identified as a key mechanism, and it has also been related to the metastatic activity of differentiated thyroid cancer. We aimed to verify the difference in the expression of Wnt3a, a canonical activator of the β-catenin signaling, and CDX-2, a transcription factor upregulated by Wnt/β-catenin pathway, in multinodular goiter and differentiated thyroid cancer and to determine their prognostic value. METHODS: We included 194 thyroid tissue surgical specimen and their clinicopathological data: study group (differentiated thyroid cancer, n=154) and control group (multinodular goiter, n=40). Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue by the primary antibodies Wnt3a and CDX-2. RESULTS: High Wnt3a expression was significantly associated with differentiated thyroid cancer (p=0.031). CDX-2 was negative in all differentiated thyroid cancer cases (100%) and also in multinodular goiter. Wnt3a expression was significantly associated with tumors ≤20 mm (p=0.044) and with the absence of capsule invasion (p=0.031). The multivariate analyses suggested that older age (≥55), independent of capsular invasion and tumor size, was an independent prognostic factor for Wnt3a expression (p=0.058). CONCLUSIONS: Wnt3a expression but not CDX-2 is correlated with differentiated thyroid cancer samples in comparison to multinodular goiter. Although its prognostic value was limited to tumor size and capsule invasion, a combined model in a panel of immune markers can add accuracy in the classification of challenging thyroid follicular-derived lesions.
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Objective:To investigate the effect of electroporation-mediated local gene therapy on the expression of Wnt3a and β-catenin in callus of distraction gap during mandibular distraction osteogenesis of rabbits.Methods:The experiments were conducted in the laboratory of the Affiliated Friendship Plastic Surgery Hospital of Nanjing Medical University from September 2019 to December 2019. Forty eight New-Zealand rabbits were randomly divided into control group (group A), gene therapy group (group B) and normal saline group (group C), with 16 rabbits in each group. After bilateral mandible osteotomy and distractors were implanted, the distractors were activated at a speed of 0.8 mm/d on 4th day, postoperatively, and lasted for 7 days, followed by consolidation period. Group A distracted only, group B was subject to local injection of recombinant plasmid pIRES-hBMP2-hVEGF165 in the distraction gap and electroporation stimulation at the beginning of activation distractors; and group C local injection of the same dose of normal saline in the distraction gap and electroporation stimulation at the beginning of activation distractors. Four animals in each group were sacrificed on the day at the end of distraction, 7th, 14th, 28th days of consolidation period, respectively. The callus in the distraction gap was taken for immunohistochemical staining and RT-PCR to detect the expression of Wnt3a and β-catenin, and image analysis was performed. SPSS 22.0 statistical software was used for data analysis.Results:Immunohistochemical staining showed that Wnt3a and β-catenin were mainly located in the cytoplasm and nuclei of fibroblasts, chondrocytes and osteoblasts in callus tissue. Immunohistochemistry and RT-PCR showed that the expression of Wnt3a and β-catenin reached a peak at the end of distraction. With the disappearance of distraction tension, the expression of Wnt3a and β-catenin gradually decreased. After gene therapy intervention, the expression of Wnt3a and β-catenin was significantly increased, and the expression of Wnt3a and β-catenin in group B was the highest at each time point, with statistically significant difference compared with groups A and C ( F=96.3, P<0.01). Conclusions:Gene therapy promotes the expression of Wnt3a and β-catenin in the callus of distraction gap, regulating the balance of the bone reconstruction system and thus promoting the formation of new bone in the distraction gap.
