Effects of sulindac on oxidative stress in an autistic model induced by prenatal exposure to valproic acid

AIM: To investigate the effects of sulindac on oxidative stress in autism. METHODS: With an autistic model induced by prenatal exposure to valproic acid (VPA), we detected the expression of the signaling molecules of canonical Wnt pathway in the prefrontal cortex (PFC) and hippocampus (HC) of autistic rats treated with sulindac. The protein expression levels of glycogen synthase kinase 3β (GSK-3β), β-catenin and 4-hydroxynonenal (4-HNE) were observed by Western blotting. The mRNA expression of thioredoxin(Trx)1 and Trx2 was assessed by semi-quantitative RT-PCR.RESULTS: The protein level of GSK-3β and mRNA levels of Trx1 and Trx2 were lower, whereas the protein expression levels of β-catenin and 4-HNE were higher in VPA group than those in control group. In contrast, the protein levels of GSK-3β were significantly higher in the animals treated with both VPA and sulindac than those in VPA group, while the levels of β-catenin and 4-HNE were decreased.CONCLUSION: Sulindac attenuates oxidative stress in the pathogenesis of autism, suggesting the up-regulation of the Wnt/β-catenin signaling pathway disrupts oxidative homeostasis and further facilitates susceptibility to autism.     

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling

AIM: To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS: The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured. The protein expression of β-catenin, glycogen synthase kinase-3β (GSK-3β), c-Myc and cyclin D1 in the ASMC was determined by Western blot. After depressing the interaction between β-catenin and p300/CBP, the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry. Meanwhile, the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS: The protein levels of β-catenin, c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P<0.05). After depressing the interaction between β-catenin and p300/CBP, the cell activity of ASMC was decreased in asthma group compared with control group (P<0.05), and the change of the cell cycle distribution in asthma group was also more obvious (P<0.05). After inhibiting P38 MAPK activity, the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P<0.05).CONCLUSION: Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1, reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

Effects of GSK-3β knockdown by RNA interference on formation of keloid in vitro

AIM: To study the suppressive effect of glycogen synthase kinase-3β (GSK-3β) knockdown by RNA interference on the formation of keloid. METHODS: Human keloid fibroblasts (KFB) in vitro were transfected with 3 pairs of specific GSK-3β small interfering RNA (siRNA). The best siRNA to inhibit the GSK-3β expression in human KFB was screen by RT-PCR and Western blot. The expression of GSK-3β and related proteins at mRNA and protein levels in the KFB was determined by RT-PCR and Western blot.RESULTS: The GSK-3β siRNA1434 remarkably inhibited the expression of GSK-3β at mRNA and proteins levels in the human KFB. After transfection with GSK-3β siRNA, the protein levels of β-catenin, p-GSK-3β, Wnt2 and cyclin D1 were all decreased. KFB growth became slow. With the extension of time, the inhibition of cell growth increased, and the cell doubling time was significantly delayed. CONCLUSION: siRNA targeting GSK-3β efficiently knocks down the expression of GSK-3β in the human KFB, and inhibits the activation of Wnt signaling pathway, thus inhibiting the growth of keloid. GSK-3β may be a potential therapeutic target for keloid.     

Thiazolidinedione improves Alzheimer-like changes in the hippocampus of type 2 diabetic rats by Wnt pathway

