| Background:Chronic myeloid leukemia(CML)is a malignant proliferative disease derived from pluripotent hematopoietic stem cells,which has the biological characteristics of highly proliferative and malignant invasion.It accounts for about 20% of adult leukemia.Most ofCML patientswill appear inthe specific cytogenetic changes of the Phchromosome,which appears ontranslocationthe long arm of chromosome 9 ABL oncogene to breakpoint cluster region(BCR)of chromosome 22 to form the BCR-ABL fusion gene.It exhibits constitutive tyrosine kinase activity that enhances cell proliferation and migration,protects cells from apoptosis,blocks cell differentiation and leads to CML via activating a variety of downstream pathways.The abnormal expression of miR-4521 is closely related to many cancers.High expression of FAM129 A is associated with the occurrence and development of various malignancies;down-regulation of FAM129 A can inhibit the proliferation,migration,invasion and apoptosis of thyroid cancer cells.However,the research about miR-4521,FAM129 A in CML and K562 cellhas rarely been reported.Objective:1.To study the expression of miR-4521 and FAM129 A in bone marrow samples from CML patients and the correlation between them.2.To study the negative regulation of mi R-4521 and FAM129 A.3.To investigate the effects of over-expression miR-4521 on the malignant behaviors of K562 and its molecular mechanism.4.To explore the effects of FAM129 A down-regulation on the malignant behaviors of K562 and its molecular mechanism.Methods:1.qRT-PCR was used to detect the expression of miR-4521 and FAM129 A in 33 cases of human CML samples,6 cases of normal donors and 7 cases of non-malignant samples;and compare the changes of miR-4521 and FAM129 A levels in CML with primary and CR and their relationship;2.MiRNA target gene prediction software(TargetScan,mi RDB)was used to analyze the direct relationship between miR-4521 and FAM129A;Constructing the expression vector ofpsi-CHECKTM2.0-WT-FAM129A-3'UTR/psi-CHECKTM2.0-MUT-FAM129A-3' UTR;The recombined vector was transfected into 293 T cells with miR-4521 mimic/NC mimicand the activity value of double luciferase was detected,and the target relationship was verified.3.Using LipofetaminTM 2000 transfection reagent,miR-4521 mimic was transiently transfected into K562,and the expression of miR-4521 and FAM129 A were detected by qRT-PCR and WB;Trypan blue excluding assay determined the effects of over-expression mi R-4521 on the proliferation ability of K562;Transwell assay measured the effects of over-expression miR-4521 on the migration and invasion capabilities of K562;qRT-PCR detected the expression of MMP-2,MMP-9,TIMP-1 on the over-expression miR-4521.4.Using LipofetaminTM 2000 transfection reagent,si-FAM129 A was transiently transfected into K562,and the expression of FAM129 A were detected by WB;Trypan blue excluding assaydetermined the effects of down-expression FAM129 A on the proliferation ability of K562;Transwell assay measured the effects of down-expression FAM129 A on the migration and invasion capabilities of K562;qRT-PCR detected the expression of MMP-2,MMP-9,TIMP-1 on the down-expression FAM129 A.Results: 1.Compared to the normal group,the expression of miR-4521 was down-regulated and FAM129 A was up-regulated;The expression of them was not different in non-malignant samples;In the same sample,compared with the initial sample,two expressions had a "reply" phenomenonin the CR phase;They were the negative correlation between miR-4521 and FAM129A(P=0.0152,R2=0.2218).2.Dual luciferase reporter gene assay showed that the detection activity of the two groups was not significantly different with NC mimics or miR-4521 mimic co-transfection in the MUT group;But in the WT group,the detection activity of the two groups was significantly different;Compared to the NC mimics group,the activity was decreased by 44% in the miR-4521 mimic group,P=0.0022.3.After transient transfection of mi R-4521 mimic,the proliferation,migration and invasion of K562 were inhibited;The gene and protein expression of FAM129 A was reduced by 57.1% and 41%,respectively;The gene expression of MMP-2,MMP-9 was decreased,TIMP-1 was increased;4.After transient transfection of si-FAM129 A,theproliferation,migration and invasion of K562 were inhibited;The gene expression of MMP-2,MMP-9 was decreased,TIMP-1 was increased;Conclusion: 1.Low expression of miR-4521 and high expression of FAM129 Awere positively correlated with CML;In CR phase,their expressions restored;the relationship of two was negative correlation in bone marrow of CML samples;miR-4521 negatively regulates FAM129 A expression by binding to FAM129 A 3 'UTR region.3.Up regulation of miR-4521 could significantly inhibit theproliferation,migration and invasion of K562,reduce the expression of FAM129 A,MMP-2 and MMP-9,induce the expression of TIMP-1.4.FAM129 A down-regulation could significantly inhibit the proliferation,migration and invasion of K562,reduce the expression of MMP-2 and MMP-9,induce the expression of TIMP-1.In conclusion,miR-4521 affects the expression of MMP-2,MMP-9,TIMP-1by negatively regulating the expression of FAM129 A,thereby affecting K562's proliferation,migration andclinical progressionof CML. |
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