Effects of miR-34a on regulating the autophagy of HEK293 cells

Objective To investigate the effects of miR-34a on regulating the autophagy of HEK293 cells.MethodsHEK293 cells were transfected with miR-34a mimics (mimics group) or miR-34a inhibitor (inhibitor group) by lipofectamine 2000.HEK293 cells were treated for 2h with the lysosomal inhibitors pepstatin A and E64d (both 10mg/ml) or DMSO as control after transfection for 48h.The expression of miR-34a and p53 were detected by real-time PCR.Western blotting was used to monitor the changes in autophagy-associated protein LC3Ⅱ and p53.The biological targets of miR-34a on autophagy-related gene (including beclin1,sestrin 2 and FoxO3) were analyzed.ResultsCompared with control group,the expression of miR-34a in mimics group was significantly up-regulated (P0.05),and in inhibitor group was significantly down-regulated (P0.05).Compared with control group,LC3Ⅱ protein levels were significantly decreased in mimics group and significantly increased in inhibitor group (P0.05).The mRNA and protein expression of p53 showed no significant difference among the 3 groups.Bioinformatics analysis revealed that beclin1,sestrin 2 and FoxO3 might be the potential target genes of miR-34a,and there might exist 3,2 and 4 miR-34a target islands in sestrin 2,beclin1 and FoxO3 mRNA,respectively.ConclusionmiR-34a may inhabit the autophagy of HEK293 cells by directly inhibiting the expression of autophagy-associated genes of HEK293 cells.