Role Of RPS3 In ATPR-induced Differentiation In Human Chronic Myeloid Leukemia K562 Cells And Its Related Mechanisms

Chronic myelogenous leukemia（CML）is a clonal dysplasia hematological malignancy of bone marrow hematopoietic cells.Its clinical symptoms are mainly peripheral leukocytosis and hepatosplenomegaly.It is generally believed that the pathogenesis of chronic myelogenous leukemia is the reciprocal translocation of chromosome 9 and chromosome 22 at the chromosome level,which leads to the formation of BCR-ABL fusion gene.The bcr-abl fusion protein encoded by this fusion gene has strong tyrosine kinase activity and plays an important role in the genesis and development of CML.Inhibition of tyrosine kinase activity has become an effective treatment for CML.However,some patients are not susceptible to tyrosine kinase inhibitors.All-trans retinoic acid（ATRA）has been widely used as a differentiation inducer in the treatment of CML patients for a long time,but its clinical application is greatly limited due to its serious adverse reactions.4-amino-2-trifluoromethylphenyl retinoate is a novel retinoic acid derivative designed and synthesized by our group on the basis of ATRA structure.Previous studies have confirmed that ATPR has an obvious effect on inducing K562cell differentiation,but its mechanism is still not completely clear,which needs to be further explored.To further enrich and improve the mechanism of ATPR-induced CML cell differentiation and the role of ribosomal protein RPS3 in ATPR-induced CML cell differentiation.In this study,K562 cell line was selected as the research object.The effect of ATPR on the expression of RPS3 and the role of RPS3 in the differentiation of K562 cells induced by ATRP were observed in vitro.The main research contents of this subject include the following four parts:1.Effect of ATPR on RPS3 expression and cell cycle in K562 cell line.In this study,K562 cells were treated with ATPR(10^-6M).Western blot analysis and Real-time PCR were used to detect the protein and RNA levels of RPS3.Meanwhile,the cell cycle of K562 cells treated with ATPR(10^-6M)was detected by flow cytometry.The results showed that ATPR could significantly reduce the expression of RPS3 in K562 cells in vitro,and ATPR could block K562 cells in G₁phase.2.Role of RPS3 in ATPR-induced differentiation of K562 cells.After RPS3-siRNA was used to down-regulate the expression of RPS3,K562 cells were treated with ATPR(10^-6M).The expression levels of RPS3,CDK4 and Cyclin D3were detected by Western blot.Flow cytometry was used to detect the expression levels of CD11b and CD133 cell surface differentiation antigens.Western blot was used to quantitatively analyze the protein of CD11b and CD133.The results showed that silencing RPS3 could promote the inhibition of ATPR on CDK4 and Cyclin D3,while silencing RPS3 could promote the up-regulation of ATPR on CD11b and CD133.To further explore the effect of RPS3 on ATPR-induced differentiation of K562cells,the overexpression plasmid pEGFP-N1-RPS3 was used to transfect K562 cells.The expression of RPS3 in K562 cells was up-regulated.The expression levels of RPS3,CDK4,Cyclin D3,CD11b and CD133 were detected by Western blot.The results showed that over-expression of RPS3 could inhibit the down-regulation of CDK4 and Cyclin D3 by ATPR,and over-expression of RPS3 could attenuate the up-regulation of CD11b and CD133 by ATPR.3.Role of Hippo signaling pathway in ATPR-induced differentiation of K562 cells.After RPS3-siRNA was used to down-regulate the expression of RPS3,K562 cells were treated with ATPR(10^-6M).The expression levels of MST,SAV,LATS and YAP were detected by Western blot,and the gene expression levels of these factors were detected by Real-time PCR.At the same time,the overexpression plasmid pEGFP-N1-RPS3 was used to transfect K562 cells.The expression of RPS3 in K562cells was up-regulated.The expression levels of MST,SAV,LATS and YAP were detected by Western blot,and the gene expression levels of these factors were detected by Real-time PCR.The results showed that ATPR could significantly reduce the expression levels of MST,SAV,LATS and YAP.Silence of RPS3 could promote the inhibition of ATPR on these proteins,while overexpression of RPS3 could significantly weaken the inhibition of ATPR.4.Effect of ATPR on RPS3 expression in NB4 and THP-1 cell lines.K562 cells were treated with ATPR（10^-66 M）,and the protein levels of RPS3 were analyzed by Western blot.The results showed that in vitro ATPR significantly reduced the expression of RPS3 in NB4 and thp-1 cells.