Methods To explore the anti-tumor effect of the zeste homolog enhancer 2 (EZH2) inhibitor EPZ011989 on retinoblastoma (RB) and its potential mechanism, and to evaluate whether it exerts an indirect neuroprotective effect on the optic nerve by regulating the epithelial-mesenchymal transition (EMT)-like process and the Wnt/β-catenin signaling pathway. MethodsWestern blotting (WB) was used to detect the expression of EZH2 protein in human retinal pigment epithelial cells (ARPE-19) and RB cells (Y-79, WERI-Rb-1). The half-maximal inhibitory concentration (IC50) of EPZ011989 on Y-79 and WERI-Rb-1 cells was determined using the Cell Counting Kit-8 (CCK-8). Y-79 and WERI-Rb-1 cells were treated with EPZ011989 at their respective IC50 concentrations and divided into untreated control groups (NC), dimethyl sulfoxide (DMSO) groups, and EPZ011989 treatment groups. Meanwhile, ARPE-19 cells were assigned to DMSO and EPZ011989 treatment groups. To verify the effect of EPZ011989 on the Wnt/β-catenin signaling pathway and its mechanism specificity, Y-79 and WERI-Rb-1 cells were respectively assigned to DMSO, EPZ011989 treatment, and EPZ011989 plus LiCl co-treatment groups. Cell proliferation, cell cycle, and apoptosis in different treatment groups were assessed by CCK-8 and flow cytometry, while WB was used to detect the expression of key proteins in the Wnt/β-catenin signaling pathway and EMT-related proteins.Animal experiment: 15 male C57BL/6J mice were randomly divided into 3 groups (n=5). A nude mouse RB subcutaneous transplantation tumor model was established by subcutaneous inoculation of Y-79 cells. The mice were orally administered with equal volume of DMSO (Control group), 100 mg/kg EPZ011989 (EPZ011989 low-dose group), and 300 mg/kg EPZ011989 (EPZ011989 high-dose group), twice a day for 4 weeks. Tumor volume and weight were monitored, and the expression levels of proliferation markers (Ki67) and EZH2 protein in the tumor-bearing tissues were detected by immunohistochemistry, and the expression levels of key proteins of the Wnt/β-catenin pathway in the tumor-bearing tissues were detected by WB. One-way analysis of variance was used for comparisons among multiple groups, and Tukey's post hoc test was used for pairwise comparisons between groups. ResultsThe expression levels of EZH2 protein in Y-79 and WERI-Rb-1 cells were significantly higher than those in ARPE-19 cells (P＜0.01). The IC50 values of EPZ011989 for Y-79 and WERI-Rb-1 cells were 1.851 μmol/L and 3.949 μmol/L, respectively. At these concentrations, EPZ011989 significantly inhibited the viability of both RB cell lines (P＜0.001) without significantly affecting the viability of ARPE-19 cells. Flow cytometry results showed that compared to the DMSO group, EPZ011989-treated Y-79 and WERI-Rb-1 cells exhibited consistent changes: the proportion of cells in the G0/G1 phase was significantly decreased (P＜0.001), while the proportions in the S and G2/M phases were significantly increased (P＜0.05); simultaneously, the apoptosis rates of both cell lines were significantly elevated (P＜0.001). Western blot analysis revealed that, compared to the DMSO group, EPZ011989 treatment significantly upregulated E-cadherin protein expression (P＜0.001) and downregulated N-cadherin and Vimentin expression (P＜0.001) in both RB cell lines, while also inhibiting the expression of key proteins in the Wnt/β-catenin pathway (Wnt1, β-catenin, c-MYC, Cyclin D1) (P＜0.01). In the rescue experiment with co-treatment of LiCl, compared with the EPZ011989 treatment group, the changes in cell cycle, increased apoptosis, inhibited Wnt/β-catenin pathway proteins (P＜0.001), and reversed EMT-like process-related proteins (down-regulation of E-cadherin, up-regulation of N-cadherin and Vimentin) caused by EPZ011989 were partially reversed in the EPZ011989+LiCl co-treatment group (P＜0.01). Tumor tissue detection showed that the tumor size in the high-dose EPZ011989 group was the smallest compared with the Control group and the low-dose EPZ011989 group. Compared with the Control group, the tumor volume and weight in the low-dose EPZ011989 group and the high-dose EPZ011989 group were significantly reduced (P < 0.001). Moreover, the reduction in tumor volume and weight in the high-dose EPZ011989 group was more significant (P＜0.001). The results of immunohistochemistry and WB assays showed that compared with the Control group, the expression of Ki67, EZH2, and key proteins of the Wnt/β-catenin pathway (Wnt1, β-catenin, c-MYC, Cyclin D1) in the low-dose EPZ011989 group and the high-dose EPZ011989 group were significantly decreased (P＜0.001), and the expression levels of these proteins in the high-dose group were the lowest. Conclusion: EPZ011989 inhibits the malignant progression of RB by suppressing the activation of the Wnt/β-catenin signaling pathway and may indirectly protect the function of the optic nerve by reducing the risk of optic nerve compression and regulating the EMT-like process and the Wnt/β-catenin signaling pathway. ConclusionEPZ011989 inhibits the malignant progression of RB by suppressing the activation of the Wnt/β-catenin signaling pathway, and may protect the optic nerve function indirectly by reducing the risk of optic nerve compression and regulating EMT-like processes and the Wnt/β-catenin signaling pathway.
