| Context:Acute myeloid leukemia((Acute myeloid leukemia))is a malignant hematological disease characterized by abnormal proliferation and differentiation of hematopoietic stem cells and myeloid progenitors,which can lead to hematopoietic system disorder and inhibit normal hematopoiesis of erythrocytic and megakaryocytic lineages.The main manifestations of the disease are infection,hemorrhage,anemia and infiltrating skin,mucous membrane,lymph nodes,cerebrospinal fluid,spleen and other important organs or tissues,with rapid onset,rapid deterioration and short natural course of disease.In recent years,due to a deeper understanding of the pathophysiological process of the disease,a large number of new drugs have emerged;the continuous progress of hematopoietic stem cell transplantation and the continuous improvement of supportive therapy have significantly improved the remission rate and cure rate of the disease.However,the overall survival rate of the disease is still less than 50%.This is mainly due to the patient’s primary drug resistance or disease recurrence and eventually develop into refractory leukemia.MircroRNA is a highly conserved class of non-coding RNA,in the process of evolution,most of which originate from introns and are about 18-22nt in length.because these non-coding RNA can bind to the mRNA of the target gene to cause the latter degiadation or translation inhibition,so they play an important role in gene post-transcriptional regulation.In the process of normal physiological metabolism,this regulation is one of the key elements in the process of cell proliferation,differentiation,apoptosis and maintaining cell homeostasis.Moreover,in recent years,it has been found that microRNAs not only has a unique expression pattern in the process of malignant evolution,but also plays the role of oncogenes or tumor suppressor genes.Therefore,it is also one of the key factors that affect the fate of tumors.As one of them,miR-204 is located in the sixth intron of human chromosome 9 q21,the gene of transient receptor potential ion channel protein 3(TRPM3).In many tumors,the microRNA mainly plays an anti-tumor effect.It has been found that patients with acute myeloid leukemia with low expression of miR-204 are closely related to poor prognosis.Therefore,the miR-204 can be used as one of the independent prognostic factors of AML.In addition,miR-204 can also target the apoptosis of AML cells induced by BIRC6 and enhance the sensitivity to chemotherapy.Therefore,miR-204 and its downstream target genes may bring new gospel to AML patients.Human hepatocyte factor(HGF)is a double-stranded glycoprotein,which can regulate the proliferation,survival,migration and morphological transformation of target cells,as well as cell renewal and tissue repair.The gene encoding this cytokine is located in the long arm(7q2111)of chromosome 7.It plays an important role in the process of cell malignant evolution.It has been reported that overexpressed HGF/c-MET pathway has been found in solid tumors such as gastric cancer,liver cancer,colon cancer,breast cancer and non-small cell lung cancer,which is closely related to tumor progression and prognosis.In AML,HGF can play a important role in PI3/AKT and MAPK/ERK signal pathways to promote the proliferation,invasion and migration of AML cells.Therefore,both miR-204 and HGF are closely related to AML.However,the relationship between them in AML and the potential molecular regulation mechanism still need to be further clarified.Objective:To explore the role of miR-204 and HGF in AML,Furthermore,to clarify the relationship between them and their potential molecular biological mechanism.providing a new theoretical basis for targeted treatment of this disease.Method:The expression levels of miR-204 and HGF in bone marrow mononuclear cells of patients with AML or normal subjects and AML cell lines were measured by RT-qPCR technique and the correlation was analyzed.The levels of proteins related to HCF/c-MET pathway such as HGF,c-MET,p-MET and c-myc in AML cell line were detected by western blot,and the expression levels of miR-204 and HGF in AML cell line were regulated by transient transfection and RNAi.The relationship between them was preliminarily predicted by online bioinformatics prediction software,and HGF3’UTR wild type and mutant luciferase reporter plasmids were constructed.The changes of luciferase activity after up-regulation of miR-204 were detected by double luciferase reporter gene assay,cell proliferation was measured by MMT method,apoptosis level was determined by Annexin V/PI flow cytometry,and cell migration and invasion were measured by Transwell method.GraphPad8,SPSS 23.0 and ImageJ were used to analyze the data.The differences