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Objective To investigate the effect of whole body vibration training on biomechanics and Wnt3a protein expression of the femur. Methods Forty-eight female SD rats were randomly divided into sham operation group, osteoporosis group and whole body vibration group, 16 in each group. The bone morphometric parameters were measured by Micro-CT, mechanical parameters of bone structure and materials were measured by three-point bending test, protein expression of Wnt3a and β-catenin was measured by Western blotting, and gene expression of Wnt3a, β-catenin, cyclin D1 and tcf1 was detected by qRT-PCR. ResultsCompared with sham operation group, bone mineral density (BMD), bone volume fraction (BVF), trabecular number, trabecular thickness and cortical bone thickness in osteoporosis group were decreased, and trabecular space was increased; compared with osteoporosis group, BMD, BVF, trabecular number, trabecular thickness and cortical bone thickness in whole body vibration group were increased, and trabecular space was decreased. Compared with sham operation group, the maximum load, elastic load and deflection of osteoporosis group were significantly reduced; compared with osteoporosis group, the maximum load, elastic load and deflection of whole body vibration group were significantly increased. Compared with sham operation group, the maximum stress, elastic stress, maximum strain and elastic modulus in osteoporosis group decreased significantly; compared with the osteoporosis group, the elastic stress, maximum strain and elastic modulus in whole body vibration group increased significantly. Compared with sham operation group, Wnt3a, β-catenin protein and gene expression decreased, cyclin D1, tcf1 gene expression also decreased; compared with osteoporosis group, Wnt3a, β-catenin protein and gene expression increased, cyclin D1, tcf1 gene expression increased as well. Conclusions Whole body vibration training can improve biomechanical properties of the femur and expression of Wnt3a protein in osteoporotic rats. The research findings provide laboratory reference data for the prevention and treatment of osteoporosis by whole body vibration training.
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BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.
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Animals , Hair Follicle/cytology , Hair Follicle/metabolism , Dermis/cytology , Wnt3A Protein/metabolism , RNA, Long Noncoding/metabolism , Biological Assay/methods , Goats , RNA, Long Noncoding/genetics , Luciferases , MethylationABSTRACT
Objective@#To investigate the Wnt3a expression in tissues of HCC and its gene knockout on effects of HepG2 cell proliferation or xenograft tumor growth.@*Methods@#Hepatic Wnt3a expressions in 87 HCC and their matched surrounding tissues were observed by tissue microarray and immunohistochemistry for analyzing its clinicopathological characteristics; Wnt3a-knockout HepG2 cell lines were established by Crispr/cas9-sgRNA system and genomic cleavage efficiency was verified at gene level by surveyor assay. The relative proteins were confirmed by Western blotting; Cell Counting Kit-8 assay was used to examine cell proliferation after knocking-out Wnt3a successfully, and the nude mice HepG2 cell xenograft tumors delete that the relationship between Wnt3a and HCC growth.@*Results@#The positive Wnt3a with brown staining particles was mainly distributed in cytosol and membrane of hepatocytes. The incidence of hepatic Wnt3a expression in cancerous tissues (95.4%) was significantly higher (χ 2 = 47.754, P < 0.001) than that in their surrounding tissues (49.4%). The high Wnt3a expression was 70.1% in the HCC and only 14.9% in the surrounding tissues. High Wnt3a expression was associated with poorly-differentiated grade, liver cirrhosis, HBV infection, portal vein invasion, TNM stage and 5-year survival rate. After knocked-out by Crispr/cas9-sgRNA system successfully, Wnt3a expression was down-regulated significantly at gene or protein level. Key molecule β-catenin in cytoplasma was obviously inhibited. HepG2 cell lines proliferation was suppressed in time-dependent manner. The nude mice HepG2 cell xenograft tumors confirmed that the knock-out of Wnt3a could significantly supressed HCC growth with slower speed (t = 6.418, P < 0.001), smaller volume(869.4 ± 222.5 mm3 vs 355.0 ± 99.9 mm3, t = 5.168, P < 0.001), and lighter weight (0.88 ± 0.20 g vs 0.35 ± 0.11 g, t = 5.628, P < 0.001)compared with the control group.@*Conclusion@#Abnormal expression of Wnt3a could be expected as a promising target for HCC gene therapy.