Objective To investigate the expressions of CXCR4 and β-catenin in pancreatic cancer, explore the relationship between them, and explore the possible biomarkers about invasion and metastasis of pancreatic cancer. Methods Forty-eight samples of pancreatic cancer and 20 samples of normal pancreas tissues were selected. The expressions of CXCR4 and β-catenin were examined by the immunohistological technique. Spearman, Chi-square， and rank test were used to analyze the relation between the protein expressions and clinical characteristics. Survival analysis was evaluated by Kaplan-Meier product limit method and Log-rank test. Variables were evaluated by Cox proportional hazards analysis. The size of test was 0.05. Results The positive expression rates of CXCR4 and β-catenin in pancreatic cancer tissues were 85.4% (41/48) and 75.0% (36/48), respectively. Co-expression rate of CXCR4 and β-catenin was 70.8% (34/48). There were significant differences between various CXCR4 staining and lymph node metastasis and TNM stage (P=0.012, 0.005, respectively). β-catenin positive expression was associated with lymph node metastasis (P=0.047). However, abnormal β-catenin positive expression could not determine the clinical survival. Kaplan-Meier estimated curves suggested that clinical prognosis was poor for patients with CXCR4 expression. Multivariate analysis showed that CXCR4, late TNM stage, and lymph node metastasis were independent prognostic factors for pancreatic cancer. Conclusions Both CXCR4 and β-catenin abnormally express in pancreatic cancer. CXCR4 may be an important marker for pancreatic cancer progression.
ObjectiveTo investigate the effects of hypoxia inducible factor 1α (HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) into vascular endothelial cells.MethodsSHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase (ALP) staining after osteogenic differentiation culture. The SHED were divided into blank control group (SHED without any treatment), empty group (SHED infected with empty lentivirus), HIF-1α overexpression group (SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group (SHED interfered by IWR-1), and combination group (HIF-1α overexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor (vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1 (VCAM-1), KDR (Kinase-inserted domain containing receptor), and VE-cadherin (VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3β (p-GSK3β) and β-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) phagocytosis.ResultsAfter identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly (P<0.05). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly higher (P<0.05), the relative expression of p-GSK3β protein was significantly lower (P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased (P<0.05) in the HIF-1α overexpression group; while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group (P<0.05). Compared with the HIF-1α overexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly lower (P<0.05), the relative expression of p-GSK3β protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups (P<0.05).ConclusionOverexpression of HIF-1α can promote SHED to differentiate into vascular endothelial cells by activating Wnt/β-catenin signaling pathway.
ObjectiveTo summarize the active changes of Wnt signaling pathway in osteoarthritis (OA) as well as the influence and mechanism of dual-targeted regulation on cartilage and subchondral bone and the role of crosstalk between them on OA process.MethodsThe relevant literature concerning the articular cartilage, subchondral bone, and crosstalk between them in OA and non-OA states by Wnt signaling pathway in vivo and vitro experimental studies and clinical studies in recent years was reviewed, and the mechanism was analyzed and summarized.ResultsWnt signaling can regulate the differentiation and function of chondrocytes and osteoblasts through the classic β-catenin-dependent or non-classical β-catenin-independent Wnt signaling pathway and its cross-linking with other signaling pathways, thereby affecting the cartilage and bone metabolism. Moreover, Wnt signaling pathway can activate the downstream protein Wnt1-inducible-signaling pathway protein 1 to regulate the progress of OA and it also can be established gap junctions between different cells in cartilage and subchondral bone to communicate molecules directly to regulate OA occurrence and development. Intra-articular injection of Wnt signaling inhibitor SM04690 can inhibit the progress of OA, and overexpression of Wnt signaling pathway inhibitor Dickkopf in osteoblasts can antagonize the role of vascular endothelial growth factor work on chondrocytes and inhibit the catabolism of its matrix.ConclusionThe regulation of metabolism and function of cartilage and subchondral bone and crosstalk between them is through interactions among Wnt signaling pathway and molecules of other signaling. Therefore, it plays an vital role in the occurrence and development of OA and is expected to become a new target of OA treatment through intervention and regulation of Wnt signaling pathway.
