二甲双胍对HaCaT细胞增殖及miR-21-5p和PDCD4表达的影响  

Effect of metformin on the proliferation of and expressions of miR-21-5p and PDCD4 in HaCaT human keratinocytes

作　　者：江萌[1] 马伟元[1] 孙青[1] 

机构地区：[1]山东大学齐鲁医院皮肤科,济南250012

出　　处：《中华皮肤科杂志》2015年第3期200-203,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81071291、81402607）;山东省自然科学基金（Y2007C003）

摘　　要：目的 探讨二甲双胍对角质形成细胞增殖的影响及其可能的作用机制.方法 以HaCaT细胞为研究对象,分别给予不同浓度二甲双胍（25、50、75、100 mmol/L）刺激24 h,CCK8法检测HaCaT细胞增殖活性;实时定量PCR法检测HaCaT细胞中miR-21-5p及其下游靶基因PDCD4 mRNA表达水平;蛋白免疫印迹法检测HaCaT细胞中PDCD4蛋白表达水平.多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验.结果 二甲双胍可抑制HaCaT细胞增殖,25、50、75、100 mmol/L二甲双胍作用于HaCaT细胞24 h后,细胞抑制率分别为5.43％±3.67％、19.61％±6.95％、45.93％±9.56％、61.91％±6.93％,各组细胞抑制率差异有统计学意义（F=246.90,P＜0.05）;25 mmol/L组与对照组（0.00％±3.00％）相比,差异无统计学意义,其余各组抑制率均较对照组显著增高（P＜0.05）;实验组不同浓度组间比较,差异也均有统计学意义（P＜0.05）.25、50、75、100 mmol/L二甲双胍实验组miR-21-5p mRNA相对表达量（2-△△Ct）分别为0.90±0.11、0.33±0.05、0.21±0.07、0.14±0.04,PDCD4mRNA相对表达量（2-△△Ct）分别为2.11±0.64、7.22±1.13、11.16±1.23、19.12±3.16,各组细胞miR-21-5p mRNA和PDCD4 mRNA的表达差异均有统计学意义（F值分别为36.99和96.26,均P＜0.05）;25 mmol/L组与对照组相比,差异无统计学意义,其余各组miR-21-5p及PDCD4 mRNA的表达均较对照组显著下调或上调（均P＜0.05）;实验组不同浓度组间比较,差异也均有统计学意义（P＜0.05）.与对照组相比,25、50、75、100 mmol/L二甲双胍实验组PDCD4蛋白表达明显升高,其相对表达量分别为1.22±0.08、2.09±0.20、2.26±0.11、2.37±0.07,组间表达差异有统计学意义（F=75.37,P＜ 0.05）.25 mmol/L组与对照组相比差异无统计学意义,其余各组均较对照组明显升高（P＜0.01）.结论 二甲双胍在体外能够显著抑制HaCaT细胞的增殖,可能通过下调miR-21-5p的表达,使其下游靶基因PDObjective 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 mefformin 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-△△Q） 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 ± 0.1 1 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

关 键 词：角蛋白细胞 二甲双胍 银屑病 细胞增殖 miR-21-5p PDCD4 HaCaT细胞 

分 类 号：R96[医药卫生—药理学]