Wnt/β-catenin signaling pathway participates in cancer cell proliferation,invasion and metastasis and effectively induces drug resistance.It is also the key signal to mediate cancer carcinogenesis.Recent studies in vitro indicate that disturbance of Wnt/β-catenin signaling pathway can increase the sensitivity of the cancer cells to chemotherapeutic drugs.In-depth researches and analysis of tumor drug resistance induced by Wnt/β-catenin will provide potential targets and possible therapeutic means for the treatment of tumors.

Objective: To investigate the effect of receptor interacting protein kinase 4 (RIPK4) gene silencing on tumor necrosis factor-a (TNF-a)-induced apoptosis of human osteosarcoma MG-63 cells. Methods: The specific siRNA targeting RIPK4 gene (RIPK4-siRNA) was transfected into MG-63 cells by liposome (named as RIPK4-siRNA group), while the MG-63 cells were transfected with negative control-siRNA (NC-siRNA) as the negative control (named as NC-siRNA group). Then the MG-63 cells transfected with RIPK4-siRNA were treated with human recombinant TNF-a (named as RIPK4-siRNA+TNF-a group), while the MG-63 cells were only treated with TNF-a as the positive control (named as TNF-a group). The proliferation of MG-63 cells was detected by 5-ethynyl-2'-deoxyuridine (EdU) method. The apoptosis of MG-63 cells was detected by Hoechst 33258 staining. The expressions of RIPK4, Bax, Bcl-xL and caspase-3 proteins in MG-63 cells were examined by Western blotting. Results: The expression level of RIPK4 protein in MG-63 cells was significantly down-regulated after transfection of RIPK4-siRNA (P < 0.05). The EdU positive rates of RIPK4-siRNA, NC-siRNA, TNF-a and RIPK4-siRNA+TNF-a groups were 60.7%, 39.6%, 43.3% and 16.7%, respectively. The proliferation of MG-63 cells in RIPK4-siRNA group was lower than that in NC-siRNA group (P < 0.05). Furthermore, the proliferation in RIPK4-siRNA+TNF-a group was lower than those in other three groups (all P < 0.05). The apoptosis of MG-63 cells in RIPK4-siRNA groups was significantly higher than that in NC-siRNA group (P < 0.05). Furthermore, the apoptotic rate in RIPK4-siRNA+TNF-a group was significantly higher than those in other three groups (all P < 0.05). The expression levels of Bax and caspase-3 proteins in RIPK4-siRNA group were significantly higher than those in NC-siRNA group (P < 0.05), but the expression level of Bcl-xL protein in RIPK4-siRNA group was significantly lower than that in NC-siRNA group (P < 0.05). The expression levels of Bax and caspase-3 proteins in RIPK4-siRNA+TNF-a group were significantly increased, but the expression of Bcl-xL protein was significantly decreased as compared with other three groups (all P < 0.05). Conclusion: RIPK4 gene silencing can induce the apoptosis of MG-63 cells, and may increase the sensitivity of apoptosis induced by TNF-a.

AIM:To explore the time-dependent change of Ski protein expression in normal and activated astrocytes in rats.METHODS:The astrocytes were obtained from rat cerebral cortex and cultured in vitro.The astrocytes were treated with LPS and scratch injury for activation.Western blot analysis was used to determine glial fibrillary acidic protein (GFAP) and Ski protein levels in activated astrocytes at a series of time points.The indirect immunofluorescence staining method was performed to detect the location of Ski protein in the astrocytes.RESULTS:The protein of GFAP was naturally expressed in the astrocytes, beginning to increase after treated with LPS and scratch injury.Little protein expression of Ski in the normal astrocytes was observed.The Ski protein expression began to increase after treated with 1 mg/L LPS, peaked at 4 d (P<0.05) and then deceased, but was stills higher than that in the normal cells.The protein expression level of Ski after scratch injury was highly consistent with above mentioned.Ski was mainly observed in the nucleus of the normal cells and the cells treated with LPS for 6 d, while it was observed in the cytoplasm 2 and 4 d after treated with LPS.CONCLUSION:The protein of Ski is expressed in the astrocytes, and the expression level is increased in activated astrocytes,mainly located in the nucelus.Ski may plays an essential roles in the processes of activation and proliferation of astrocytes.

Objective:To investigate the extracellular signal-regulated kinase 5 (ERK5) and matrix metallo proteinase-9 (MMP-9) expres-sion levels in osteosarcoma tissues and their clinical significance. Methods:The ERK5 and MMP-9 expression levels in 71 specimens of osteosarcoma tissue and 40 specimens of normal bone tissue were detected by immunohistochemistry. The relationship between ERK5 and MMP-9 expression levels, their clinical characteristics, and prognosis of patients with osteosarcoma were analyzed. Results:The positive expression of ERK5 and MMP-9 in osteosarcoma tissues was 85.9%(61/71) and 74.65%(53/71), respectively, which were significantly higher than those in normal bone tissues at 12.5%(5/40) and 10.0%(4/40) (all P<0.05). The positive expression of ERK5 and MMP-9 was associated with Enneking stage and metastasis (all P<0.05). Kaplan-Meier analysis showed that the survival duration of patients with positive ERK5 and MMP-9 expression levels was shorter than those of the patients in the negative expression groups (all P<0.05). Univariate analysis of COX proportional hazards regression model revealed that tumor size, Enneking stage, metastasis, and positive ERK5 and MMP-9 expression levels are relevant to the overall survival of patients with osteosarcoma (all P<0.05). Multi-variate analysis of COX proportional hazards regression model confirmed that Enneking stage, metastasis, and positive ERK5 and MMP-9 expression levels can act as independent prognostic factors for osteosarcoma patients (all P<0.05). Conclusion:The ERK5 and MMP-9 expression levels are high in osteosarcoma tissues and are related to the clinical characteristics and prognosis of patients with osteo-sarcoma. Thus, ERK5 and MMP-9 expression levels may play important roles in osteosarcoma development and progression.