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@# Objective: To explore the effects of noscapine (Nos) on the expression of cadherin 17 (CDH17) in colon cancer SW480 cells and the mechanism of Nos on cell migration. Methods: SW480 cells were divided into the control group, empty vector (si-EV) group, CDH17 interference (si-CDH17) group, Nos treatment group, and CDH17 interference+Nos treatment (si-CDH17+Nos) group. Small interfering RNA (siRNA) was used to knockdown CDH17, and the selected concentration of Nos was (55.30±2.21) µg/ml (IC50). The mRNA expression of CDH17 was detected by qPCR; the apoptosis and migration abilities of SW480 cells were observed by Hoechst33258 staining and Transwell assay; the contents of VEGF, MMP2 and MMP9 in SW480 cells were measured by ELISA, and the protein expressions of CDH17, Wnt3a and β-catenin were determined by WB. Results: Compared with the control group, mRNA and protein expressions of CDH17 obviously decreased, cell apoptosis and migration significantly reduced, while the contents of VEGF, MMP2 and MMP9 as well as the protein expressions of Wnt3a and β-catenin significantly decreased in Nos treatment group, siCDH17 group and si-CDH17+Nos treatment group (all P<0.01).The effect of si-CDH17+Nos treatment was more significant than that of si-CDH17 (P<0.01). Conclusion: Nos induces apoptosis and inhibits the migration of human colon cancer SW480 cells, which may be related to the down-regulation of CDH17 expression and inhibition of the Wnt3a/β-catenin signaling pathway.
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Objective To investigate the expressions of Wnt3a and Wnt5a in papillary thyroid carcinoma (PTC) and their clinical significances. Methods Immunohistochemical SABC method was used to detect the expressions of Wnt3a and Wnt5a proteins in PTC tissues and their paracancerous tissues collected from 79 patients in Dandong First Hospital from January 2014 to June 2018, and the relationships between the expressions of Wnt3a and Wnt5a proteins and clinicopathological features of PTC patients were analyzed. The expressions of Wnt3a and Wnt5a proteins in 10 pairs of fresh PTC tissues and paracancerous tissues were detected by Western blot. Results The results of immunohistochemistry showed that the positive expression rates of Wnt3a and Wnt5a proteins in PTC tissues were significantly higher than those in paracancerous tissues [69.6％ (55/79) vs. 25.3％ (20/79), 60.8％ (48/79) vs. 20.2％ (16/79)], and the differences were statistically significant (χ 2 values were 31.092 and 26.894, both P < 0.01). The results of Western blot showed that the expressions of Wnt3a and Wnt5a proteins in 10 pairs of fresh PTC tissues was significantly higher than those in paracancerous tissues(1.61±0.40 vs. 0.43±0.14, 1.38±0.291 vs. 0.36±0.13), and the differences were statistically significant (t values were 16.234 and 13.493, both P < 0.01). The expressions of Wnt3a and Wnt5a in PTC tissues were correlated with TNM stage, differentiation, extramembranous invasion and lymph node metastasis (Wnt3a: χ2 values were 6.645, 15.945, 8.783 and 11.220; Wnt5a: χ2 values were 21.525, 7.611, 17.880 and 12.581, all P < 0.05), but not with patients'age, sex and tumor diameter (all P > 0.05). There was a positive correlation between Wnt3a and Wnt5a proteins expressions in PTC (r = 0.597, P < 0.01).Conclusion The abnormal expressions of Wnt3a and Wnt5a proteins in PTC may be related to the development of PTC.
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Aim: To explore the effects of microRNA- 27a-3p(miR-27a-3p) on collagen type I (Col I) and collagen type III (Col III) synthesis in pulmonary fibroblasts and the underlying mechanisms. Methods: Human pulmonary fibroblasts MRC-5 were cultured and then transfected with miR-27a-3p mimic or its inhibitor. qPCR and Western blot were used to detect miR- 27a-3p levels and nuclear β-catenin content, respectively. The expression of Col I, Col III and Wnt3a was measured using qPCR and Western blot. Bioinformatics predicted the potential of miR-27a-3p bound to Wnt3a 3'-untranslated region (3'-UTR). Dual luciferase reporter gene analyzed the effects of miR-27a-3p mimic transfection on luciferase activity of wild type and mutant Wnt3a 3'-UTR. MRC-5 cells were treated with the Wnt3a/β-catenin signaling pathway inhibitor Dkkl, followed by transfection with miR-27a-3p mimic or its inhibitor. Col I and Col III expression was detected by qPCR and Western blot. Results: miR-27a- 3p mimic markedly increased miR-27a-3p levels and decreased Col I, Col III, Wnt3a and β-catenin expression (P 0. 05), revealing Wnt3a as a target of miR-27a-3p. In addition, Dkkl pretreatment almost fully reversed the inductive effect of miR-27a-3p inhibitor on Col I and Col III expression in MRC-5 cells (P < 0. 05). Conclusions: miR-27a-3p inhibits the biosynthesis of Col I and Col III in pulmonary fibroblasts, which is attributed to inactivated Wnt3a/β-catenin signaling pathway.
