LncRNA CRNDE Regulates AML Progression By Sponging Mir-136-5p

ObjectiveTo investigate the molecular mechanism of lnc RNA CRNDE regulating the proliferation,apoptosis and cell cycle of AML cells.Methods1.The expression level of CRNDE in the bone marrow cells was analyzed by the GEPIA database.The expression of CRNDE m RNA in AML cell lines were detected by qRT-PCR.2.Bioinformatics software Lnc Locator was used to predict cell localization of CRNDE.3.Knockdown of CRNDE in U937 and KG-1a cells were achieved by transfecting lentivirus.The effects of CRNDE on the proliferation were detected by CCK-8 assay and cell counting assay.The cell cycle and apoptosis were detected by flow cytometry and western blot.The effect of CRNDE on the expression of miR-136-5p and MCM5 was detected by qRT-PCR and Western blot.4.The expression of miR-136-5p and MCM5 m RNA in AML cell lines were detected by qRT-PCR.Overexpression and inhibition of miR-136-5p were achieved by transfecting miR-136-5p mimic and inhibitor.The effects of miR-136-5p on the proliferation were detected by CCK-8 assay.The cell cycle and apoptosis were analysed by flow cytometry.The regulation of miR-136-5p on the expression levels of CRNDE and MCM5 was detected by qRT-PCR and Western blot.5.Bioinformatics software Starbase V2.0 predicted binding sites between CRNDE and miR-136-5p.CRNDE wild-type and mutant plasmids were constructed and co-transfected into 293 T cells with miR-136-5p mimic and NC mimic,respectively.Dual-Luciferase assay was used to verify the binding sites between the two.The enrichment effect of AGO2 antibody on CRNDE was detected by RIP.6.Bioinformatics software Target Scan 7.0 predicted binding sites between MCM5 and miR-136-5p.MCM5 3'UTR wild-type and mutant plasmids were constructed and co-transfected into 293 T cells with miR-136-5p mimic and NC mimic,respectively.Dual-Luciferase assay was used to verify the binding sites between the two.7.Mi R-136-5p inhibitor was transfected into sh-CRNDE cells,and the change of MCM5 expression was detected by qRT-PCR and Western blot.Results1.CRNDE was highly expressed in the bone marrow cells of AML patients and AML cell lines.2.CRNDE was mainly located in the cytosol.3.CRNDE knockdown can inhibit cell proliferation,promote apoptosis and arrest cell cycle in G1 phase.It can also upregulate miR-136-5p and inhibit the expression of MCM5 m RNA and protein.4.The expression of miR-136-5p was low in AML cell lines,while MCM5 was high.Overexpression of miR-136-5p can inhibit cell proliferation,promote apoptosis and arrest cell cycle in the G1 phase.It can also downregulate the expression of CRNDE m RNA and MCM5 m RNA and protein.Inhibition of miR-136-5p had the opposite effect.5.Bioinformatics software Starbase V2.0 and Dual-Luciferase assay showed the binding effect between CRNDE and miR-136-5p.RIP assay showed that compared with the IgG group,the enrichment products of AGO2 antibody group contained more CRNDE.6.Bioinformatics software Target Scan 7.0 and Dual-Luciferase assay showed the binding effect between MCM5 and miR-136-5p.7.Inhibition of miR-136-5p can reverse the downregulation of MCM5 induced by CRNDE knockdown at m RNA and protein levels.Conclusion1.CRNDE was highly expressed in the bone marrow cells of AML patients and AML cell lines and mainly located in the cytosol.CRNDE promotes the progression of AML.2.The expression of miR-136-5p was low in AML cell lines and miR-136-5p played a tumor suppressor role.3.CRNDE acts as a competing endogenous RNA of miR-136-5p,thereby regulating the expression of MCM5.