| Erythroid differentiation refers to the process in which hematopoietic stem cells pass through pluripotent hematopoietic stem cells,myeloid progenitor cells,and erythroid progenitor cells,and then form mature red blood cells.It is an important way for the body to produce mature red blood cells.Once the development of erythroid cells is stagnated at a certain stage and cannot eventually differentiate into mature red blood cells,it will cause leukemia.Chronic myeloid leukemia(CML)is a malignant clonal proliferative disease of pluripotent hematopoietic stem cells that originates in bone marrow.It has the characteristics of uncontrolled proliferation,arrest of apoptosis,inhibition of hematopoietic function,and differentiation disorders.Therefore,leukemia cells being induced to differentiate in the terminal direction is an important strategy for studying leukemia treatment.Therefore,the in-depth study on the differentiation mechanism of leukemia cells and the discovery of differentiation-related genes and new therapeutic targets provide a new theoretical basis for the treatment of leukemia.Signal adaptor protein CRKL is a member of the CRK family.Like other connexins,it can recruit signal transduction molecules through its SH2 and SH3 domains,phosphorylate tyrosine-rich regions and act as signal switches.In recent years,studies have shown that the abnormal expression of CRKL is closely related to the occurrence and development of malignant tumors,and may be a potential target molecule to affect the treatment and prognosis of tumors.Previous studies in this group showed that CRKL affected the biological functions of K562,such as proliferation,migration,invasion and megakaryocyte differentiation,and we screened and identified the increased expression of molecules related to erythroid differentiation by using K562 cells stably down-regulated by CRKL through gene chip and proteomics,suggesting that CRKL is related to erythroid differentiation of K562 cells.MiR-429 is a member of the miR-200 family,which includes miR-141,miR-200 c,miR-200 a,miR-200 b and miR-429.The abnormal expression of miR-429 is closely related to tumorigenesis,metastasis,apoptosis and drug resistance.The preliminary results of this group showed that there was a targeted regulatory relationship between miR-429 and CRKL in the study of migration and invasion of Hep G2 cells,and the expression levels of the two were negatively correlated,and they mainly played a role through ERK pathway.but the role and mechanism of miR-429 in erythroid differentiation of leukemic cells are not clear.In this thesis,we mainly study the molecular mechanism of miR-429 targeting CRKL on erythroid differentiation of leukemic cells,and provide new clues and ideas for molecular targeted therapy of leukemia.Objective:1.To investigate the expression and correlation between the difference of miR-429 and CRKL expression in patients with CML.2.Screening CRKL expression related erythroid differentiation related molecules3.To determine the effects of miR-429 and CRKL on erythroid differentiation of K562 cells;4.To explore the molecular mechanism of erythroid differentiation of K562 cells regulated by miR-429-CRKL axis.Methods:1.The relative expression of miR-429 and CRKL in CML clinical samples and the correlation between them were detected by qRT-PCR.2.The erythroid differentiation-related molecules expressed in K562 cells after stable CRKL knockout were screened by gene chip.3.Benzidine staining was used to detect the erythroid differentiation positive cells of K562 cells treated with Hemin.4.qRT-PCR was used to detect the expression of erythroid molecules in K562 cells treated with Hemin for 24 and 48 h.5.qRT-PCR and Western blot(WB)were used to detect the expression of miR-429 and CRKL in K562 cells induced by Hemin for 24 h and 48 h.6.Plasmid PCDH-CRKL,negative control PCDH,miR-429 inhibitor and negative control miR-NC inhibitor were transiently transfected into K562 cells by Lipofectamine TM 2000 and Letran-R methods,respectively.WB and qRT-PCR methods were used to detect the expression levels of CRKL and miR-429,respectively.7.The effect of miR-429 and CRKL levels on erythroid differentiation was detected by benzidine staining.8.The effects of miR-429 and CRKL levels on the expression of K562 erythroid differentiation molecules were detected by qRT-PCR.9.The effect of miR-429 and CRKL levels on the proliferation of K562 cells was detected by CCK-8 assay10.The effect of miR-429 on the expression of erythroid differentiation molecules GPA and CD71 in K562 cells was detected by flow cytometry11.WB method was used to detect the effects of high and low expression of miR-429 and high expression of CRKL on the expression of CRKL and p-CRKL in K562 cells and the expression of ERK pathway proteins Raf,p-Raf,MEK,p-MEK,ERK1/2,p-ERK1/2 and Ras.12.After inhibiting ERK pathway with PD98059,the effects of miR-429 and CRKL on the expression of ERK and p-ERK in K562 cells were detected by WB assay,on erythroid differentiation of K562 was detected by benzidine staining,on the expression of erythroid molecules was detected by