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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.
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Humans , Cell Line, Tumor , Cell Movement , Cell Proliferation , Curcumin , Pharmacology , Stomach Neoplasms , Drug Therapy , Pathology , Wnt Signaling Pathway , Wnt3A Protein , Metabolism , beta Catenin , MetabolismABSTRACT
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.
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Humans , Cell Line, Tumor , Cell Movement , Cell Proliferation , Phenanthrenes , Stomach Neoplasms , Wnt Signaling Pathway , Wnt3A Protein , beta CateninABSTRACT
Hematopoietic stem cells(HSCs)senescence is an important reason of hematopoietical and immunologi-cal function senescence.It also is play a key role during aging-related diseases development.Under certain condi-tions,the activation of classical Wnt 3a/β-catenin is in favour of maintains polarity and young states of HSCs,self-renewing,proliferation and differentiation potency.Switching to the non-classical Wnt5a pathway,further activation of Cdc42 protein and others can promote HSCs ageing,and indirectly inhibits Wnt3a/beta-catenin pathway.The in-tervention of two Wnt signaling pathways switching and mechanism,not only can illustrate the mechanism of HSCs aging,but also clear how to slow down ageing.This could provide a new strategy on the solution of age-related dis-eases and keeping a young state.
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Objective To investigate the effects of CHIR99021 and Wnt3a, Wnt/β-catenin signaling pathway acti-vators, on cardiac differentiation of mouse embryonic stem cells ( mESCs ) . Methods The embryonic bodies ( EBs) were formed through suspension culture method, CHIR99021 or Wnt3a was added into differentiated medi-um from day 2 to 5, named CHIR99021 group or Wnt3a group, respectively. In addition, there was a control group in which EBs were automatically differentiated. The expression levels of Brachyury, the mesoderm specific target gene, and Nkx2. 5, cardiac-precursor marker, as well as the transcripts of cardiomyocyte markers,α-myosin heavychain (α-MHC ) , cardiac troponin T ( cTnT ) and connexin-43 ( Cx43 ) were analyzed through quantitative RT-PCR. Besides, the cardiac-specific proteins including α-MHC, cTNT and CX43 were detected by immunofluores-cence and Western blot. Results The mESCs in every group did differentiate into cardiomyocytes. The expression of Brachyury was substantially augmented by treatment with CHIR99021 and Wnt3a, showing a peak of expression at day 7. Similarly, CHIR99021 and Wnt3a dramatically increased the expression levels of Nkx2. 5,α-MHC, cT-nT and Cx43 with the time of differentiation, with the expression of target genes in CHIR99021 group and Wnt3a group was greater than that in the control group and CHIR99021 group was higher than Wnt3 a group at day 15 ( P<0. 05, P < 0. 01 ). Western blot analysis suggested that the expressions of α-MHC, cTNT and CX43 in CHIR99021 group and Wnt3a group were greater than those in the control group, and CHIR99021 group was higher than Wnt3 a group at day 15 . Conclusion Both CHIR99021 and Wnt3 a could improve cardiogenesis from mESCs through activate Wnt/β-catenin signaling pathway at the early stage of differentiation while the former is better than the latter.
