Abstract:

Li Jingrong*, Wang Jianqin, Fang Ruihua, Zeng Renshan, Mo Jinxue, Guo Yunlong, Jia Shuqing. *Department of Dermatology, Nansha Central Hospital of Guangzhou, Guangzhou 511457, China Corresponding author: Wang Jianqin, Email: jianqinwang @tom.com 【Abstract】 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.