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.
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.
ObjectiveTo investigate the effects of telmisartan on the proliferation, migration and apoptosis of non-small cell lung cancer A549 and the mechanism of regulating Wnt signaling pathway.MethodsNon-small cell lung cancer cell line A549 was cultured in vitro. Cell counting kit-8 (CCK-8) assay was used to detect the effect of telmisartan at different concentrations on the proliferative activity of A549 cells. The survival fraction of A549 treated with different concentrations of telmisartan was determined by colony-formation assay. The effect of telmisartan at different concentrations on the migration ability of A549 cells was examined in the wounding healing assay. Hoechst staining was used to detect the effects of telmisartan at different concentrations on the apoptosis of A549. Western bloting was used to detect the expressions of β-actin, proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, Wnt-3a, Beta-catenin (β-catenin), serine protein kinase 3β (p-GSK-3β), glycogen synthase kinase-3β (GSK-3β) and c-myc.ResultsDifferent concentrations of telmisartan treatment inhibited the proliferation activity, colony-formation rate and migration of A549 cells, and reduced the expression of PCNA in a concentration-dependent manner. Telmisartan treatment promoted the apoptosis of A549 cells, significantly increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2. The expression levels of Wnt-3a, β-catenin, p-GSK-3β, and c-myc in A549 cells increased after treatment with telmisartan, while the expression levels of GSK-3β decreased.ConclusionTelmisartan may play a role in the proliferation, migration and apoptosis of non-small cell lung cancer A549 cells, and inhibiting the Wnt/β-catenin signaling pathway may be one of the mechanisms.