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Objective To investigate the protective effect of Wnt3a on the oxidative stress damage of melanocytes and its mechanism. Methods The melanocytes were divided into 4 groups:control group,Wnt3a group,H2O2treatment group,Wnt3a treatment group.The melanocytes were treated with 750 μmol/L H2O2for oxidative stress damage.The activity of cells was detected by MTT assay.The apoptosis rate was examined by flow cytometry.The ROS production was observed by fluorescence microscopy and flow cytometry. The Nrf2/ARE pathway activation was determined with an ARE-driven luciferase reporter construct. The expressions of Nrf2 and HO-1 were examined by Western blot. Results Compared with control group,cell activity decreased(P<0.01),the ratio of apoptosis increased(P<0.01),ROS production was raised in H2O2treatment group(P<0.01). While compared with H2O2treatment group,cell activity was relieved(P<0.05), the ratio of apoptosis decreased(P<0.05), ROS production was de-clined in Wnt3a treatment group(P<0.05).Wnt3a up-regulated the transcriptional activity of Nrf2 as determined with improving the relative activity of luciferase regulated by the antioxidant response element ARE. Wnt3a also increases the expression of Nrf2 and HO-1 protein levels. Conclusions Wnt3a protects the melanocytes against oxi-dative injury by H2O2. The mechanism would be related to the activation of Nrf2/ARE pathway.
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Objective To investigate the regulating effect of fire needling on Wnt andβ-catenin genes of Wnt/β-catenin pathway in neural stem cells in spinal cord injury.Methods Sixty female SD rats were randomly allocated, including 5 rats to a blank group, 5 rats to a sham operation group, 25 rats to a model group and 25 rats to a fire needling group. The model and fire needling groups of rats were again separately divided into 1 d, 3 d, 7 d, 10 d and 14 d groups. A model was made using modified Allen's method in the model and fire needling groups. The BBB score was recorded and the expressions of Wnt andβ-catenin genes were determined in every group.Results In the model and fire needling groups, the BBB scores were significantly higher at 7 and 10 days than at the previous time point and there was a statistically significant difference (P<0.05). The BBB score at every time point after treatment was significantly higher in the fire needling group than in the model group and there was a statistically significant difference (P<0.05). Wnt3a gene expressions increased in the model and fire needling groups at 7 and 10 days compare with the previous time point (P<0.05). At 7 and 10 days, Wnt3a gene expression was higher in the fire needling group than in the model group and there was a statistically significant difference (P<0.05). The changing tendency ofβ-catenin gene expression levels was basically the same as that of Wnt3a's.Conclusion Fire needling can modulate the expressions of Wnt3a andβ-catenin genes in neural stem cells in spinal cord injury.
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Objective To investigate the protective effect of Wnt3a on the oxidative stress damage of melanocytes and its mechanism. Methods The melanocytes were divided into 4 groups:control group,Wnt3a group,H2O2treatment group,Wnt3a treatment group.The melanocytes were treated with 750 μmol/L H2O2for oxidative stress damage.The activity of cells was detected by MTT assay.The apoptosis rate was examined by flow cytometry.The ROS production was observed by fluorescence microscopy and flow cytometry. The Nrf2/ARE pathway activation was determined with an ARE-driven luciferase reporter construct. The expressions of Nrf2 and HO-1 were examined by Western blot. Results Compared with control group,cell activity decreased(P<0.01),the ratio of apoptosis increased(P<0.01),ROS production was raised in H2O2treatment group(P<0.01). While compared with H2O2treatment group,cell activity was relieved(P<0.05), the ratio of apoptosis decreased(P<0.05), ROS production was de-clined in Wnt3a treatment group(P<0.05).Wnt3a up-regulated the transcriptional activity of Nrf2 as determined with improving the relative activity of luciferase regulated by the antioxidant response element ARE. Wnt3a also increases the expression of Nrf2 and HO-1 protein levels. Conclusions Wnt3a protects the melanocytes against oxi-dative injury by H2O2. The mechanism would be related to the activation of Nrf2/ARE pathway.
