| Background:Acute myeloid leukemia(AML)is a malignant blood disease derived from myeloid progenitor cells.AML is characterized by abnormal proliferation of primary and early myeloid cells in the bone marrow and peripheral blood.Its invasive progression and high mortality rate make AML one of the major causes of cancer death in young and middle-aged patients worldwide.In recent years,great progress has been made in the pathogenesis,diagnosis and treatment of AML,but the prognosis of AML patients is still not optimistic.Therefore,it is of great significance to find new biomarkers of AML malignancy and study its mechanism.CRKL is a signal adaptor protein with molecular weight of 39 k D,which is one of the CRK family and contains one SH2 and two SH3 domains.CRKL can combine with different proteins to form complexes and play important biological functions.As a proto-oncogene,CRKL can affect the proliferation,migration and invasion,clonogenesis and apoptosis of tumor cells,and play an important role in a variety of cancers.Micro RNAs(miRNAs)are a class of highly conserved non-coding regulatory small Rnas composed of 19～25 nucleotides(nt),whose main function is to negatively regulate the stability of mRNA of protein-coding target genes.It inhibits or promotes mRNA degradation by binding to the 3’-non-coding region(3’-UTR)or 5’-UTR of the target gene mRNA,and interacts with the target gene mRNA to degrade or inhibit correct translation initiation.miR-124-3p is a highly conserved miRNA with a length of 22 nt.The decreased expression level of miR-124-3p is related to the occurrence and development of tumors,and can regulate tumor growth and metastasis.In our previous study,miR-124-3p was found to have a targeted negative regulatory relationship with CRKL,and was associated with malignant behaviors such as proliferation,migration and invasion,cloning and apoptosis of liver cancer cells HCCLM3 and Huh7 and chronic myelotic leukemia cells K562.In this paper,we further studied the regulation and mechanism of malignant behavior of acute myeloid leukemia cells by miR-124-3p-CRKL axis.Objective:1.To analyze the influence of the expression level of miR-124-3p on the expression level of CRKL in U937 cells;2.To determine the effect of CRKL on the proliferation,migration,invasion,and apoptosis malignant phenotypes of U937 cells in vitro;3.To determine the effect of miR-124-3p on the proliferation,migration,invasion,and apoptosis malignant phenotypes of U937 cells in vitro;4.To explore the regulatory mechanism of miR-124-3p targeting CRKL on the malignant behavior of acute myeloid leukemia cells.Methods:1.The expressions of miR-124-3p in AML patients were analyzed in GEO gene database;2.The mRNA expression levels of miR-124-3p and CRKL were detected by qRT-PCR after instantaneous transfection of NC-mimic,miR-124-3p-mimic,NC-inhibitor and miR-124-3p-inhibitor 48 h.CRKL protein was detected by Western blot;3.The ability of U937 cells to proliferate in vitro after changes in miR-124-3p and CRKL levels was detected by Trypan blue counting method;4.The ability of U937 cells to migration and invasion after changes in miR-124-3p and CRKL levels was detected by Transwell assay;5.The ability of U937 cells to apoptosis of after changes in miR-124-3p and CRKL levels was detected by flow cytometry;6.Rescue experiment was conducted to study whether the inhibition effect of CRKL up-regulation and reversal of miR-124-3p on the migration ability of U937 cells could be reversed;7.The protein levels of RAF/MEK/ERK,EMT and key apoptotic molecules in U937 cells were determined by Western blot after changes in miR-124-3p and CRKL levels;8.After the ERK pathway was inhibited by ERK inhibitor PD98059,the protein levels of CRKL,ERK,p-ERK,EMT and apoptosis-related molecules in U937 cells were detected by