The Role And Mechanism Of MiR-141-5p Via Targeting RAB32 In Chronic Myeloid Leukemia

Chronic myeloid leukemia(CML)is one of the most extensively studied neoplasms.The cytogenetic hallmark of CML is the Philadelphia chromosome(Ph),created by a reciprocal translocation between chromosomes 9 and 22(t [9];[22] [q34;q11]).This translocation results in the formation of a hybrid BCR-ABL oncogene on chromosome 22,which codes for a deregulated tyrosine kinase.Imatinib is the first BCR–ABL tyrosine kinase inhibitor(TKI)to be used for the treatment of CML.Due to its unique mechanism of action and high efficiency and specificity,it is widely used in CML treatment.However,resistance and intolerance to TKIs prevent a full therapeutic benefit in 20% of patients.Therefore,a better understanding of the tumor biology of CML and alternative therapeutic strategies is urgently needed.MicroRNAs(microRNA,miRNA)are small(18-22 nucleotides)non-coding RNAs,and widely expressed in prokaryotic and eukaryotic cells.MiRNAs regulate gene expression by specifically interacting with 3’ untranslated region(3’-UTR)of target gene mRNAs.By regulating the expression of target genes,miRNAs participate in a variety of biological processes.As one important member of the miR-200 family,miR-141 is aberrantly expressed in many human malignant tumors,participating in various cellular processes including epithelial-mesenchymal transition(EMT),proliferation,migration,invasion,and drug resistance.Edurne San et al.found that the expression of hsa-mir-141 was decreased in drug-resistant CML patients.In the previous study,we found that the expression of miR-141-5p was low in CML and K562 cells,and were up-regulated remarkably after patients were treated.The knowledge of biological effects of miR-141-5p in CML is still unclear.RAB32 is the predicted target gene of miR-141-5p through bioinformatics software.RAB32,a member of the Ras superfamily of small GTPases,regulates various intracellular membrane-trafficking events in many cell types.Notably,high RAB32 expression shortens neurite length,alters mitochondria morphology,and accelerates apoptosis/necroptosis of human primary neurons and cell lines.However,the biological role and underlying mechanisms of miR-141-5p via targeting RAB32 in CML remain poorly understood.In this study,we investigated whether miR-141-5p is a tumor suppressor in CML and aimed to further explore the potential mechanisms of action in vitro and in vivo.Therefore,it provides new clues for the diagnosis and new Targeted therapy of CML.The main research contents are summarized as follows: 1.Expression of miR-141-3p and miR-141-5p in CML patients and CML K562 cellsFirstly,the expressions of miR-141-3p and miR-141-5p in newly diagnosed CML patients,healthy controls,K562,K562/G cells and CD34+ cells were detected by q-PCR.Q-PCR confirmed the low expression of miR-141-3p and 5p in CML patients,K562 and K562/G cells,and the low expression of miR-141-5p was more significant.18 newly diagnosed CML patients were treated by nilotinib or imatinib,we have found by return visit,significant declines of BCR-ABL copy numbers were detected in 14 patients after one year,while 4 CML patients showed any declines.The expression of miR-141-3p and miR-141-5p were up-regulated remarkably when patients were treated with nilotinib or imatinib for 3 months.It suggested that low expression of miR-141 may play a role in the occurrence and development of CML.2.Biological effects of miR-141-5p in CML K562 cell linesGiven that lower expression miR-141-5p in CML patients,miR-141-5p was chose as research object for further investigation.Using cationic liposome method respectively in K562 cells transfection with miR-141-5p inhibitor& Negative control or miR-141-5p mimics& Negative control to down-regulate or up-regulate the expression of miR-141-5p,Western blotting and q-PCR were used to detect the expression of related genes in K562 cells,PI staining after transfection and then flow cytometry was used to analyze the cell cycle,and Annexin V/PI method was used to detect the apoptosis of K562 cell lines.The results showed that after transfection with miR-141-5p inhibitor,the expression of miR-141-5p in K562 cells was significantly decreased,and the protein and mRNA levels of c-Myc,Cyclin D1,MMP-3,MMP-9 in K562 cells were significantly increased.The proportion of K562 cells in S phase and G2/M phase were increased,suggesting that the low expression of miR-141-5p could enhance the proliferation and migration of K562 cells.Conversely,after transfection with miR-141-5 p mimics,the expression of miR-141-5p in K562 cells was significantly increased.c-Myc,Cyclin D1,MMP-3,MMP-9 protein and mRNA expression levels were decreased significantly,the protein expression level of cleaved caspase 3 was up-regulated,S phase and G2/M phase cells proportion reduced,early and late cell apoptosis ratio increased significantly.It suggested that over-expression of miR-141-5p can significantly inhibit the proliferation and migration abilities of K562 cells,promote cell apoptosis.3.Effect of miR-141-5p on tumorigenesis of xenograft with nude miceMouse tumor xenografts were used to assess the effect of miR-141-5p in vivo.Used lentivirus vector to establish the stable expression of miR-141-5p mimics&negative control in K562 cell,and inoculated it subcutaneously to female nude mice,observed and measured volume and weight of transplanted tumor.The results showed that tumor volume and weight of miR-141-5p mimics group were significantly lower than the control group.It suggested that miR-141-5p may act as a tumor suppressor in CML.4.The mechanism of miR-141-5p via targeting RAB32 in CMLBioinformatics analysis predicted that RAB32 was the direct target gene of miR-141-5p.Through the construction and transfection of RAB32 3’ UTR-WT and miR-141-5p mimics/NC into K562 cell,which caused the fluorescence changes,identified the potential target of miR-141-5p was RAB32.The protein and mRNA levels of RAB32 were up-regulated or decreased in K562 after inhibition or overexpression of miR-141-5p.The expression of RAB32 was significantly up-regulated in CML patients and down-regulated in healthy controls and CML after treatment.The above results suggested that miR-141-5p could regulate the expression of RAB32 in CML.Transfected with RAB32 siRNA inhibited the proliferation and migration and promoted the apoptosis of K562 cells.Co-transfection with miR-141-5p mimics and empty plasmid led to the significant down-regulation of the mRNA and protein levels of c-Myc,Cyclin D1,MMP-3 and MMP-9.However,they were unchanged with miR-141-5p mimics and RAB32 over plasmid(RAB32-N1)in K562 cells.It suggested that RAB32-N1 could not alter the effect of miR-141-5p mimics on proliferation,migration and apoptosis of K562 cells.Taken together,all the results suggested that miR-141-5p may play a role in CML via targeting RAB32.