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Objective To investigate the regulating effect of fire needling on Wnt andβ-catenin genes of Wnt/β-catenin pathway in neural stem cells in spinal cord injury.Methods Sixty female SD rats were randomly allocated, including 5 rats to a blank group, 5 rats to a sham operation group, 25 rats to a model group and 25 rats to a fire needling group. The model and fire needling groups of rats were again separately divided into 1 d, 3 d, 7 d, 10 d and 14 d groups. A model was made using modified Allen's method in the model and fire needling groups. The BBB score was recorded and the expressions of Wnt andβ-catenin genes were determined in every group.Results In the model and fire needling groups, the BBB scores were significantly higher at 7 and 10 days than at the previous time point and there was a statistically significant difference (P<0.05). The BBB score at every time point after treatment was significantly higher in the fire needling group than in the model group and there was a statistically significant difference (P<0.05). Wnt3a gene expressions increased in the model and fire needling groups at 7 and 10 days compare with the previous time point (P<0.05). At 7 and 10 days, Wnt3a gene expression was higher in the fire needling group than in the model group and there was a statistically significant difference (P<0.05). The changing tendency ofβ-catenin gene expression levels was basically the same as that of Wnt3a's.Conclusion Fire needling can modulate the expressions of Wnt3a andβ-catenin genes in neural stem cells in spinal cord injury.
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Objective To construct a eukaryotic vector of chicken-derived Wnt3a tagged with EGFP (pCAG-MCs-Wnt3a-EGFP) and investigate the influence to the proliferation and axonal formation of neural precursor cells when Wnt3a was overexpressed during the development of chick embryonic spinal cord. Methods Wnt3a gene was amplified from the total RNA obtained from chick embryonic spinal cord using molecular techniques, then connected with pCAG-MCs-EGFP to construct pCAG-MCs-Wnt3a-EGFP, which was identified by digestion and genetic sequencing. At embryonic day (E) 2.5-3.0, pCAG-MCs-Wnt3a-EGFP (experimental group) and pCAG-MCs-EGFP (control group) were transfected into the chick embryonic spinal cord using in vivo electroporation, respectively. Samples were collected at E4 (5 simples of each groups) and then conducted frozen section. The immunofluorescent staining was performed to detect the expression of Wnt3a and proliferating cell nuclear actigen (PCNA) for analyzing the relationship between Wnt3a and cell proliferation, and observe the axonal formation of neural precursor according to the green fluorescence of Wnt3a protein. Results pCAG-MCs-Wnt3a-EGFP was obtained and its gene sequencing was identical with the Gene bank. Green fluorescence was observed at E4 after pCAG-MCs-Wnt3a-EGFP transformed to chick spinal cord. In transversal section of chick embryonic spinal cord, the results of immunofluorescent staining showed Wnt3a was successfully overexpressed. Meanwhile, the amount of neurons projecting axons was dramatically decreased (n=3, P < 0.01), compared to the control group, concomitant with the significant elevation of PCNA level (n =3, P < 0.01). Conclusion pCAG-MCs-Wnt3a-EGFP is successfully constructed and our study confirmed that Wnt3a plays a vital role in the proliferation and axonal formation of neural precursor cells in the developing chick spinal cord.
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Objective To investigate the effect of geraniin on expression of Wnt3a protein and mRNA in bone marrow stromal cell (BMSC) from osteoporotic rats. Methods The model of osteoporosis (OP) was duplicated by ovariectomy in rats. BMSCs were isolated and cultured. BMSCs from shamed rats were routinly cultured and taken as normal control, and BMSCs from OP rats were divided into model group, 1μmol/L simvastatin positive group, and geraniin group (0.01, 0.1, 1, 10, 100 μmol/L), respectively. The methods of realtime-PCR and western blot were used to assay the protein and mRNA expression of wnt3a, respectively.Results As compared with normal control group, the protein and mRNA expression of wnt3a in model group were significantly suppressed;Compared with model group, 1 μmol/L simvastatin, and 0.1, 1, 10 and 100 μmol/L geraniin significantly increased the expression of wnt3a protein and mRNA. Conclusion It is suggested that geraniin activates wnt/β-catenin pathway though increasing the expression of signaling protein wnt 3a in BMSCs from OP rats. It would be beneficial to osteogenic differentiation of BMSCs and osteogenesis.