0.05),but the ratio of testosterone/estradiol was significantly higher in patients than that of the controls(P

Objective To investigate the regulatory effects of miR-145 on the proliferation,cell cycle and apoptosis of a human keratinocyte cell line HaCaT.Methods miR-145 mimics and negative control (NC) mimics were chemically synthesized and then transiently transfected into HaCaT cells respectively.After additional culture for different durations,real-time PCR was performed to determine the expression level of miR-145,MTS assay to estimate cell proliferation,and flow cytometry to detect cell apoptosis and cycle.Luciferase assay,real-time PCR and Western blot were conducted to determine whether NRAS was the target gene of miR-145.Results The miR-145 expression level in miR-145 mimic-transfected cells increased by 85.00 ± 1.21 folds compared with NC mimic-transfected cells (t =115.90,P ＜ 0.0001).The transfection with miR-145 mimics significantly inhibited the proliferation of HaCaT cells (F =8.76,P =0.008),and the inhibitory effect significantly varied with the duration (24-96 hours) of culture after transfection,with no interaction effect between the transfection with miR-145 mimics and culture duation (F =1.21,P =0.18).Compared with NC mimic-transfected cells,those transfected with miR-145 mimics showed a significant increase in the proportion of early apoptotic cells (18.9％ ± 4.1％ vs.4.3％ ± 1.2％,t =7.126,P ＜ 0.01),late apoptotic cells (9.3％ ± 2.3％ vs.3.6％ ± 1.6％,t =12.38,P ＜ 0.01),G1-phase cells (85.83％ ± 5.2％ vs.62.08％ ± 6.23％,t =11.78,P =0.007),but a significant decrease in the percentage of G2-phase cells (6.26％ ± 1.2％ vs.19.36％ ± 3.45％,t =7.610,P =0.017) and S-phase cells (7.91％ ± 1.3％ vs.18.56％ ± 5.23％,t =7.230,P=0.019).As luciferase assay showed,luciferase activity was significantly lower in HaCaT cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-wild carrying the wild-type 3'UTR of NRAS than in those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-wild (t =11.09,P =0.008),but similar between cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-mut carrying the mutant-type 3'UTR of NRAS and those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-mut (P ＞ 0.05).Real-time PCR and Western blot revealed that the overexpression of miR-145 mimics had no significant effect on NRAS mRNA expression (P ＞ 0.05),but significantly inhibited NRAS protein expression (1.52 ± 0.07 vs.0.20 ± 0.02,t =28.43,P＜ 0.01).Conclusion miR-145 might inhibit proliferation and promote apoptosis of HaCaT cells by influencing cell cycle via NRAS.