RESUMEN
AIM: To investigate the effects of bone morphogenetic protein 7 ( BMP-7 ) on the expression of transcription factor E2A and inhibitor of differentiation 2 (Id2) in the renal tubule epithelial cells(NRK-52E)exposed to high glucose, and to explore its possible mechanism of improving renal tubular fibrosis induced by high glucose.METH-ODS:The NRK-52E cells were divided into control group, high glucose (HG) group and high glucose with different doses of BMP-7 (10μg/L and 20μg/L) group.The cells in HG group and BMP-7 group were cultured for 12 h, 24 h and 48 h. The protein expression of Id2, E2A, E-cadherin,α-smooth muscle actin (α-SMA) and collagen-I was detected by Western blot.In addition, the mRNA expression of Id2 was detected by real-time PCR.RESULTS:Compared with control group, the mRNA and protein levels of Id2 and the protein level of E-cadherin were down-regulated, while the protein levels of E2A,α-SMA and collagen-I were up-regulated in HG group (P<0.05).Compared with HG group, the mRNA and pro-tein levels of Id2 and the protein level of E-cadherin were significantly up-regulated, while the protein expression of E2A,α-SMA and collagen-I was significantly down-regulated in 20 μg/L BMP-7 group ( P<0.05 ) .The correlation analysis showed that the Id2 protein level was negatively correlated with the E2A protein level (P<0.05).CONCLUSION:BMP-7 may intercept the process of renal tubule fibrosis induced by high glucose via promoting the expression of Id2 and inhibi-ting the expression of E2A at protein level.
RESUMEN
Purpose To study the expression of inhibitor of differentiation 2 (Id2) in Ewing sarcoma of bone as well as the correlation with cell cycle proteins. Methods The expression of Id2, c-Myc, Cyclin D1, p53, p16 and p27 in 45 cases of Ewing sarcoma were detected by immunohistochemical EnVision methods, the relationship between the expression of Id2 and clinicopathologic characteristics and the correlation with cell cycle proteins were analyzed. Results Total expression of Id2 was found in 88. 9% of Ewing sarcoma. Strong expression of Id2 was detected in 33. 3% cases. Higher frequencies of c-Myc and Cyclin D1 immunostaining were observed (66. 7% and 71. 1%, respectively), but lower frequencies of p53, p16 and p27 expression were present (20. 0%, 35. 6% and 28. 9%, respectively). Id2 overexpression displayed a negative relationship with p27 expression (P<0. 05). Among the follow-up of 24 cases, overexpression of Id2 was found more frequency in dead and metastases cases (41. 2%) when compared to that of progres-sion-free survival cases (14. 3%). Conclusions Id2 was extensively expressed in Ewing sarcoma of bone. Overexpression of Id2 and its correlation with p27 expression suggest that this is an important pathway involved in development of Ewing sarcoma. Moreover, Id2 overexpression may predict poor prognosis.
RESUMEN
Objective To construct short hairpin RNA (shRNA) expression plasmids against the inhibitor of differentiation 2 (Id2) gene and establish a suitable cell model for the role of Id2 in proliferation and differentiation of human Ewing sarcoma cell.Methods Three shRNA sequences targeting Id2 gene were designed and inserted into the pGPU6/GFP/Neo (-shRNA-Id2) expression vectors.The recombinant pGPU6/GFP/Neo-shRNA-Id2 plasmids were introduced into Ewing sarcoma RD-ES cells by liposome-mediated transfection.The knock-down efficiency of Id2 in infected RD-ES cells was verified by Western blot assay.The cell growth and cell cycle changes were evaluated by cell counting and flow cytometry between transfected cells and control cells.Results The Id2 expression decreased 54 ％ and 57 ％,respectively,in RD-ES cell line which were transfected with the shRNA-Id2-543 and shRNA-Id2-593 plasmids compared with the control group cells by Western blot analysis.The cell growth assay demonstrated that the cell number in transfected cells was significantly decreased during 6-7 d compared with the control group (P ＜ 0.05).The cells at the S-phase of cell cycle were increased [(36.60±1.53) ％ and (44.89E2.46) ％ vs (29.73±2.03) ％,P ＜ 0.05],and no significant changes at the G2 phase or even reduction in the transfected cells.Conclusions Id2 stable knock-down cell lines are successfully established.The reduced expression of Id2 is related with decreased cell growth and cell cycle arrest in the S-phase.Id2 maybe plays an important role in proliferation of Ewing sarcoma cell.