ObjectiveTo investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism.MethodsTen patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the in situexpression of β-catenin was detected by immunohistochemistry, and the protein expressions of collagen type Ⅱ and Aggrecan were detected by Western blot. The NPC were isolated and cultured from degenerative nucleus pulposus tissues. RES treated the third-passage NPC with (group B) or without IL-1β (group C), to further determine the protein expressions of collagen type Ⅱ and Aggrecan by Western blot, the unstimulated cells were set up as blank control group (group A). Moreover, NPC treated with small interfering RNA (siRNA) targeted silent SIRT1 or β-catenin were used to determine the protein and gene expressions of β-catenin and SIRT1 by Western blot and real-time fluorescence quantitative PCR. In addition, the third-passage NPC treated with complete medium (group 1), IL-1β (group 2), RES+IL-1β (group 3), and SIRT1-siRNA+RES+IL-1β (group 4) for 24 hours were used to detect the nuclear translocation of β-catenin by cell immunofluorescence staining. Finally, the third-passage NPC treated with complete medium (group Ⅰ), IL-1β (group Ⅱ), IL-1β+β-catenin-siRNA (group Ⅲ), IL-1β+RES (group Ⅳ), and IL-1β+RES+SIRT1-siRNA (group Ⅴ) for 24 hours were used to detect the protein expressions of collagen type Ⅱ and Aggrecan by Western blot.ResultsImmunohistochemical staining and Western blot detection showed that when compared with control group, the cell proportion of expression of β-catenin were significantly increased in degenerative group (t=4.616, P=0.010); the protein expression of β-catenin was also significantly increased and the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased (P<0.05). In cytology experiments, the protein expression of β-catenin in group B was significantly higher than that in groups A and C, and the protein expressions of collagen type Ⅱ and Aggrecan in group B were significantly lower than those in groups A and C (P<0.05). After transfection of siRNA, the protein expressions of SIRT1 and β-catenin significantly decreased (P<0.05). The results of cell immunofluorescence staining further confirmed that when compared with group 3, after the SIRT1 was silenced by siRNA in group 4, the attenuated nuclear translocation of β-catenin by RES treatment was aggravated. Western blot results showed that the protein expressions of collagen type Ⅱ and Aggrecan in group Ⅱ were significantly lower than those in group Ⅰ(P<0.05); after transfection of β-catenin-siRNA in group Ⅲ, the degradation of ECM by IL-1β was obviously inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly increased when compared with group Ⅱ (P<0.05); after transfection of SIRT1-siRNA in group Ⅴ, the protective effect of RES on the degradation of ECM was inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased when compared with group Ⅳ (P<0.05).ConclusionRES regulates the ECM expression of NPC via Wnt/β-catenin signaling pathway, which provide a new idea for intervertebral disc degeneration disease treatment.
Objective To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process. MethodsThe identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot. ResultsWhen compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A (P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D (P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased (P<0.05), while NSE, MAP-2, and GFAP genes significantly increased (P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E (P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E (P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E (P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E (P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E (P<0.05). Conclusion Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.
Objective To explore the expressions of Galectin-3, Fascin-1, and β-catenin protein in colorectal adenocarcinoma and the relations to clinicopathologic characteristics. Methods The expressions of Galectin-3, Fascin-1, and β-catenin protein were detected in 60 cases of colorectal adenocarcinoma, 30 cases of adenoma, and 30 cases of normal mucosa by microwave-EliVisionTM immunohistochemistry method, and analyzed the expressions of them and the relations to clinicopathologic characteristics. Results The expression rate of Galectin-3, Fascin-1, and β-catenin protein in CRC was 68.3% （41/60）, 53.3% （32/60）, and 81.7% （49/60） respectively, which was 46.7% （14/30）, 30.0% （9/30）, and 43.3% （13/30） respectively in adenoma, and 20.0% （6/30）, 3.3% （1/30）, and 13.3% （4/30） respectively in normal mucosa, the differences had statistical significance （P＜0.05）. The expressions of Galectin-3, Fascin-1, and β-catenin protein had statistically significant correlation with the TNM stage, invasive degree, and lymph node metastasis of colorectal adenocarcinoma （P＜0.05, P＜0.01）. The expressions of Galectin-3 and β-catenin protein had statistically significant correlation with the different differentiation degree of colorectal adenocarcinoma （P＜0.05）, but the expression of Fascin-1 protein was not related to differentiation degree of colorectal adenocarcinoma （P＞0.05）.The expressions of Galectin-3, Fascin-1, and β-catenin protein had not statistically significant correlation with the patient’s age and gender, and tumour size （P＞0.05）.There were positive correlations between the Galectin-3 and Fascin-1 or β-catenin （r=0.728，P＜0.01；r=0.696，P＜0.01）, and there was positive correlation between β-catenin and Fascin-1 （r=0.507，P＜0.01）. Conclusions The high expressions of Galectin-3, Fascin-1, and β-catenin protein in colorectal adenocarcinoma tissues are some extent correlated to the high invasive ability and lymph node metastasis, which could be used for the indexes to predict the invasion and metastasis in colorectal carcinoma potentially.
