The Role And Mechanism Of MiR-139-5p In Acute Myeloid Leukemia By Targeting Tspan3

Acute myeloid leukemia is a malignant leukemia,which is characterized as the accumulation of blood and bone marrow immature bone marrow cells.Malignant cells in AML have completely lost ability to mature,and often exhibit chromosome,signal transduction,and cell cycle control abnormalities.Although some progress has been made in the prognosis of AML,the relapse rate in AML patients is still high,and the overall survival rate is very low.And for,it is difficult to assess the survival rate and prognosis of most AML patients lacking typical cell inheritance or molecular changes.Therefore,studying the pathogenesis of AML at the levels of molecular biology and cell biology is of great significance to find more valuable biomarkers and targeted therapies,and to improve the therapeutic efficacy for AML.It is reported that the abnormal expression of microRNA is related to the development of AML.MicroRNAs(miRNAs)are small non-coding RNAs that regulate target gene expression by targetinging the mRNA of the target gene at the transcriptional level.Inceasing evidence prove that miRNAs play a key role in multiple processes of cancers,including cell proliferation,apoptosis,differentiation,invasion and tumor angiogenesis.Recently,studies have shown that the abnormal expression of miR-139-5p is related to the occurrence and development of cancers,including gastric cancer,breast cancer,colorectal cancer,liver cancer and glioblastoma.However,the specific roles and mechanisms of miR-139-5p in AML are unknown.Tetraspanin-3(Tspan3)is a member of the Tetraspanins family.Tetraspanins is a large family of membrane proteins with four transmembrane spirals,including two extracellular domainss and two intracellular domains.Tetraspanins express in many types of cells and tissues.Tetraspanins is related to many cell processes,such as cell adhesion,signal transduction,cell proliferation,migration,fusion and immune response.Studies have shown that Tspan3 is an important regulator of invasive leukemia.Tspan3 is the target gene of RNA binding protein Musashi2(Msi2),a key regulator of leukemia and play an important role in the development and dissemination of AML.In order to explore the roles and mechanism of miR-139-5p in AML by targeting Tspan3,we first detected the expression of miR-139-5p and Tspan3 in BMMC(bone marrow mononuclear cells)in people with AML andnormal people,and carried out the correlational analysis of the expression of miR-139-5p and Tspan3.In addition,the target genes of miR-139-5p were predicted by online bioinformatics softwares DINAN,miRDB and TargetScan.Lent-miR-139-5p and Lent-Tspan3 vectors were constructed.And then LV-miR-139-5p and LV-Tspan3 lentivirus particles infected HL-60 and OCI-AML3 cells to establish stable cell lines overexpressing miR-139-5p and co-expressing miR-139-5p and Tspan3.The relationship between miR-139-5p and Tspan3 was determined by dual-luciferase reporter system assay.Then the effects of miR-139-5p on cell proliferation,apoptosis,cycle,invasion,migration and the PI3K/AKT pathway by targeting Tspan3 were detected by CCK-8,flow cytometry,Transwell assay and Western blot,respectively.Finally,HL-60 mouse models of AML were constructed,and the effect of miR-139-5p on the growth of AML xenografts in animals by targeting Tspan3 was further investigated.This study aimed to explore the role and mechanism of miR-139-5p in AML by targeting Tspan3,which will provide cancer new treatment target and safe and effective treatment method for AML.This study is divided into three parts:The first part:the expression of miR-139-5p and Tspan3 in AML and its significance;The second part:the effect of miR-139-5p on the biological function of AML cells by targeting Tspan3 and its molecule mechanism;The third part:the effect of miR-139-5p on the growth of AML xenograft by targeting Tspan3 and its molecule mechanism.Main content:The first part:The expression of miR-139-5p and Tspan3 in AML and its significanceMethods1.qRT-PCR was used to detect the expression of miR-139-5p and Tspan3 in BMMC of people with AML and normal people.2.Correlation analysis of the expression of miR-139-5p and Tspan3 was carried out by graphpad prism software.3.The online bioinformatics softwares DINAN,miRDB and TargetScan were used to predict the target genes of miR-139-5p.Results1.Compared with bone marrow monouclear cells of normal people,the expression of miR-139-5p was significantly decreased in bone marrow monouclear cells of people with AML,while the expression of Tspan3 was significantly increased.2.The expression of miR-139-5p and Tspan3 was