AIM: The abnormal level of insulin and glycemia in type 2 diabetes mellitus(T2DM) are important risk factors of Alzheimer's disease (AD). To explore the mechanism that thiazolidinedione (TZD) decreases the incidence of AD in T2DM, we use TZD on T2DM rats for an intervention and detect the change of Wnt pathway before and after the intervention.METHODS: To establish a T2DM model, the rats were fed with high glucose, high fat and high protein for 8 weeks, and then injected with STZ. TZD was administered intragastrically for 2 and 4 weeks and the rats were divided into TZD2W and TZD4W groups, respectively. Plasma insulin level was measured by RIA method, and the plasma glucose was detected by glucose-oxidase method. Total tau level, the phosphorylation level of tau at individual phosphorylation sites and the level of amyloid β precursor protein(APP), β-catenin, glycogen synthase kinase-3β (GSK-3β) and PPARγ in rat hippocampus were analyzed by Western blotting. The activity of GSK-3β in the hippocampus of rats was determined using γ- -ATP and the specific peptide substrate. The level of APP was also determined by immunochemistry. The insulin resistant (IR) degree was valued by HOMA-IR.RESULTS: Glycemia level in T2DM and TZD2W groups was obviously higher than that in control group. No significant difference of glycemia level between TZD4W and control group was observed. Plasma insulin levels in all groups were evidently higher than that in control group. The IR degree in T2DM and TZD2W groups increased significantly as compared to control group, but no obvious difference between TZD4W and control group was observed. The level of phosphorylated tau protein at site Ser199/202 and Ser422, and APP level in hippocampus of T2DM rats were found to be notably raised as compared to control group, but the level of phosphorylated tau protein at those sites and APP level were decreased gradually. No change of the PPARγ level was found in the hippocampus in T2DM and control group, but a notable increase was observed after TZD intervention. There was a decrease in β-catenin level in hippocampus of T2DM rats, which increased after TZD intervention for 2 and 4 weeks. There was a rise of GSK-3β activity in T2DM rats, which decreased after intervention.CONCLUSION: These findings suggest that TZD may improve the AD-like changes in the hippocampus of T2DM rats by up-regulation of Wnt pathway, which acts before the insulin signal transduction in the contribution of AD-like changes in T2DM rats.     

Overexpression of HBXIP in HepG2 cells induces cell migration and regulates expression of β-catenin

AIM: To investigate the effects of hepatitis B virus X-interacting protein(HBXIP) in hepatic cancer cells on the cell migration and expression of β-catenin. METHODS: Transwell assay was used to assess the cell migration. Gelatin zymography was used to observe the activity of matrix metalloproteinase 9 (MMP-9). The expression of MMP-9, glycogen synthase kinase 3β(GSK-3β), p-GSK3β, β-catenin and p-β-catenin in HepG2 cells was determined by Western blotting. RESULTS: HepG2 cells which stably overexpressed HBXIP (HepG2-HBXIP) exhibited higher migration ability than the control cells. The results of the gelatin zymography assay showed that HBXIP overexpression increased the activity of MMP-9 in HepG2 cells. The results of Western blotting indicated that HBXIP increased the expression of MMP-9 and β-catenin, inhibited the phosphorylation of β-catenin and promoted the phosphorylation of GSK-3β (Ser9). CONCLUSION: HBXIP regulates the GSK-3β/β-catenin signaling pathway, resulting in a significant improvement of hepatocellular carcinoma cell migration.     

Expression changes of Wnt/β-catenin signaling pathway in diabetic ulcer

AIM: To observe the effect of Wnt/β-catenin signaling pathway on diabetic ulcer. METHODS: Diabetic animal model was established in the female Wistar rats by intraperitoneal injection of low-dose streptozotocin following high-fat diet feeding. A circular wound was made on the dorsum of the rats in both control group and diabetic group. The condition of wound healing was recorded and the structures of the wound tissues were observed by HE staining in the 2 groups at 3, 7 and 14 d after wounding. The expression of β-catenin, GSK-3β and Rspo-3 at mRNA and protein levels in the wound tissues was detected by RT-PCR and ELISA. RESULTS: In diabetic group, the wound healing rate was lower (P<0.05), and the inflammatory cells, fibroblast cells and new capillaries in the wound tissues were fewer than those in control group. The expression of β-catenin and Rspo-3 at mRNA and protein levels in the wound tissues in control group was significantly higher than those in diabetic group, and the expression of GSK-3β was exactly the opposite (P<0.05). CONCLUSION: The down-regulation of Wnt/β-catenin probably resultes from the decreased level of Rspo-3, which may be one of the reasons for delaying the diabetic ulcer healing.     

