Expression Of UHRF1 In Ewing Sarcoma And Its Target Therapy

Objective: Ewing sarcoma is a rare malignant tumor that occurs in adolescent bone and soft tissues.The tumor has a short course,rapid recurrence,and high metastatic rate.The formation of EWS-ETS fusion protein is the main initiating factor,of which 85% is EWS-Fli1 fusion protein.Since the fusion protein is not a rigid molecule,it is difficult to find a specific targeted drug.In recent years,its epigenetic research and immune escape have been in the ascenda nt.As a tumor that often occurs in children and adolescents,mutations are often rare in Ewing sarcoma.Although high-dose chemotherapy may contribute to tumor delay,it is often accompanied by neurodevelopmental insufficiency,mental retardation,depression and other adverse consequences,so we hope to explore new ideas for the treatment of Ewing Sarcoma by epigenetics into consideration.UHRF1 is an important epigenetic regulator that participates in DNA methylation and histone modifications,so that the two are in dynamic equilibrium to maintain the normal cell cycle.Based on previous experiments,we also found that CX-5461 can degrade UHRF1 by promoting ubiquitination and promote protein degradation of DNMT1.Methods: In this study,the expression of UHRF1 in Ewing's sarcoma tissues and cell lines(SK-N-MC,SK-ES-1,A673)was firstly detected by immunohistochemistry and Western blot,and then in vitro experiments were performed using relevant cell lines.The control group and the experimental group were transfected with Srambled Si RNA(Si Scr)and Si UHRF1 respectively to detect cell proliferation and cell cycle changes.Protein and m RNA were extracted to detect UHRF1,DNMT1 and other protein and m RNA changes;Si Scr and Si EWS-Fli1/Si Fli were transfected respectively to explore the effect of EWS-Fli1 on UHRF1 and DNMT1;after treatment with CX-5461 on cell lines,the changes of UHRF1 and DNMT1 were detected.CX-5461 was found to significantly reduce UHRF1 in protein level,further exploration of CX-5461 with 293 T cells was used to explore the mechanism.293 T cell line was transfected with UHRF1+Scramble,UHRF+HA-Ubiquitin,UHRF1+HA-Ubiquitin related plasmids and after which,CX-5461 and MG-132 were added in order to explore whether CX-5461 degrades UHRF1 through the ubiquitination pathway.Results: UHRF1 protein was highly expressed in Ewing's sarcoma and cell lines,which was involved in maintaining cell cycle and proliferation,and maintained the stability of DNMT1 in Ewing's sarcoma.EWS-Fli1 can maintain the stability of UHRF1 and DNMT1;Induction of Ewing sarcoma autophagy and G2/M cell cycle arrest,CX-5461 can also reduce the protein levels of UHRF1 and DNMT1,further verifying that CX-5461 degrades by promoting ubiquitination of UHRF1.Conclusion: EWS-Fli1 can maintain the malignant degree of Ewing sarcoma by maintaining the stability of UHRF1 protein.CX-5461 has the potential to be a new targeted drug for UHRF1 treatment of Ewing sarcoma.