negatively correlated in BMMC of people with AML.3.Several bioinformatics softwares predicted that Tspan3 was the target gene of miR-139-5p.The second part:The effect of miR-139-5p on the biological function of AML cells by targeting Tspan3 and its molecule mechanism Methods1.The Lent-miR-139-5p and Lent-Tspan3 lentivirus vector were constructed.Lent-miR-139-5p and Lent-Tspan3 lentivirus vectors were packaged.LV-miR-139-5p and LV-Tspan3 lentivirus particles were titrated.HL-60 and OCI-AML3 cells were infected with LV-miR-139-5p lentiviru particle,and HL-60-miR-139-5p and OCI-AML3-miR-139-5p stable cell lines were screened by purinamycin.HL-60-miR-139-5p and OCI-AML3-miR-139-5p stable cell lines were infected with LV-Tspan3lentiviruparticle,andHL-60-miR-139-5p+Tspan3and OCI-AML3-miR-139-5p+Tspan3 cell lines were obtained.2.The relationship of miR-139-5p and Tspan3 was verified by dual luciferase reporter assay3.CCK-8 assay was used to detect the effect of miR-139-5p on the proliferation of HL-60 and OCI-AML3 cells by targeting Tspan3.4.The effect of miR-139-5p on the cycle and apoptosis in HL-60 and OCI-AML3 cells by targeting Tspan3 were detected by flow cytometry assay.5.Transwell assay was used to detect the effects of miR-139-5p on the invasion and migration in HL-60 and OCI-AML3 cells by targeting Tspan3.6.Western blot was used to detect the effect of miR-139-5p on the PI3K/AKT pathway in HL-60 and OCI-AML3 cells by targeting Tspan3.Results1.Monoclonal PCR and sequencing results of Lent-miR-139-5p and Lent-Tspan3 lentivirus vectors were consistent with data from NCBI,indicating that recombinant lentivirus vectors were constructed successfully.The titration value of LV-miR-139-5p and LV-Tspan3 virus was 2×10~8 TU/mL.2.The Result from dual luciferase reporter assay showed that Tspan3 was the target gene of miR-139-5p.3.In HL-60 and OCI-AML3 cells,miR-139-5p overexpression inhibited cell proliferation,migration and invasion.miR-139-5p overexpression decreased the proportion of cells in S phase,imposed cell cycle block in G2/M phase,and promoted apoptosis through inhibiting the activation of the PI3K/AKT pathway by targeting Tspan3.The third part:The effect of miR-139-5p on the growth of AML xenograft by targeting Tspan3 and its molecule mechanism Methods1.HL-60 mouse models of AML were constructed,and we detected the volume and weight of the xenografts of HL-60 mouse models of AML.2.The expression of proliferation related protein Ki67 was detected by immumohistochemical staining in xenografts of HL-60 mouse models of AML.3.The expression of miR-139-5p and Tspan3 in the xenografts of HL-60 mouse models of AML was detected by qRT-PCR.4.The expression of Tspan3,Akt and p-Akt protiens in the xenografts of HL-60mouse models of AML was detected by Western blot assay.Results1.We successfully constructed the HL-60 mouse models of AML,(HL-60,HL-60-miR-139-5p,and HL-60-miR-139-5p+Tspan3 groups).2.Our results showed that miR-139-5p overexpression could significantly inhibit the growth of AML xenografts,while Tspan3 overexpression could restore the growth of AML xenografts.3.Immumohistochemical staining results showed that expression of proliferation related protein Ki67 was significantly decreased in HL-60-miR-139-5p xenograft tissue,compared with HL-60 group,while Tspan3 overexpression reversed the inhibitory effect.4.qRT-PCR and Western blot assays revealed that miR-139-5p overexpression obviousy hindered the expression of Tspan3 in AML xenograft tissue.5.Western blot results showed that miR-139-5p overexpression could inhibit the expression of p-Akt in AML xenograft tissue,compared with HL-60 group,while Tspan3 overexpression reversed the inhibitory effect.Conclusions1.Compared with BMMC of normal people,the expression of miR-139-5p was significantly decreased in BMMC of people with AML,while the expression level of Tspan3 was significantly increased.The expression levels of miR-139-5p and Tspan3was negatively correlated.In addition,several bioinformatics softwares predicted that Tspan3 was the target gene of miR-139-5p.2.Dual-luciferase reporter assay showed that Tspan3 was the target gene of miR-139-5p.In HL-60 and OCI-AML3 cells,miR-139-5p overexpression inhibited cell proliferation,migration and invasion.miR-139-5p overexpression decreased the proportion of cells in S phase,imposed cell cycle block in G2/M phase,and promoted apoptosis through inhibiting the activation of the PI3K/AKT pathway by targeting Tspan3.3.In vivo,experiments further showed that miR-139-5p overexpression inhibited the growth of AML xenografts through through inhibiting the activation of the PI3K/AKT pathway by targeting Tspan3.