MiR-638 Down-regulate The Bone Marrow Angiogenesis By Suppressing VEGFA In Acute Myeloid Leukemia

BackgroundAcute leukemia is a common hematological malignant diseases.Significant improvements in diagnosis and treatment of leukemia have been presented in the past decades,at the same time,its overall incidence rate is still on the rise.It is an important task to research its pathogenesis and therapeutic strategies.Previously,most of researchers payed close attention to the changes of leukemia stem cells,such as cytogenetics,gene recombination,mutation.In recent years,several studies have showed that the hematopoietic microenvironment played important roles in the he development of leukemia.Leukemia cells proliferate in the hematopoietic microenvironment,and continuously promote its alteration,correspondingly,the development of the hematopoietic microenvironment provides better and better growing conditions for leukemia cells.Bone marrow angiogenesis is an important pathological process in the hematopoietic microenvironment,a great deal of participants involved in it,including vascular endothelial cells,cytokines,signal transduction pathways,and leukemia stem cells themselves.The loss of balance between the promoter and the inhibitor contributes to this pathological process.Among the numerous contributing factors,VEGF is thought to play a key role in the development of tumors angiogenesis.In the 90s of last century,researchers first discovered bone marrow angiogenesis in patients with multiple myeloma,the following scholars found that this phenomenon also exists in acute leukemia.With the rapid development of molecular biology,the goal of treatment for tumor is no longer focus on the regulation of protein expression and cytokine levels.The regulation of pathognomonic gene expression at the molecular level has become a great challenge for medical researchers,and the discovery of miRNA has brought new hope to this subject.MicroRNAs(miRNAs)are highly conserved 18-25 nucleotides non-protein-coding small RNAs,which bind to imperfect matches to the 3' untranslated region(UTR)and other regions of target mRNAs/RNAs molecules,and thus repress their translation and/or stability.MiRNAs are involved in almost all biological mechanisms,more than half of the miRNAs are located in the oncogenes or anti-oncogenes,that's also an important mechanism for tumor development as well as the maintenance of homeostasis,and its abnormal expressions are important biological markers for various diseases.miRNAs play an important role in the development of tumor,proliferation,differentiation,apoptosis,stress response and metastasis.Many miRNAs can be detected in the plasma.with the rapid development of molecular biology technology,it would not only become biological markers for special disease,but also be helpful to evaluate the efficacy and prognosis.Active angiogenesis and metastasis are the main causes of tumor recurrence and low survival rate.There is increasing evidence that miRNA is involved in this process.However,to determine the relationship between miRNA and angiogenesis is a huge challenge,because one miRNA may correspond to hundreds or even thousands of genes,one gene can be regulated by numerous miRNAs.According to the basic data and our preliminary study on the expression of miRNAs in AML patients.We found that miR-638 may be involved in the process of tumor angiogenesis with the role of anti-oncogene,but its expression in acute myeloid leukemia and the specific mechanism is still not clear.ObjectiveTo observe the levels of VEGFAmRNA and miR-638 in the mononuclear cells and analyse their roles in bone marrow angiogenesis in acute myeloid leukemia.Further,the expressions of VEGFA mRNA and protein were observed in leukemia cell lines,as up-regulating and down-regulating the level of miR-638.and last,to explore the roles of miR-638 and VEGFA in bone marrow angiogenesis,to lay a foundation for finding new biomarkers and new therapeutic methods.MethodsClinical research77AML patients and 21 volunteers are observed in this research.Immunohisto-chemistry method was used to detect the bone marrow microvessel density(MVD)with Anti-vWF antibody.The expressions of miR-638 and VEGFAmRNA in bone marrow mononuclear cells were detected by qTR-PCR method.Bivariable analysis was performed to analyze the correlations of MVD,VEGFAmRNA,miR-638 levels and clinicopathological parameters of AML.After standard chemotherapy,evaluate the efficacy and detect the expression levels of MVD,miR-638 and VEGFAmRNA in bone marrow again.Fundamental researchLeukemia cell lines K562,HL60 and HUVECs were