Wnt signaling pathway is involved in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells

AIM：To investigate the changes of Wnt signaling pathway in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：The BMSCs were isolated from SD rats, purified by differential time adherent method and divided into control group and catalpol (1.0 mg/L) group. Flow cytometry was used to detect the proliferation index of BMSCs. The mRNA levels of Wnt3a, Wnt5a, Wnt11 and β-catenin was evaluated by real-time PCR. In addition, the protein expression level of β-catenin was determined by Western blotting. RESULTS：Prolife-ration index was increased from 8.90%±0.46% to 17.93%±1.68% after treatment with catalpol (P<0.01). Compared with control group, the mRNA expression of Wnt5a, Wnt11 and β-catenin was all increased with catalpol treatment. No difference of Wnt3a mRNA expression between control group and catalpol group was observed. Meanwhile, the protein expression of β-catenin was increased in catalpol group compared with control group. CONCLUSION：Catalpol promotes BMSCs going into the cell cycle. Classical and non-classical Wnt signaling pathways are activated in this process.     

Intracerebroventricular injection of streptozotocin induces dysfunction of insulin signaling in brain and cognitive deficits in rats

AIM：To investigate the effect of insulin signaling on the development of Alzheimer disease (AD) and to explore its potential mechanisms. METHODS：The rats were treated with streptozotocin (STZ, 3 mg/kg) intracerebroventricularly (ICV) at the 1st day and the 3rd day of the experiment to induce dementia model. Twenty-one days after the injection of STZ at the 1st day, spatial learning and memory of the rats were determined by Morris water maze test. The expression levels of insulin-degrading enzyme (IDE), glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β (p-GSK-3β), tau and phosphorylated tau (p-tau) were measured by Western blotting. The levels of amyloid β-proteins (Aβ1-40 and Aβ1-42) in the brain of the rats were detected by the method of immunohistochemistry. The mRNA levels of insulin, insulin receptor, tau and IDE were measured by real-time RT-PCR. RESULTS：ICV-STZ deteriorated the abilities of spatial learning and memory of the rats and reduced the activity of IDE and the mRNA levels of insulin and insulin receptor. STZ treatment enhanced GSK-3β activity and tau phosphorylation. The levels of Aβ1-40 and Aβ1-42 in cerebral cortex were significantly increased in the rats treated with STZ. CONCLUSION：ICV-STZ results in AD-like behaviors and pathological changes via damaging the brain insulin signaling, indicating that insulin signaling may play important roles in the AD pathogenesis.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Effects of GSK-3β inhibitor on proliferation and apoptosis of SW480 cells

AIM: To investigate the mechanism and the effect of glycogen synthase kinase 3β (GSK-3β) inhibitor (2’Z,3'E)-6-bromoindirubin-3'-oxime (BIO) on the protein expression of β-catenin and Bcl-2, and proliferation and apoptosis in colon carcinoma SW480 cells.METHODS: The immunohistochemical staining and Western blotting were performed to detect the protein expression of β-catenin, cyclin D1 and Bcl-2. The cell cycle distribution and apoptotic rate were detected by flow cytometry. The morphologic features of SW480 cells before and 24 h after BIO exposure at different concentrations were observed under microscope with HE staining.RESULTS: Compared with the untreated SW480 cells, the protein expression of β-catenin significantly increased and some β-catenin positive nuclear staining positive cells appeared in BIO treated cells. and The cells exposed to BIO showed that the cyclin D1 protein and the cells in S stage and G₂/M stage moderately increased, the protein level of Bcl-2 moderately decreased, and the cell apoptosis rate was significantly lower than those in control cells. Furthermore, the morphological changes of the SW480 cells were observed 24 h after BIO treatment. CONCLUSION: Our results indicate that GSK-3β inhibitor BIO participates in the cellular processes of promoting proliferation and inhibiting apoptosis in colon carcinoma cells. The mechanisms are mainly associated with activating the β-catenin pathway and regulating the balance of Bcl-2 pathway, and the up-regulation of β-catenin is most likely the possible factor for SW480 cell regression.     