Objective
To investigate the effect of Wnt/β-catenin signal pathway on the apoptosis in steroid-induced avascular necrosis of femoral head (SANFH) in rats.
Methods
Seventy-two male Sprague Dawley rats (weighing, 200-230 g) were randomly divided into the control group (group A, n=24), the model group (group B, n=24), and the intervening group (group C, n=24). The rats in groups B and C were injected with lipopolysaccharide and methylprednisolone (MPS) to establish the SANFH model. The rats in group C were injected intramuscularly with human recombinant secreted frizzled related protein 1 (SFRP1) [1 μg/(kg·d)] at the first time of MPS administration for 30 days. The rats in group A received saline injection at the same injection time of group B. The general condition of rats in groups B and C was observed during modeling and after modeling. At 2, 4, and 8 weeks after last injection of MPS, 8?rats were sacrificed to harvest the femoral head. Histological staining was performed to evaluate osteonecrosis. Apoptosis was detected via TUNEL staining. The expressions of Wnt/β-cate nin pathway signaling molecules (activated β-catenin and c-Myc) were detected by immunohistochemistry and Western blot.
Results
Six rats were added in groups B and C because of 6 deaths. The other rats survived to the end of experiment. Normal bone structure was observed in group A; osteonecrosis of bone structure disturbance and disruption of the trabecula were found with time in groups B and C. Group C had the highest empty lacuna rate and apoptosis rate, followed by groups B and A, showing significant difference between groups (P < 0.05). The expression levels of activated β-catenin and c-Myc were significantly lower in group C than groups A and B (P < 0.05), and in group B than group A (P < 0.05).
Conclusion
Wnt/β-catenin signal pathway is involved in the pathogenesis in early SANFH model and its?possible mechanism?is to affect the cell cycle and cell apoptosis by the regulation of c-Myc expression.
Objective
To review the progress of the mechanisms of Wnt/β-catenin and nuclear factor-kappa B (NF-кB) pathways in the process of the intervertebral disc degeneration.
Methods
The related literature about the mechanisms of Wnt/β-catenin and NF-кB pathways in the process of the intervertebral disc degeneration was reviewed, analyzed, and summarized.
Results
Wnt/β-catenin and NF-кB pathways are both activated in the process of the intervertebral disc degeneration, and exist interaction. However, the specific mechanisms and interactive mediums of Wnt/β-catenin and NF-кB pathways in the process of the intervertebral disc degeneration are still unclear.
Conclusion
The mechanisms of Wnt/β-catenin and NF-кB pathways in the process of the intervertebral disc degeneration have to be studied deeply.
ObjectiveTo investigate the effect of eukaryotic initiation factor 6 (eIF6) expression on invasion and metastasis of colorectal cancer cells and Wnt/β-catenin signaling pathway.MethodsImmunohistochemistry was used to detect the expressions of eIF6 protein in colorectal cancer tissues and paracancerous tissue. Sw837 cell lines with overexpression/knockdown eIF6 were constructed by transfection and divided into control group, empty plasmid group, overexpression group and knockdown group respectively. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blotting (WB) were used to verify the overexpression/knockdown of eIF6 in sw837 cells. WB was used to detect the expressions of β-catenin, glycogen synthase kinase-3β (GSK-3β), anaphase promoting complex (APC), zinc finger transcription factor SNAI (Snail), E-cadherin and Vimentin. Transwell invasion test, Scratch test and subcutaneous tumorigenesis test were used to detect the migration ability, invasion ability and tumorigenesis ability in vivo of cells. The pathological changes of the transplanted tumor were observed by HE staining.ResultsThe positive expression rate of eIF6 protein in cancer tissues was significantly higher than that in adjacent tissues (P<0.05). Compared with those in the control group, the protein expression levels of β-catenin, Snail and Vimentin in the overexpression group were higher, the protein levels of GSK-3β, APC and E-cadherin were lower, the ability of cell migration and invasion, and tumorigenicity in vivo were enhanced, the difference were statistically significant (P<0.05). The protein expression levels of β-catenin, Snail and Vimentin were lower in knockdown group, the protein levels of GSK-3β, APC and E-cadherin were higher, the ability of cell migration and invasion, and tumorigenicity in vivo were reduced, the difference were statistically significant (P<0.05).ConclusioneIF6 may promote the invasion and metastasis of colorectal cancer cells by activating Wnt/β-catenin signaling pathway.
