The Effect And Mechanism Of HnRNP U On Proliferation And Apoptosis Of Chronic Myeloid Leukemia Cells

ObjectiveThe fusion gene BCR-ABL encodes the BCR-ABL protein.The tyrosine kinase activity of BCR-ABL protein continuely and abnormally activates downstream signaling pathways,leading to the pathogenesis of CML.Although tyrosine kinase inhibitors(TKIs)such as imatinib(IM)and other drugs prolong the survival of patients,about 25% of patients develop drug resistance,thus new treatments are urgently needed in clinical.Heterogeneous ribonucleoprotein(hnRNP)is a class of RNA-binding proteins which participate in the processes of shearing,maturation,transportation,stabilization and translation regulation of mRNA in cells.In recent years,many members of the hnRNP family have been confirmed to play an important role in chronic myeloid leukemia(CML).hnRNP U discovered in 1992 has been found to closely related to the pathogenesis and development of solid tumors.However,the studies of hnRNP U in CML have not been reported.In order to make this research universal for CML,the two clinical status of IM sensitivity and IM resistance were covered in this study.In this project,IM sensitive cell line K562 and IM resistant cell line K562/G01 cells were used as models for exploring the role of hnRNP U and possible mechanism in CML.This study aimed to provide an experimental basis for exploring the pathogenesis and new therapy strategies of CML.Methods1.The expression of hnRNP U in bone marrow specimens of normal control and CML patients,and in BCR-ABL negative cell lines TK6,THP1 and BCR-ABL positive cell lines K562,KCL22,K562/G01 were detected by RT-qPCR and Western blot.The proteins level of BCR-ABL phosphorylation and hnRNP U were detected through Western blot after K562 and K562G01 cells treated with different concentrations of imatinib for 48 h.2.K562 and K562/G01 cells were infected with the sh-hnRNP U lentivirus for 72-96 h,then the knockdown effect of hnRNP U mRNA was detected by RT-qPCR.The knockdown effect of hnRNP U protein was detected by Western blot after infected-cells treated with puromycin for four days.K562 and K562/G01 cells were infected with the sh RNA-1 and sh RNA-2 lentiviruses for 96 h,then CCK8 and clone formation assay were used for detecting their proliferation ability.The cells infected with the sh RNA-1 and sh RNA-2 lentiviruses were treated with puromycin for four days,then puromycin withdrawed and continuely cultured for four or five days,then the apoptosis was detected by flow cytometry.3.The potential target genes of hnRNP U were predicted by bioinformatics technology.The changes of mRNA,protein level and mRNA half-life of the corresponding target gene were detected by RT-qPCR or Western blot after hnRNP U knockdown.RIP assay was used for detecting the binding ability of hnRNP U and target gene mRNA.Results1.Compared with normal human bone marrow specimens and BCR-ABL negative cell lines,the expression of hnRNP U were upregulated in both bone marrow specimens of CML patients(P<0.001)and CML cell lines(P<0.05).The expression of hnRNP U was not affected by BCR-ABL phosphorylation level.2.The hnRNP U mRNA was knocked down to below 50% in cells infected with the lentivirus for 72-96 h.The hnRNP U protein was significantly reduced in infected-cells treated with puromycin.CCK8 results showed that the cell proliferation rate of sh-hnRNP U group slowed down significantly at 48 h(P<0.05).The clone formation results showed that the number of clones in sh-hnRNP U group decreased about 50% and the clone morphology was obviously smaller.The results showed that the proliferation of CML cells was significantly inhibited because of hnRNP U knockdown(P<0.001).Flow cytometry results showed that CML cells had undergone apoptosis.3.The target genes c-Myc and BCL-2 were screened out by the databases of GEPIA and STARBASE3.0.The target genes are positively related to hnRNP U and the mRNA may bind to hnRNP U.The mRNA expression level and mRNA stability of c-Myc and BCL-2 were reduced after hnRNP U knockdown.The expression level of c-Myc and BCL-2protein were also reduced.RIP assay founded that hnRNP U combined with c-Myc and BCL-2 mRNA.Conclusion:This study confirmed that the expression of hnRNP U was increased in bone marrow specimens of CML patients and CML cell lines K562,KCL22,K562/G01.The upregulated expression of hnRNP U promoted the proliferation of CML cells and inhibited their apoptosis.The possible mechanism was that hnRNP U combined with c-Myc and BCL-2 mRNA enhanced their mRNA stability thus promoted the expression of c-Myc and BCL-2 protein.This study indicated that hnRNP U was equally important for the survival of IM-sensitive and IM-resistant CML cell lines,and was the experimental basis for deeply revealing the role of hnRNP U in the pathogenesis of CML.