Effect of EZH2 regulating Wnt/β-catenin signaling pathway on apoptosis of brain glioma cells

AIM: To investigate the effect of enhancer of zeste homolog 2 (EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87, H4 and U251 and normal human astrocytes (NHA) was detected by RT-qPCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway, the apoptosis rate was measured by flow cytometry, and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87, H4 and U251 were significantly higher than those in NHA (P<0.05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells (P<0.05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells, the apoptotic rate was significantly increased, and the activity of caspase-3 was significantly increased in the cells (P<0.05). Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2, and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Effects of stachydrine hydrochloride on experimental acute cerebral infarction in rats

AIM: To observe the therapeutic effect of stachydrine hydrochloride on experimental acute cerebral infarction in rats and to explore the underlying mechanisms. METHODS: SD rats (n=75) were randomly divided into 5 groups: sham group, cerebral infarction model group, and stachydrine hydrochloride (10 mg/kg, 20 mg/kg and 40 mg/kg) treatment groups. After the establishment of cerebral infarction model, the rats were given stachydrine hydrochloride at dose of 10 mg/kg, 20 mg/kg or 40 mg/kg by gavage daily for 14 d. The impairment of neurological function in each group was scored. The cerebral infarction volume and brain water content were measured. Moreover, the protein levels of β-catenin, cyclin D1, glycogen synthase kinase 3β (GSK-3β) and p-GSK-3β in the brain tissues were detected by Western blot. RESULTS: Compared with cerebral infarction group, the score of neurological function impairment, cerebral infarction volume and brain water content were significantly decreased in stachydrine hydrochloride treatment groups. In addition, the protein levels of β-catenin, cyclin D1 and p-GSK-3β were markedly increased after stachydrine hydrochloride treatment. CONCLUSION: Stachydrine hydrochloride protects against experimental acute cerebral infarction through activation of Wnt/β-catenin signaling pathway.     

Promoting cell viability and migration of gastric cancer cells by PRRX2 via activation of Wnt/β-catenin signaling pathway

AIM: To study the effect of paired-related homeobox 2 (PRRX2) gene on the viability and migration ability of gastric cancer cells, and to analyze the underlying mechanism of regulating Wnt/β-catenin signaling pathway.METHODS: The expression of PRRX2 in gastric cancer and normal gastric tissue and the correlation between PRRX2 expression in gastric cancer tissues with the overall survival rate of gastric cancer patients were analyzed by bioinformatics. The small interfering RNA (siRNA) and over-expressed plasmids of PRRX2 were transfected into gastric cancer cells MGC-803 and SGC-7901, respectively. MTT assay and Transwell assay were used to detect the viability and migration ability of gastric cancer cells. Western blot and TOPflash/FOPflash dual-luciferase reporter gene assay were used to detect the activity of Wnt/β-catenin signaling pathway. Co-immunoprecipitation was used to detected the interaction between PRRX2 and β-catenin proteins.RESULTS: Knockdown of PRRX2 attenuated the viability and migration ability of gastric cancer cell line MGC-803 (P<0.05). Over-expression of PRRX2 enhanced the viability and migration ability of SGC-7901 cells (P<0.05), increased the protein levels of β-catenin, c-Myc and cyclin D1 (P<0.05) and the activity of TOPflash/FOPflash dual-luciferase reporter gene (P<0.05). PRRX2 interacted with β-catenin protein in gastric cancer cells.CONCLUSION: PRRX2 promotes the viability and migration ability of gastric cancer cells, which may be related to Wnt/β-catenin signaling pathway.     