used as the research objects,and the expression of miR-638 and VEGFA was detected by qRT-PCR method.Liposome transfected miR-638mimics and its inhibitor into K562 and HL60 cell lines,and detect the miR-638,VEGFAmRNA and VEGFA protein levels respectively in transfected cells.Dual-luciferase reporter gene assay was constructed using the dual-Luciferase reporter assay system to measure the reporter activity according to the manufacturer's protocol.For VEGF 3'-untranslated region(UTR)luciferase reporter assay,wild-type or mutant reporter constructs(pmirGLO-VEGF-3'UTR-WT or pmirGLO-VEGF-3'UTR-MUT)were co-transfected into K562 and HL60 cells in plates with miR-638 and renilla plasmid by using lipofectamine 2000.Reporter gene assays were performed 48 hour after transfection.To observe the effects of leukemia cells that transfected with miR-638 and its inhibitor on endothelial cell proliferation,HUVECs was co cultured with K562 and HL60 using transwell chamber(Pore size of the pemeable membrane was 0.4?m),and the proliferation of HUVECs was detected by MTT method.ResultsClinical findingsThe MVD in primary AML patients was significantly higher than that in the control group,especially in patients with extramedullary infiltration.After standard chemotherapy,paired t test was used for analysis the patients before and after treatment.The bone marrow MVD was still high despite complete remission(CR).CR group and non-remission(NR)group had no statistical difference.The relative expression level of VEGFA mRNA in primary AML patients was significantly increased compared with the normal group,in contrast to this,miR-63 8 level was declined,and this phenomenon is particularly significant in the patients whose bone marrow-blasts>50%.After treatment,VEGFA mRNA level was decreased and miR-638 level was increased compared with before.When analyze the correlations between the three observation indicators,the results showed a significant positive correlation between MVD and VEGFA mRNA(r=0.583,P<0.05),miR-638 was negatively correlated with VEGFA mRNA(r=-0.389 P<0.05).The survival rate of patients with low MVD in bone marrow was significantly higher than that in the high group,and the relative expressions of VEGFA mRNA and miR-638 had no significant correlation with survival.Fundamental resultsThe expression level of miR-638 in leukemia cell lines K562 and HL60 was significantly lower than that in normal mononuclear cells.As miR-638mimics and miR-638 inhibitor were transfected into K562 and HL60 cell lines we detected miR-638 level by qTR-PCR method.In cell lines transfected with miR-638-mimics,the miR-638 expression level was increased,whereas in miR-63 8-inhibitor transfected cell lines was decreased.Meanwhile we detect the VEGFA mRNA and VEGFA protein with qRT-PCR and Western blot methods respectively.In transfection of miR-63 8-mimics cell lines,the levels of VEGFA mRNA and VEGFA protein were significantly down-regulated,but there were no significant differences in miR-638 inhibitor cell lines compared with the negative control cells.Luciferase reporter gene assay:In K562 and HL60 cell lines,when co-transfecte the wild-type VEGFA-3'UTR with miR-638,the luciferase activity was significantly lower than that of control group and mutation group,and in the mutant VEGFA-3'UTR group,the luciferase activity was not significantly different from that of the control group.The results confirmed that the 3 'UTR of VEGFA mRNA is a direct target of miR-638,and that miR-638 regulates the expression of its protein by targeting VEGFA-3 'UTR.The 560nm OD values represent the proliferation ability of HUVECs,in the two cell lines which transfected with miR-638 mimics,the OD values were significantly decreased compared with the negative control and inhibitor groups.There was no significant difference in K562 cells transfected with miR-638 inhibitor and negative control,whereas,in HL60 cell lines,the OD value was increased when transfected with miR-638 inhibitor.ConclusionsBone marrow angiogenesis is an important pathological process in acute myeloid leukemia.The detection of bone marrow microvessel density is important for the prognosis of leukemia.The expression of miR-638 in acute myeloid leukemia was down-regulated,which was negatively correlated with vascular endothelial growth factor.The VEGFAmRNA 3'-UTR was a direct target of miR-638,and VEGFAmRNA was the target gene of miR-638.MiR-638 can down-regulate the bone marrow angiogenesis by suppressing VEGFA and may be a biological marker for the detection of bone marrow angiogenesis and a new target for anti-angiogenesis therapy of leukemia.