qRT-PCRResults:1.Compared with normal peripheral blood samples,the expression of miR-429 in mononuclear cells of bone marrow samples from patients with initial CML was 1.1 times lower(P=0.0269),and the expression of CRKL was 4.45 times higher(P=0.0208),indicating a negative correlation between the two(R2=0.424,P=0.0301)2.After the stable down-regulation of CRKL,the microarray data showed that the levels of SPTA1,SPTB,TFRC,PKLR,EPB41L3,AHSP,RHD,RHCE were increased by 1.50,1.64,1.76,1.82,1.90,2.20,1.94 and 2.06 times in K562 respectively.3.The erythroid differentiation rate of K562 cells induced by 40 μM Hemin for 24 and 48 h was increased by 36.5%(P=0.0036)and 85.9%(P=0.0034),respectively.Compared with 0 h,the expressions of erythroid molecules γ-globin,ε-globin,GPA andα-globin were increased by 3.2 times(P=0.0152),1.0 times(P=0.0416),2.4 times(P=0.0064)and 1.9 times(P=0.0095)at 24 h;and 3.5 times(P=0.0020),0.86 times(P=0.0107),2.1 times(P =0.0040)and 1.5 times(P =0.0008)at 48 h,respectively.4.qRT-PCR results showed that miR-429 expression was increased by 43.5%(P=0.0279)and 123.0%(P=0.0011)in K562 treated with 40 μM Hemin for 24 and 48 h,respectively.WB results showed that CRKL decreased by 28.7%(P=0.0008)and 37.7%(P=0.0072),respectively.5.Compared with miR-NC inhibitor transfected cells,the erythroid differentiation rate of K562 cells transfected with miR-429 inhibitor decreased by 51.4%(P<0.0001);The erythroid differentiation rate of K562 cells transfected with PCDH-CRKL was47.2% lower than that of PCDH group(P<0.0001).6.Compared with miR-NC inhibitor transfected cells,γ-globin,ε-globin and HBA were decreased by 61.3%(P=0.0005),78.9%(P=0.0020)and 44.6%(P=0.0226)in miR-429 inhibitor transfected cells.Compared with PCDH transfected cells,γ-globin,ε-globin and HBA were decreased by 55.3%(P=0.0034),76.8%(P<0.0001)and 92.1%(P<0.0001)in PCDH-CRKL transfected cells.7.CCK-8 analysis showed that compared with miR-NC inhibitor transfected cells,the proliferation capacity of miR-429 inhibitor transfected cells at 24 h,48 h and 72 h was increased by 10.3%(P=0.0020),16.5%(P<0.0001)and 61.1%(P<0.0001),respectively;Compared with PCDH transfected cells,the proliferation capacity of PCDH-CRKL transfected cells was increased by 18.2%(P=0.0010),40.4%(P<0.0001) and 25.7%(P<0.0001),respectively.8.Flow cytometry results showed that the proportion of CD71 and CD235A(GPA)double positive cells in miR-429 mimic transfected cells was 42.5% higher than that in miR-NC mimic transfected cells.9.Compared with miR-NC mimic,CRKL expression in K562 cells transfected with miR-429 mimic was decreased by 39.3%(P=0.0006),p-CRKL was decreased by42.0%(P=0.0058);p-Raf,p-MEK and p-ERK were increased by 71.3%(P=0.0034),47.5%(P=0.0306)and 67.7%(P=0.0004),respectively.Compared with miR-NC inhibitor transfected cells,CRKL was increased by 34.7%(P<0.0001),p-CRKL was increased by 83.0%(P=0.0041);p-Raf,p-MEK and p-ERK expressions were decreased by 33.0%(P=0.0071),35.3%(P=0.0045)and 46.3%(P=0.0025),respectively.Compared with PCDH transfected cells,CRKL expression was increased by 116.3%(P=0.0004),p-CRKL expression was increased by 81.0%(P=0.0047);p-Raf,p-MEK and p-ERK expression were decreased by 35.7%(P=0.0147),40.7%(P=0.0039)and41.0%(P=0.0005)in PCDH-CRKL transfected cells.There was no difference in the expression of Raf,MEK,ERK and Ras in the three groups.10.After PD98059 inhibited ERK pathway,the erythroid differentiation rate of K562 cells transfected with miR-429 mimic was 67.0% higher than that transfected with miR-NC mimic(P=0.0032).The erythroid differentiation rate of cells transfected with miR-429 mimic+PD98059 was 34.5% lower than that of cells transfected with miR-429mimic(P=0.0088).Compared with si-NC transfected cells,the erythroid differentiation rate of si-CRKL transfected cells increased by 72.0%(P=0.0002).Compared with si-CRKL transfected cells,the erythroid differentiation rate of si-CRKL+PD98059transfected cells decreased by 55.5%(P=0.0002).Correspondantly,after blocking ERK pathway,the γ-globin,ε-globin and GPA in cells transfected with miR-429 mimic were increased by 74.8%(P=0.0022),43.5%(P=0.0020)and 42.5%(P=0.0011),respectively,compared with those transfected with miR-NC mimic.Compared with cells transfected with miR-429 mimic,γ-globin,ε-globin and GPA decreased by 56.2%(P=0.0015),63.6%(P=0.0022)and 23.1%(P=0.0286),respectively.Compared with si-NC transfected cells,γ-globin,ε-globin and GPA in si-CRKL transfected cells increased by24.2%(P<0.0001),28.7%(P=0.0338)and 48.5%(P=0.0164),respectively.Compared with si-CRKL transfected cells,γ-globin,ε-globin and GPA in si-CRKL +PD98059transfected cells decreased by 46.0%(P=0.0052),30.9%(P=0.0172)and 44.5%(P=0.0059),respectively.Conclusion:1.The low expression of miR-429 and high expression of CRKL in clinical samples of CML were negatively correlated2.MiR-429 promoted erythroid differentiation of K562 cells.3.CRKL inhibited erythroid differentiation of K562 cells.4.MiR-429 targeted CRKL regulates erythroid differentiation of K562 cells through activation of ERK signaling pathway... |
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