Scutellaria barbata flavonoids inhibits NFT aggregation and regulatory mechanism in rats induced by composited Aβ

AIM: To investigate the effects of Scutellaria barbata flavonoids (SBF) on neurofibrillary tangle (NFT) aggregation, tau protein phosphorylation and the regulated mechanism of glycogen synthase kinase (GSK) 3β and protein phosphatase (PP) 2A in the rats induced by amyloid β protein 25-35 (Aβ_25-35) in combination with AlCl₃ and recombinant human transforming growth factor (RHTGF)-β1(composited Aβ). METHODS: The male SD rats were used to establish the simulated Alzheimer disease (AD) model by intracerebroventricular injection of composited Aβ. The Morris water maze was applied for screening the successful model rats with learning and memory deficits. The successful model rats were daily and orally administrated with SBF at doses of 35, 70 and 140 mg/kg or positive control drug Ginkgo biloba leaves flavonoids (GLF) at 140 mg/kg for 37 d. The silver nitrate staining was used to determine the cortical NFT. The protein levels of total tau, phosphorylated protein of tau at Ser199 and Ser214 sites, GSK3β and PP2A in hippocampus and cortex were determined by Western blot. The mRNA expression of GSK3β and PP2A in the hippocampus and cortex was detected by RT-PCR. RESULTS: Compared with sham group, the cell number of positive NFT with silver nitrate staining in model rat cerebral cortex was significantly increased. The protein levels of phosphorylated tau protein at Ser199 and Ser214 sites, GSK3β in the hippocampus and cerebral cortex in the model rats dramatically elevated, and PP2A was marked decreased as compared with the sham group rats. Meanwhile, the mRNA expression of GSK-3β significantly increased but PP2A was decreased. However, these above abnormalities were differently attenuated by treating with SBF at different doses or GLF at 140 mg/kg for 37 d. CONCLUSION: SBF suppresses the NFT aggregation by inhibition of the regulatory functions of GSK-3β and PP2A, thus reducing the phosphorylation of tau protein.     

Role of Wnt signaling pathway in development of radioresistance in esophageal cancer

AIM:To investigate the role of Wnt signaling pathway in the development of radioresistance in esophageal cancer cells. METHODS:The mRNA expression of the genes involved in Wnt signaling pathway was determined by real-time RT-PCR. The protein levels involved in Wnt signaling pathway were also confirmed by Western blotting and immunofluorescence method. Xenograft assay was performed to compare the proliferation ability and tumorigenicity of the cell lines. RESULTS:Up-regulation of Wnt1 induced the activation of Wnt signaling pathway and the variation of the downstream genes. β-catenin was accumulated and nuclear-transferred. The expression of downstream targeted genes such as cyclin D1 and WISP1 was increased. The radioresistant esophageal cancer cells showed higher proliferation ability and tumorigenicity in nude mouse xenograft. CONCLUSION:Wnt signaling-induced acceleration of proliferation is important for the development of radioresistance in esophageal cancer cells.     

Over-expression of SCUBE2 suppresses epithelial-mesenchymal transition through Wnt/β-catenin signaling pathway in colorectal cancer cells

