Abstract:

Jiang Meng, Ma Weiyuan, Sun Qing. Department of Dermatology, Qilu Hospital of Shandong University, Jinan 250012, China Corresponding author: Sun Qing, Email: sunqing7226@163.com 【Abstract】 Objective To evaluate the effect of metformin on the proliferation of keratinocytes, and to investigate its possible mechanism. Methods HaCaT human keratinocytes were divided into several groups to remain untreated （control group） or be treated with different concentrations （25, 50, 75, 100 mmol/L） of metformin for 24 hours （intervention groups）. Subsequently, CCK8 assay was conducted to evaluate the proliferation of HaCaT cells, real-time quantitative PCR to measure the mRNA expressions of miR-21-5p and its downstream target gene PDCD4, and Western blot to detect the expression of PDCD4 protein in HaCaT cells. Statistical analysis was done by using one-way analysis of variance for multiple group comparisons and SNK-q test for paired comparisons. Results After 24-hour treatment, the proliferation of HaCaT cells was inhibited by （5.43 ± 3.67）%, （19.61 ± 6.95）%, （45.93 ± 9.56）% and （61.91 ± 6.93）% by metformin of 25, 50, 75 and 100 mmol/L, respectively, with significant differences observed in cell proliferation inhibition rates among these intervention groups （F = 246.90, P < 0.05）. Cellular proliferative activity was similar between the control cells （0.00 ± 3.00%） and those treated with 25 mmol/L metformin, but significantly higher in the control cells than in the other 3 metformin-treated groups （all P < 0.05）, and significantly different between the 4 metformin-treated groups （all P < 0.05）. The relative mRNA expression level （2－△△Ct） of miR-21-5p was 0.90 ± 0.11, 0.33 ± 0.05, 0.21 ± 0.07 and 0.14 ± 0.04 （F = 36.99，P < 0.01）, while that of PDCD4 was 2.11 ± 0.64, 7.22 ± 1.13, 11.16 ± 1.23 and 19.12 ± 3.16 （F = 96.26, P < 0.05）, and the expression level of PDCD4 protein was 1.22 ± 0.08, 2.09 ± 0.20, 2.26 ± 0.11 and 2.37 ± 0.07 （F = 75.37, P < 0.05）, respectively, in HaCaT cells treated with metformin of 25, 50, 75 and 100 mmol/L. Similarly, no significant difference was observed between the control cells and those treated with 25 mmol/L metformin in the expression level of miR-21-5p mRNA, PDCD4 mRNA or protein, but decreased expression of miR-21-5p mRNA and increased expression of PDCD4 mRNA and protein were noted in cells treated with the other 3 concentrations of metformin compared with the control cells （all P < 0.05）, and significant differences were also found in the expression levels of miR-21-5p mRNA as well as PDCD4 mRNA and protein among the 4 intervention groups （all P < 0.05）. Conclusion Metformin can markedly inhibit the proliferation of HaCaT cells in vitro, likely by downregulating miR-21-5p expression and upregulating PDCD4 expression.