| Background: Chronic myeloid leukemia(CML)accounts for about 20 % of all adults leukemia,and it only is second to acute myeloid and acute lymphocytic leukemia and in the third place.It has the characteristic of BCR-ABL Ph chromosome and which can cause disease of malignant clonal-proliferation of infiltration,and cause excessive growth of cells,inhibition of apoptosis,changes in migration and invasion ability,interference of cell differentiation and so on.K562 cells were separated from the red blood cells in CML patients,which had properties of tumor stem cells,and the cells stop to differentiate.K562 cells are the ideal cell model to study CML.The CRK adaptor protein was found in avian sarcoma virus CT10(chicken tumor 10),it contained SH2 and SH3 domains and was a product of oncogene v-CRK.CRK family included CRKⅠ,CRKⅡ and CRKL,which were expressed in various tissues.The CRK family regulated the transcription,proliferation,differentiation and apoptosis of the cells via tyrosine kinases and small G proteins.As members of the CRK family,CRKL and CRKⅡ had high homologous in structure,and can recruit signaling molecules through SH2 and SH3 domains.The abnormal expression of CRKL and CRK were related to cancers,but there were little research on the effect of both of them.Our previous study showed that CRKL affected proliferation,migration and invasion of megakaryocyte differentiation and other biological functions of K562 and the expression of CRKⅡ increased when down regulation of CRKL,but the specific mechanismare was not clear.This study focused on the effect of CRKL on erythroid differentiation in K562,CRKⅡ interference and CRKL+CRKⅡ double knock-down to study the effect of cell differentiation,proliferation,migration and invasion,and to explore the function,mechanism and connection of CRKⅡ and CRKL.Objective: 1.To investigate the effect of CRKL on the erythroid differentiation and its molecular mechanism of K562.2.To analyse the proliferation,migration and differentiation of K562 when CRKⅡ down-regulation.3.To discuss the relationship of CRKⅡ and CRKL in function and mechanism.Methods: 1.qRT-PCR assay detected the expression of CRKL and CRKⅡ in 14 patients with non-malignant,and 33 patients which was in the primary stage and 5 pairs(primary cancer-complete remission)and the normal group;2.Hemin induction,benzidine staining,the gene chip and proteomics assays detected the role of CRKL in erythroid differentiation of K562;3.To detect the expression of the Raf/MEK/ERK/Elk-1 pathway when CRKL down-regulation by WB;4.WB and q RT-PCR detected the expression level of CRKⅡ down-regulation;5.CCK-8 assay was used to detect the effect on the proliferation of K562 when CRKⅡ knock-down,transwell assay analysed the migration and invasion of K562,q RT-PCR detected the influence on erythroid differentiation of K562;6.WB assay was used to detect the expression of p130Cas/CRK/Rac1 and Raf/MEK/ERK/Elk-1 pathway after down-regulation of CRKⅡ;7.Immunoprecipitation assay detected the interaction of CRKL and CRKⅡ in K562;8.Trypan blue staining detected the proliferation of K562 when CRKL+CRKⅡ double down-regulation,transwell assay studied the migration and invasion ability of cells,q RT-PCR detected the effect of erythroid differentiation when CRKL+CRKⅡ double down-regulation;9.WB assay detected the expression of Rac1,ERK and p-ERK after CRKL+CRKⅡ double knock-down.Results: 1.The q RT-PCR results revealed that the expression level of CRKL in primary stage of marrow samples was high significantly,and it was 6.2 times higher than normal samples(P=0.009),and in 5 paired samples,the expression of CRKL was high in primary cancer patients,and was low in association CR samples in 5 pair samples (P=0.0165).Compared with normal,the level of CRKL was declinced in CR(P=0.0258).The expression of CRKⅡ was slightly high in primary patient samples,and it was 1.8 times higher than normal(P=0.0855).In 5 paired samples,3 of CML in primary cancer was high expression,it was low expression in association CR samples(P=0.1014).Compared with normal,the level of CRKⅡ had no difference in CR(P=0.1051);2.The expression of CRKL was decreased after hemin induced by K562,the protein level decreased by 52.8 %(P=0.0007)and 54.6 %(P=0.0004)in 1 and 2 day after inducing,and the expression of CRKⅡ was not obvious;When the ratio of positive cells of NC was 3.4 %,the cell positive rate of CRKL down-regulation was increased by 10.5 %(P=0.0239),the expression of GPA and γ-globin was increased by 59.4 %(P=0.0096)and 96.9 %(P=0.0006)respectively,and there were HBA and HBD and other similar proteins;3.CRKL knock-down increased the expression of GATA-1 and HMGB2;4.CRKL knock-down activated the Raf/MEK/ERK/Elk-1 pathway;5.CRKⅡ decreased the proliferation ability of K562 in vitro,and decreased the migration and invasion ability of cells by 54.7 %(P=0.0221)and 56.1 %(P=0.013),there was no influence in differentiation;6.CRKⅡ inhibited the p130Cas/CRK/Rac1 pathway,and had little effect on the Raf/MEK/ERK/Elk-1 pathway;7.CRKL and CRKⅡ can interact with each other in K562;8.CRKL+CRKⅡ double knock-down decreased the proliferation of K562 and the migration and invasion ability of the cells were decreased by 35.9 %(P=0.0024)and 44.8 %(P=0.0115)respectively in vitro,but had little effect on differentiation compared with CRKL single-silence;9.CRKL+CRKⅡ double knock-down inhibited the expression of Rac1,but had little effect on ERK and p-ERK compared with CRKL single down-regulation.Conclusion: 1.CRKL and CRKⅡ were highly expressed in the primary stage of CML marrow samples,and the expression level of CRKL was higher than that of CRKⅡ,and the level of them was lower in CR samples.Compared with normal,the expression level of CRKL was declined,but CRKⅡ had no change;2.CRKL knock-down regulated K562 erythroid differentiation by activating the Raf/MEK/ERK/Elk-1 pathway,while the down-regulation of CRKⅡ had little effect on erythroid differentiation of K562;3. CRKⅡ knock-down inhibited proliferation,migration and invasion of K562 in vitro;4.CRKⅡ influenced the malignant behavior of K562 by regulating p130Cas/CRK/Rac1 signaling pathway;5.CRKL+CRKⅡ double knock-down inhibited the proliferation,migration,invasion and Rac1 expression of K562 in vitro;6.CRKL+CRKⅡ regulated the proliferation,migration and invasion of K562 cells via p130Cas/CRK/Rac1 signaling pathway. |
