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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Changes of canonical Wnt signaling pathway in brains of rats with autism induced by prenatal exposure to valproic acid

AIM：To investigate the roles of the canonical Wnt pathway in autism. METHODS：Using an autistic model induced by prenatal exposure to valproic acid (VPA), we detected the expression of the signaling molecules of the canonical Wnt pathway in the prefrontal cortex (PFC) and hippocampus formation (HF) of autistic rats. The expression levels of glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β, β-catenin and phosphorylated β-catenin were observed by Western blotting. The mRNA expression of GSK-3β, β-catenin, c-Myc and cyclin D1 was assessed by semi-quantitative RT-PCR. RESULTS：The results of Western blotting showed that inactivated GSK-3β (Ser9) phosphorylation was significantly increased, and inhibitory β-catenin (Ser33/37/Thr41) phosphorylation was obviously decreased compared with control group. The results of RT-PCR showed that the mRNA levels of β-catenin, c-Myc and cyclin D1 increased, and GSK-3β was significantly enhanced in VPA-exposed rats compared with the controls. CONCLUSION：Increased activity of canonical Wnt pathway in the PFC and HF of autistic rats may contribute to the susceptibility to autism.     

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.     

Role of HMGA2 in epithelial-mesenchymal transition of gastric cancer cells

AIM:To investigate the role of HMGA2 in the epithelial-mesenchymal transition (EMT) in gastric cancer cells. METHODS:The expression of HMGA2 in human gastric cancer cell lines with different degrees of differen-tiation (MKN45, MKN28 and SGC7901) and immortalized human gastric epithelial cell line GES-1 was determined by Western blot and RT-qPCR. pcDNA3.0-HMGA2 plasmid was transfected into the MKN28 cells by liposome method. Transfection of si-HMGA2 interference fragments into MKN45 cells was also performed. The transfection efficiency was evaluated by Western blot and RT-qPCR. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the cell viability were measured by CCK-8 assay. The effects of HMGA2 over-expression in the MKN28 cells on the cell migration and invasion abilities were detected by wound healing and Transwell invasion assays. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the expression of EMT-related markers E-cadherin, N-cadherin, vimentin at mRNA and protein levels were determined by RT-qPCR and Western blot. The changes of Wnt/β-catenin signaling pathway-related molecules in the MKN28 cells with HMGA2 over-expression were also determined by RT-qPCR. RESULTS:The expression levels of HMGA2 were quite different in different differentiation levels of gastric cancer cells (P<0.05). The increased expression level of HMGA2 in MKN28 cells inhibited the cell viability (P<0.05), while the decreased expression level of HMGA2 in MKN45 cells promoted the cell viability (P<0.05). The increased expression level of HMGA2 in MKN28 cells promoted cell migration and invasion (P<0.05), changed the expression of EMT-related markers (P<0.05), while the decreased expression level of HMGA2 in the MKN45 cells changed the expression of EMT-related markers (P<0.05). The increased expression level of HMGA2 in the MKN28 cells significantly increased the mRNA levels of β-catenin in the Wnt/β-catenin pathway and the downstream molecules c-Myc and cyclin D1 (P<0.05). CONCLUSION:HMGA2 is closely related to the migration and invasion abilities of gastric cancer cells. Moreover, it promotes the EMT process of gastric cancer cells by activating Wnt/β-catenin pathway.     

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.     

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.     

Influence of paricalcitol on renal tubulointerstitial fibrosis in diabetic nephropathy

AIM: To investigate the effect of paricalcitol (P) on renal tubulointerstitial fibrosis and the underlying mechanisms in diabetic nephropathy (DN).METHODS: DN rat model was induced by a single intraperitoneal injection of streptozotocin after fasting. The animals were randomly divided into 2 groups:the DN rats in paricalcitol-intervened group (group P) were injected intraperitoneally with paricalcitol dissolved in propylene glycol after the day when the model was induced successfully at a dose of 0.4 μg/kg (3 times a week); the DN rats in DN group (group D) were given isopyknic propylene glycol. Normal control group (group C) was also set up. The samples of blood, urine and renal tissue were collected after intervention of paricalcitol for 12 weeks. The biochemical indexes were measured. The renal tissues were used for pathologic observation and determining the expression of transforming growth factor-β1 (TGF-β1), Wnt-4, β-catenin and Klotho by immunohistochemistry and Western blotting. In addition, the correlation among the above indexes was analyzed.RESULTS: (1) Scr, BUN and 24 h urine protein increased significantly in group D compared with group C, while decreased in group P compared with group D (P<0.05). (2) The area of renal tubulointerstitial fibrosis increased in group D compared with group C, while decreased in group P compared with group D (P<0.05). (3) The expression of Klotho decreased, while the expression of TGF-β1, Wnt-4 and β-catenin increased in group D compared with group C (P<0.05). Compared with group D, the expression of Klotho increased, while the expression of TGF-β1, Wnt-4 and β-catenin decreased in group P (P<0.05). (4) The expression of Klotho was negatively correlated with the fibrosis area, TGF-β1, Wnt-4 and β-catenin (P<0.05).CONCLUSION: Paricalcitol inhibits renal tubulointerstitial fibrosis in DN by promoting the expression of renal Klotho, and inhibiting Wnt/β-catenin signaling pathway activation and TGF-β1 synthesis.     

Expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and its clinical significance

AIM: To evaluate the expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and the significance. METHODS: The patients with breast cancer (n=150) in our hospital from January 2015 to January 2017 were selected as study object. The tumor tissue samples of these patients were obtained from paraffin section of breast cancer by surgical resection with complete clinicopathological data. The corresponding paracancerous tissue sam-ples were taken from the non-tumor tissue samples from the above breast cancer patients, which were 0.5~1 cm away from the tumor tissue. The methods of real-time PCR and Western blot were performed to examine the expression of Wnt-1 and β-catenin at mRNA and protein levels. Human breat cancer MCF-7 cells were divided into 3 groups:control group (MCF-7 cells without treatment), agonist group[MCF-7 cells+Wnt3a (1 mg/L)] and antagonit group[MCF-7 cells+DKK1 (16 μmol/L)]. The expression of Wnt-1 and β-catenin at mRNA and protein levels was detected by real-time PCR and Western blot. RESULTS: Compared with the paracancerous tissues, the expression levels of Wnt-1 and β-catenin were higher in tumor tissues at mRNA and proteins levels (P<0.05). Notably, the positive expression rates of Wnt-1 and β-catenin were significantly higher in tumor tissues than that in the paracancerous tissues. Furthermore, Wnt-1 expression was associated with tumor metastasis (χ²=5.352, P=0.021), tumor stage (χ²=9.412, P=0.002) and tumor size (χ²=9.412, P=0.002). In addition, β-catenin expression was also associated with tumor metastasis (χ²=9.851, P=0.002) and tumor stage (χ²=5.661, P=0.017). Compared with control group, the expression of Wnt-1 and β-catenin at mRNA and protein levels in agonist group was increased (P<0.05),while that in antagonist group was decreased (P<0.05). CONCLUSION: The expression levels of Wnt-1 and β-catenin related with Wnt/β-catenin signaling pathway are increased in the breast cancer, which are closely related to the malignant state of the tumor.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Effects of human bone morphogenetic protein 2 and 9 on proliferation,apoptosis and migration of human gastric carcinoma MNK-45 cells

AIM: To investigate the effects of human bone morphogenetic protein 2 (BMP2) and BMP9 on the proliferation, apoptosis and migration of human gastric carcinoma cell line MNK-45. METHODS: Immunocytochemical staining, MTT assay, wound-healing test, Transwells migration test, Hoechst 33258 staining and flow cytometry (FCM) were used to determine the infection of AdBMP2 and AdBMP9 on the proliferation, apoptosis and migration of MNK-45 cells. The expression of GSK-3β (including p-GSK-3β and total GSK-3β) and β-catenin in MNK-45 cells was also detected by Western blotting. RESULTS: The proliferation of MNK-45 cells was inhibited from the third day on and in a time-dependent manner after infected with AdBMP2 and AdBMP9. The results of Hoechst 33258 staining and FCM proved that apoptosis rates in BMP2 group and BMP9 group were higher than that in GFP group. Both wound-healing test and Transwell experiment indicated that up-regulating the expression of BMP2 and BMP9 inhibited the migration of MNK-45 cells. The phosphorylation levels of GSK-3β in BMP2 group and BMP9 group were higher than that in GFP group. However, no significant change of β-catenin among groups was observed. CONCLUSION: Up-regulation of BMP2 and BMP9 expression inhibits the proliferation of MNK-45 cells.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Effects of hydrogen sulfide on high glucose-induced injury of mouse glomerular podocytes

AIM: To investigate the effect of hydrogen sulfide (H2S) on high glucose （HG）-induced injury of the mouse podocyte cell line MPC5. METHODS: The cultured MPC5 cells were randomly divided into 4 groups: HG group, normal glucose (NG) group, NG+DL-propargylglycine (PPG) group, and HG+NaHS group. After treated for a certain time, the cells were collected for further detection. The expression of zonula occludens-2 (ZO-2), nephrin, β-catenin and cystathionine γ-lyase (CSE) was determined by Western blotting. RESULTS: High glucose significantly reduced the expression of nephrin, ZO-2 and CSE (P<0.05), while the level of β-catenin was elevated obviously (P<0.05), all in a time-dependent manner. NG+PPG inhibited the levels of ZO-2 and nephrin significantly (P<0.05), and increased the level of β-catenin (P<0.05), all in a PPG concentration-dependent manner. HG+NaHS induced a more significant increase in the levels of ZO-2 and nephrin as compared with HG group (P<0.01), whereas a severe reduction of β-catenin in HG+NaHS group was observed as compared with HG group. Compared with NG group, the expression of ZO-2 and nephrin was decreased obviously, and the level of β-catenin was increased in HG+NaHS group. CONCLUSION: Down-regulation of CSE contributes to hyperglycemia-induced podocyte injury. Exogenous H2S protects against hyperglycemia-induced podocyte injury, possibly through up-regulation of ZO-2 and subsequent suppression of Wnt/β-catenin pathway.