AIM: To study the effect of SCUBE2 on epithelial-mesenchymal transition (EMT) in colorectal cancer cells and its mechanism. METHODS: The expression of SCUBE2 in human colorectal cancer cell line HCT116 and normal colonic cell line FHC was detected by real-time PCR and Western blot. HCT116 cells were transfected with GV144-SCUBE2 to over-express SCUBE2, and then the cell viability, migration, and apoptosis were determined by MTT assay, Transwell assay and flow cytometry, respectively. The expression of EMT markers (E-cadherin, vimentin, and Snail), β-catenin, c-Myc and cyclin D1 in the HCT116 cells was analyzed by real-time PCR or Western blot after transfection with GV144-SCUBE2 for 6 h, followed by the stimulation of 10 μg/L recombinant TGF-β1 protein for 48 h. Additionally, the EMT process of HCT116 cells, which were stimulated by TGF-β1, over-expressed SCUBE2, and treated with Wnt/β-catenin pathway activator lithium chloride (LiCl) or inhibitor XAV93920, was analyzed by Western blot. RESULTS: Compared with FHC cells, the expression of SCUBE2 in the HCT116 cells was significantly decreased. The viability and migration ability of the HCT116 cells were suppressed by SCUBE2 over-expression, but the apoptosis was not markedly changed. Elevated expression of SCUBE2 increased E-cadherin expression, and decreased the expression of vimentin, Snail, β-catenin, c-Myc and cyclin D1 induced by TGF-β1. Treatment with LiCl blocked but treatment with XAV93920 enhanced the effects of SCUBE2 on EMT. CONCLUSION: Over-expression of SCUBE2 may inhibit the cell growth and migration, and suppress EMT through Wnt/β-catenin signaling pathway.     

Dominant negative human epidermal growth factor receptor induces G0/G1 arrest in human gastric cancer cells

AIM：To explore the effect of dominant negative epidermal growth factor receptor (DNEGFR) on the cell cycle of human gastric cancer cells. METHODS：Two human gastric cancer cell lines were used in the study. The cells were divided into 6 groups, including untreated SGC-7901 cells (US group), SGC-7901 cells stably transfected with pEGFP-N1 (ES group), SGC-7901 cells stably transfected with pEGFPN1-DNEGFR (DS group), untreated NCI-N87 cells (UN group), NCI-N87 cells stably transfected with pEGFP-N1 (EN group), and NCI-N87 cells stably transfected with pEGFPN1-DNEGFR (DN group). The cell cycle was determined by flow cytometry. The protein levels of cyclin-dependent kinase 2 (CDK2), cyclin D1, phosphorylated glycogen synthase kinase 3 beta at Ser9 ［p-GSK-3β (Ser9)］, p21 and p27 were detected by Western blotting. RESULTS：Transfection of the human gastric cancer cells with pEGFPN1-DNEGFR led to G0/G1 arrest, and down-regulated CDK2, cyclin D1, p-GSK-3β (Ser9) and up-regulated p21 and p27 as well. CONCLUSION：DNEGFR down-regulates cyclin D1 by activating GSK-3β, down-regulates CDK2, and up-regulates p21 and p27, which induce G0/G1 arrest in human gastric cancer cells in the end.     

Effects of RARG on viability and migration ability of gastric cancer cells

AIM:To investigate the effect of retinoic acid receptor gamma (RARG) on the viability and migration ability of gastric cancer cells. METHODS:The expression of RARG in gastric cancer and normal gastric tissues and its correlation with the overall survival rate of gastric cancer patients were analyzed by bioinformatics. The expression of RARG was promoted and inhibited by over-expression plasmid transfection and RNA interference technique in gastric can-cer cells in vitro, respectively. MTT and Transwell assays were used to detect the effect of RARG on the viability and migration ability of gastric cancer cells. The effect of RARG on regulating the Wnt/β-catenin signaling pathway was evaluated by Western blot and TOP/FOP dual-luciferase reporter assay. The protein interaction of RARG and β-catenin was determined by co-immunoprecipitation and immunofluorescence co-localization assay. RESULTS:Over-expression of RARG enhanced the viability and migration ability of gastric cancer SGC7901 cells (P<0.05). Knockdown of RARG attenuated the viability and migration ability of gastric cancer MGC-803 cells (P<0.05). At the same time, RARG over-expression increased the protein expression levels of β-catenin, c-Myc, cyclin D1, Twist and Snail (P<0.05), and the activity of TOP/FOP dual-luciferase reporter gene (P<0.05). In addition, RARG interacted with β-catenin protein in the gastric cancer cells. CONCLUSION:RARG promotes the viability and migration ability of gastric cancer cells via activating the Wnt/β-catenin signaling pathway, thus playing an important role in the development of gastric cancer.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.