The Effects Of MiR-15b On The Differentiation And Proliferation Of Acute Promyelocytic Leukemia Cells And Its Mechanism

Objective: To investigate the effects of miR-15 b on differentiation and proliferation of acute promyelocytic leukemia lines NB4 and HL-60 cells.Methods: NB4 and HL-60 cells were treated with ATRA(1μM)for 0h,24 h,48h,72 h respectively,the relative expression of miR-15 b was detected by qRT-PCR.After transfection with miR-15 b mimics/scramble or anti-15b/anti-con into NB4 and HL-60 cells,cellular morphology were observed by microscopy after Wright-Giemsa staining.The percentage of CD11b-positive cells was examined by flow cytometry.The mRNA expression of CD11 b and CSF3 R were detected by qRT-PCR.Cell proliferation and cell cycle were evaluated by CCK-8 assay and flow cytometry respectively.Bioinformatics predicted the binding site between cyclinE1 and miR-15 b.Western blot detected the protein expression of cyclinE1,Rb and p-Rb.The binding of miR-15 b and the 3’UTR of cyclinE1 was detected by double luciferase reports assay.CyclinE1 was knocked down by siRNA technology to detect relevant indicators of cell proliferation and differentiation.Rb and p-Rb protein expressions were detected by western blot.Results: we observed that ATRA induced the expression of miR-15 b in a time-dependent manner.The combined treatment of ATRA and miR-15 b mimics significantly increased cell differentiation in comparison with ATRA,alone evaluated by flow cytometry Wright-Giemsa staining and qRT-PCR.After transfected with anti-15 b,the contrary results were observed.Results of CCK-8 assay showed that miR-15 b markedly inhibited cell proliferation.Flow cytometry assay showed miR-15 b could arrest cell cycle at G0/G1 phase.Bioinformatics predicted that cyclinE1 was a target gene of miR-15 b.Overexpression of miR-15 b inhibited the expression of cyclinE1 and p-Rb.And knockdown of miR-15 b promoted the expression of cyclinE1 and p-Rb.Double luciferase reporter assay showed that miR-15 b could directly bind to the 3’UTR of cyclinE1.Inhibiting the expression of cyclinE1 promoted the differentiation of NB4 and HL-60 cells and inhibited cell proliferation.Western blot indicated the expression of p-Rb was decreased after knocking down the expression of cyclinE1.Conclusion: miR-15 b can promote NB4 and HL-60 cells differentiation and inhibits cells proliferation by regulating the protein expression of cyclinE1.