The Molecular Mechanism For Involvement Of PML-V Protein In Acute Promyelocytic Leukemia By Treatment Of Arsenic Trioxide

Objective:（1）To study the PML-V protein stability and its ubiquitination,and the involvement of PML-V protein in regulating the sensitivity of acute promyelocytic leukemia（APL）to arsenic trioxide（As₂O₃）.（2）To investigate the relationship between the expression level of PML-V protein and the degradation rate of PML-RARαfusion protein induced by As₂O₃.Meanwhile,the relationship between the expression level of PML-V protein and the recovery of normal PML nuclear bodies in NB4 cells.（3）To investigate the effect of the 7ab region of PML-V C-terminus on the function of PML-V protein.（4）Besed on the clinical progression of APL patients,exploring the statistical relationship between the PML-V gene espression level and the survival rate of APL patients.Methods:（1）Homologous recombination method was used to construct the plasmids with mutated and deleted sequences.（2）Western-blot was used to detect the expression level of target proteins in the nucleus（the soluble part of the RIPA lysate）and the nuclear matrix（the insoluble part of the RIPA lysate）.（3）The expression and morphological changes of target proteins within the cell were observed by immunofluorescence.（4）Co-immunoprecipitation was used to reveal the protein-protein interactions.（5）The m RNA level of genes was detected by real-time quantitative PCR.（6）Lentiviral transfection method was used to construct stable transfected cell lines.（7）The liposome-mediated method delivered the gene of interest into the cells.Result:（1）Among the seven PML protein isoforms,PML-V protein was found to be most sentive PML isoform to arsenic trioxide,for instance,it has shown fastest degradation rate,the shortest half-life,the lowest protein stability,the strongest ubiquitination modification ability by As₂O₃treatment.（2）When the inceasing of PML-V protein level,the degradation of PML-RARαfusion protein was significantly accelerated by As2O3.Otherwise,the rise of the expression level of PML-V protein in NB4 cells leads to the reform of the PML nuclear bodies from a abnormal diffused pattern to an normal form.（3）For PML-V isoform with mutated or deleted 7ab region,it has prolonged protein half-life,and increased protein stability as well as reduced sensitivity to arsenic.Moreover,it was further weaken or even completely inhibited the its proteins ubiquitination.Meanwhile,mutated PML-V lost the ability to degradate the PML-RARαfusion protein by As2O3 as the wild type did.（4）Compared to the healthy person,the expression of PML-V protein level in APL patients is relatively low.Moreover,there is an inverse relationship between the expression level of PML-V protein and the PML-RARαfusion protein in APL patients.For instance,in APL patient the higher the expression level of PML-V protein,the lower the expression level of PML-RARαfusion protein,and vice versa.Moreover,interferon alpha could enhance the expression of endogenous PML protein in APL cells,it can reform of PML nuclear bodies from a diffused pattern to an normal PML-NBs form.Conclusion:Based on the analysis and summary of the experimental data,among the seven PML protein isoforms,the PML-V protein was found to be the shortest half-life,the lowest protein stability,the strongest modification with ubiquitination,and the most sensitive to As₂O₃-induced degradation.Increasing of PML-V protein expression,PML-V dramatically accelerated the degradation of PML-RARαfusion protein by As₂O₃.Besides,PML nuclear bodies reformed from a diffused state to an normal state,which can alleviate the symptoms of APL.When the PML-V 7ab region is mutated or deleted,the PML-V protein loses its normal biological function,indicating that the C-terminal 7ab region of PML-V protein plays a vital role in maintaining its normal biological function.Patients with relatively high expression of PML-V protein had a milder condition and had faster remission with As₂O₃treatment.In contrast,patients with relatively low PML-V protein expression comes with more severe symptoms and slower remission after using As₂O₃.