摘要
Objective Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21（TRIM21） in osteosarcoma cell proliferation.Methods 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide（MTT） assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation（BIFC） were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.Results TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However,overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.Conclusion Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.
Objective Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21（TRIM21） in osteosarcoma cell proliferation.Methods 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide（MTT） assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation（BIFC） were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.Results TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However,overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.Conclusion Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.
基金
partially supported by Guangzhou Science and Technology Project[20160701175
201707010263]
Natural Science Foundation of Guangdong Province[2016A030313083
2016A030313420]
Team Project of Natural Science Foundation of Guangdong Province[S2013030013315]
Fundamental Research Funds for the Central Universities[Grant No.21609317
ZX20170413]
Guangdong Province Science and Technology Project YUEKEGUICAI[(2015)110-0024]
