MicroRNA-196a Overexpression Inhibits Apoptosis In Hemin-induced K562 Cells

ObjectiveK562 cells were induced to differentiate into erythrocytes by hemin,and the cell model was constructed.With the help of this cell model,the effect of mir-196 a on apoptosis of red blood cells and its mechanism were investigated.MethodsAfter K562 cells were induced by hemin,QPCR was used to analyze the content of ?-globin and CD235 a quantitatively,benzidine staining was used to analyze the expression of hemoglobin in these cells qualitatively.Then,vectors of mi R-196 a mimics and micro RNA-small hairpin negative control?mi RNA-Sh NC?were constructed,packaged with viruses and transfected into hemin-induced K562 cells,and corresponding stable transduction cell lines were established and verified.After that,the viability of the transfected hemin-induced K562 cells was tested by CCK-8 assay,the alteration of cell cycle and apoptosis rate were detected by flow cytometry,and the expression of some proteins associated with cell cycle and apoptosis was tested by Western blotting assays.To study the mechanism,the bioinformatics analysis predicted the target gene of mi R-196 a,and dual-luciferase report system verified it.After overexpression of the target gene,cell proliferation and apoptosis were analyzed again.All data were statistically analyzed using SPSS 16.0 software.ResultsIt was found that K562 cells could successfully differentiate into erythrocytes after 24 h induction with 25 ?M hemin,the content of ?-globin and CD235 a,and the positive cell rate of benzidine staining were more than the control group.After overexpressing mi R-196 a,the proliferation of hemin-induced K562 cells was promoted while the apoptosis inhibited,and the expression results of related proteins were also consistent.In addition,bioinformatics predicted that p27kip1 was one of the target genes of mi R-196 a,and it was verified that mi R-196 a directly bound to the 3'UTR of p27kip1,and the two were negatively correlated..After restoring the expression of p27kip1,the growth rate of hemin-induced K562 cells was not as high as before and the inhibition of apoptosis was alleviated.ConclusionThe present study validates that mi R-196 a overexpression inhibits apoptosis in hemin-induced K562 cells through downregulating p27^kip1.