miR-17-3p对角质形成细胞增殖的影响

Effect of miR-17-3p on the proliferation of keratinocytes

目的研究miR-17-3p在银屑病组织来源的角质形成细胞中的表达及其对角质形成细胞增殖的影响和机制。方法分离培养银屑病和正常皮肤组织来源的角质形成细胞,实时定量聚合酶链反应(RT-qPCR)测定miR-17-3p表达。在正常皮肤组织来源的角质形成细胞中分别转染mimics control、miR-17-3p mimics、inhibitor control、miR-17-3p inhibitor,命名为第1转染组、第2转染组、第3转染组、第4转染组。在正常皮肤组织来源的角质形成细胞中共转染miR-17-3p mimics、pcDNA 3.1和miR-17-3p mimics、pcDNA 3.1-缺氧诱导因子1α(HIF-1α),分别命名为第5转染组和第6转染组;在正常皮肤组织来源的角质形成细胞中共转染miR-17-3p inhibitor、siRNA control和miR-17-3p inhibitor、HIF-1αsiRNA,分别命名为第7转染组和第8转染组。用噻唑蓝(MTT)检测细胞增殖活性,用蛋白质免疫印迹法(Western Blot)检测细胞角蛋白10(Keratin10)、细胞角蛋白14(Keratin14)和HIF-1α蛋白蛋白表达。结果miR-17-3p在银屑病来源的角质形成细胞、正常皮肤组织来源的角质形成细胞中表达量分别为0.35±0.05和1.00±0.09,差异有统计学意义(P<0.05)。第1,2,3,4,5,6,7,8转染组细胞存活率分别为(100.00±10.23)%,(74.29±6.81)%,(100.00±9.80)%,(136.51±12.07)%,(75.61±8.15)%,(96.32±8.12)%,(137.54±12.05)%和(110.32±10.20)%;miR-17-3p mRNA水平分别为1.00±0.11,3.26±0.28,1.00±0.10,0.25±0.03,3.22±0.23,2.15±0.15,0.26±0.02和0.84±0.08;Keratin10蛋白相对表达量分别为1.00±0.09,1.45±0.13,1.01±0.15,0.48±0.06,1.48±0.14,0.86±0.08,0.45±0.05和0.89±0.09;Keratin14蛋白相对表达量分别为1.00±0.12,3.25±0.32,1.02±0.11,0.66±0.05,3.01±0.32,1.26±0.14,0.70±0.10和2.25±0.23;HIF-1α蛋白相对表达量分别为1.00±0.08,0.45±0.04,1.00±0.11,2.03±0.23,0.48±0.06,0.96±0.10,1.99±0.17和1.02±0.13。上述指标,第2转染组与第1转染组比较,第4转染组与第3转染组比较,第6转染组与第5转染组比较,第8转染组与第7转染组比较,差异均有统计学意义(均P<0.05)。结论miR-17-3p在银屑病组织来源的角质形成细胞中表达下调,其可以促进角质形成细胞增殖并改变细胞分化状态。

Objective To study the expression of miR-17-3p in keratinocytes derived from psoriatic tissues and its effect on the proliferation of keratinocytes.Methods Keratinocytes derived from psoriasis and normal skin tissue were isolated and cultured,and quantitative real-time polymerase chain reaction(qRT-PCR)was used to determine miR-17-3p expression.Transfected mimics control,miR-17-3p mimics,inhibitor control,miR-17-3p inhibitor in keratinocytes derived from normal skin tissue,and name them as Transfection 1 group,Transfection2 group,Transfection 3 group,Transfection 4 group.Co-transfection of miR-17-3p mimics,pcDNA 3.1 and miR-17-3p mimics,pcDNA 3.1-hypoxia inducible factor-1α(HIF-1α)in keratinocytes derived from normal skin tissue,respectively,and name them as Transfection 5 group,Transfection 6 group;co-transfected miR-17-3p inhibitor,siRNA control and miR-17-3p inhibitor,HIF-1αsiRNA in keratinocytes derived from normal skin tissue,respectively,and name them as Transfection 7 group,Transfection 8 group.The cell proliferation activity was detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT),and the protein expression of Keratin10 and Keratin14 were detected by Western blot.Results The expression level of miR-17-3p in keratinocytes derived from psoriasis and normal skin tissue were 0.35±0.05 and 1.00±0.09,comparing the two,the difference was statistically significant(P<0.05).The cell survival rates of Transfection 1 group,Transfection 2 group,Transfection 3 group,Transfection 4 group,Transfection 5 group,Transfection 6 group,Transfection 7 group,and Transfection 8 group were(100.00±10.23)%,(74.29±6.81)%,(100.00±9.80)%,(136.51±12.07)%,(75.61±8.15)%,(96.32±8.12)%,(137.54±12.05)%,(110.32±10.20)%;The miR-17-3p mRNA levels were1.00±0.11,3.26±0.28,1.00±0.10,0.25±0.03,3.22±0.23,2.15±0.15,0.26±0.02,0.84±0.08;The relative expression levels of Keratin10 protein were 1.00±0.09,1.45±0.13,1.01±0.15,0.48±0.06,1.48±0.14,0.86±0.08,0.45±0.05,0.89±0.09;The relative expression levels of Keratin14 protein were1.00±0.12,3.25±0.32,1.02±0.11,0.66±0.05,3.01±0.32,1.26±0.14,0.70±0.10,2.25±0.23;The relative expression levels of HIF-1αprotein were 1.00±0.08,0.45±0.04,1.00±0.11,2.03±0.23,0.48±0.06,0.96±0.10,1.99±0.17,1.02±0.13.There were statistically significant difference in the above indices between the transfection 2 group and transfection 1 group,between the transfection 4 group and transfection 3 group,between the transfection 6 group and transfection 5 group,between the transfection 8 group and transfection 7 group(P<0.05).Conclusion The expression of miR-17-3p was down-regulated in keratinocytes derived from psoriasis tissue,it can promote the proliferation of keratinocytes and change the state of cell differentiation.