Western blot.Results:1.The differential expression results of GEO gene database showed that,compared with healthy controls,the expression of miR-124-3p in AML patients was significantly down-regulated(P<0.001),but the expression of CRKL was not significantly changed;2.U937 up-regulated miR-124-3p: the expression level of miR-124-3p was up-regulated by 53,740 times(P=0.0012),the mRNA level of CRKL was down-regulated by 16%(P=0.0027),and the protein level of CRKL was down-regulated by 31%(P=0.0002).Down-regulated miR-124-3p: miR-124-3p was down-regulated by 60%(P=0.0038),CRKL mRNA level was up-regulated by 128%(P=0.0009),CRKL protein level was up-regulated by 37%(P < 0.0001);3.Trypan blue counting method showed that U937 cell proliferation was enhanced after up-regulation of CRKL.The proliferation ability of U937 cells was decreased after up-regulation of miR-124-3p,and increased after down-regulation of miR-124-3p;4.Transwell method showed that the migration and invasion ability of U937 cells was enhanced after up-regulation of CRKL;The migration and invasion ability of U937 cells was decreased after up-regulation of miR-124-3p,and increased after down-regulation of miR-124-3p;5.Flow cytometry showed that the apoptosis capacity of U937 cells was weakened after up-regulation of CRKL.The apoptotic ability of U937 cells was increased after up-regulation of miR-124-3p,and decreased after down-regulation of miR-124-3p;6.Rescue test results showed that compared with the miR-124-3p-mimic group,the mRNA expression of CRKL in U937 group of miR-124-3p-mimic+PCDH-CRKL was increased 90 times(P=0.0007),and the protein level was increased by 40%(P=0.0140).Correspondingly,the in vitro migration ability of miR-124-3p-mimic+PCDH-CRKL group was enhanced by 40%(P=0.0304).;7.Both miR-124-3p and CRKL can affect RAF/MEK/ERK,EMT and key molecules of apoptosis.Up-regulation of CRKL increased the protein levels of p-RAF,p-MEK,p-ERK,Bcl-2,Vimentin and N-cadherin in U937 cells by 96%(P=0.0002)、157%(P=0.0012)、49%(P=0.0142)、123%(P=0.0039)、104%(P=0.0002)和 45%(P=0.0371),respectively The expression levels of BAX and E-cadherin were decreased by 52%(P=0.0004)and 47%(P=0.0002).The up-regulation of miR-124-3p reduced the expression levels of p-RAF,p-MEK,p-ERK,Bcl-2,Vimentin and N-cadherin in U937 cells by 34%(P=0.0055)、32%(P=0.0010)、44%(P=0.0003)、21%(P<0.0001)、31%(P=0.0096)and 30%(P=0.0008),the expression levels of BAX and E-cadherin increased by 39%(P=0.0324)and 26%(P=0.0084),respectively.Down-regulation of miR-124-3p increased the expression levels of p-RAF,p-MEK,p-ERK,Bcl-2,Vimentin and N-cadherin in U937 cells by 75%(P=0.0372)、63%(P=0.0048)、 40%(P<0.0001)、 27%(P<0.0001)、 51%(P=0.0217)and 26%(P=0.0166),the expression levels of BAX and E-cadherin were decreased by 37%(P=0.0164)and 25%(P=0.0048),respectively.There were no significant changes in the expression levels of RAF,MEK and ERK in the three groups;8.After ERK inhibitor PD98059 blocked RAF/MEK/ERK pathway,the promoting effects of miR-124-3p and CRKL on ERK pathway were inhibited;In addition,the promotion effects of CRKL up-regulation and miR-124-3p down-regulation on EMT and key molecules of apoptosis were inhibited,indicating that miR-124-3p-CRKL axis regulates malignant behaviors of U937 cells through RAF/MEK/ERK pathway,EMT and key molecules of apoptosis.Conclusion:1.miR-124-3p negatively regulates the expression of CRKL in acute myeloid leukemia cells U937;2.CRKL promote the proliferation,migration and invasion ability of U937 cells in vitro,and inhibit cell apoptosis ability;3.miR-124-3p inhibit the proliferation,migration and invasion ability of U937 cells in vitro,and promote the apoptosis ability of cells ability;4.miR-124-3p targeted CRKL inhibits the malignant behavior of acute myeloid leukemia cells U937 through RAF/MEK/ERK pathway. |