between the two groups and more than two groups were analyzed by Student’s t-test and one-way ANOVA,and the correlation was analyzed by pearson method.When the p value was less than 0.05,the difference was considered to be statistically significant.Result:1.The expression of miR-204 and HGF in newly diagnosed AML patients:1.1 The relative expression of miR-204 in newly diagnosed AML patients(1.16643)was significantly lower than that in normal controls(2.231967).1.2 The relative expression of HGF in newly diagnosed AML patients(1.712266)was significantly higher than that in normal controls(1.0285).1.3 The expression of miR-204 and HGF in bone marrow mononuclear cells of newly diagnosed AML patients was analyzed by Pearson method,and it was found that there was a negative correlation between them,and the difference was statistically significant.(r=-0.3666,p<0.05).2.Expression level of miR-204 and HGF in AML cell line.2.1 The expression of miR-204 in Kasumi-1 and HL-60 cell lines was significantly lower than that in human bone marrow stromal cells(HS-5)(p<0.05).2.2 Compared with human bone marrow stromal cells(HS-5),the gene and protein expression levels of HGF in Kasumi-1 and HL-60 cell lines were significantly higher than those in HS-5(p<0.05).3.The role of miR-204 and HGF in AML cells3.1 The results of real-time fluorescence quantitative PCR showed that miR-204 mimic could significantly up-regulate the relative expression of miR-204 in AML cells,and sh-HGF interference vector could inhibit the gene and protein expression of HGF in AML cells.3.2 The results of MMT assay showed that up-regulation of miR-204 could inhibit the proliferation activity of kasumi-1 and HL-60,and the inhibition rates were 44.7%and 40.8%respectively after 72 h culture.Down-regulation of HGF could weaken the proliferation activity of AML cells and affect the survival of AML cells,and the survival rates after 72 h culture were 53.5%and 57.4%,respectively.3.3 The apoptosis of AML cells was detected by Annexin V/PI double staining and flow cytometry.The results showed that up-regulation of miR-204 or down-regulation of HGF could promote the apoptosis of AML cells.3.4 The results of Transwell assay for the migration and invasion trend of AML cells confirmed that up-regulation of miR-204 or down-regulation of HGF could weaken the migration and invasion ability of Kasumi-1 and HL-60 cells.4.Verification of the targeting relationship between miR-204 and HGF4.1 The results of Western blot showed that the expression of HGF protein decreased significantly after up-regulation of miR-204 in AML cells,while the down-regulation of miR-204 by miR-204 inhibitor could increase the expression of HGF protein.4.2 The on-line bioinformatics prediction software was used to predict the targeting relationship between miR-204 and HGF.The wild type and mutant luciferase reporter gene plasmids of HGF 3 ’UTR were successfully constructed and double luciferase reporter gene assay was carried out in human embryonic kidney 293T cells.The results showed that there was a predictive target for binding to miR-204 at HGF 3’ UTR.5.Effect of miR-204 targeting HGF on biological behavior of AML cells5.1 The results of MMT assay confirmed that miR-204 could target HGF to inhibit the proliferation of AML cells.5.2 The results of pre-staining with Annexin V/PI double staining and flow cytometry showed that miR-204 could target HGF to promote the apoptosis of AML cells.5.3 The results of Transwell assay confirmed that miR-204 could target the ability of HGF to inhibit the migration and invasion of AML cells.6.In AML cells,the molecular biological mechanism of miR-204 targeting HGF was showed by the results of western blot assay that miR-204 targeted down-regulated the expression of HGF,but did not affect the expression of c-MET receptor,but inhibited the phosphorylation level of c-MET receptor and the expression of downstream proteins CyclinD1,c-myc,Bcl-2,Bax,MMP2 and MMP9,and promoted the expression of pro-apoptosis protein BAX.On this basis,up-regulation of HGF can restore the expression level of the above mentioned proteinsConclusion1.MiR-204 is lowly expressed in bone marrow of patients with AML,which exerts its anti-leukemia effect properly by inhibiting the proliferation,migration and invasion of AML cells and inducing cell apoptosis.2.HGF is highly expressed in bone marrow samples of patients with AML,which may be related to the occurrence of AML.3.HGF is the target gene of miR-204,and miR-204 can down-regulate the expression of HGF and its downstream proteins of HGF/c-MET pathway.4.MiR-204 targeted inhibition of HGF expression and regulation of HGF/c-MET pathway may be one of the key elements of its anti-leukemia effect. |
