miR-128-3p靶向沉默瘦素抑制银屑病角质形成细胞增殖及炎症反应

miR-128-3p inhibits the proliferation of keratinocytes in psoriasis via repressing leptin

目的·探究miR-128-3p/瘦素(leptin,LEP)轴对银屑病中角质形成细胞过度增殖及炎症反应的调控作用。方法·选取BALB/c小鼠,分为对照组(n=10)及模型组(n=10)。模型组小鼠背部行咪喹莫特软膏连续涂抹以构建银屑病小鼠模型。构建miR-128-3p过表达和干扰质粒、LEP干扰质粒分别转染人永生化角质形成细胞HaCaT细胞。经实时定量聚合酶链反应检测miR-128-3p及LEP的mRNA,Western blotting检测LEP蛋白表达;酶联免疫吸附实验检测培养液中肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、IL-1β的含量;MTT实验检测细胞相对活力;EdU实验检测细胞增殖;采用双荧光素酶报告基因实验验证miR-128-3p与LEP的靶向关系。结果·与对照组小鼠比,模型组小鼠miR-128-3p表达下调,Lep的基因表达及蛋白水平升高(均P<0.05)。双荧光素酶报告基因实验证实LEP是miR-128-3p下游靶点。与模拟物阴性对照(negative control of mimic,NC mimic)组相比,miR-128-3p模拟物(miR-128-3p mimic)组miR-128-3p表达上调,LEP的基因表达及蛋白降低;miR-128-3p mimic组TNF-α、IL-6、IL-1β显著低于NC mimic;miR-128-3p上调后细胞相对活力、EdU阳性细胞率均降低(均P<0.05)。与抑制物阴性对照(negative control of inhibitor,NC inhibitor)组相比,miR-128-3p抑制物(miR-128-3p inhibitor)组miR-128-3p表达下调,LEP的基因表达及蛋白水平增加;miR-128-3p下调后细胞TNF-α、IL-6、IL-1β升高;miR-128-3p表达下调导致细胞相对活力和EdU阳性细胞率增加(均P<0.05)。进一步实验结果显示:与LEP抑制物(LEP inhibitor)组相比,miR-128-3p inhibitor+LEP inhibitor组LEP表达上调,而TNF-α、IL-6、IL-1β水平升高,细胞相对活力及EdU阳性细胞率增加(均P<0.05)。结论·miR-128-3p靶向沉默LEP抑制角质形成细胞增殖及炎症反应,抑制银屑病发生发展。

Objective·To explore the role of miR-128-3p/leptin(LEP)axis in the proliferation and inflammation of keratinocytes in psoriasis.Methods·BALB/c mice were randomly divided into a control group(n=10)and a model group(n=10).Mice in the model group were given imiquimod on the back.miR-128-3p overexpression and interference plasmids,as well as LEP interference plasmids,were constructed and transfected into HaCaT cells,respectively.miR-128-3p and LEP mRNA were quantified by real-time quantitative polymerase chain reaction,and LEP protein levels were detected by using Western blotting.Enzyme-linked immunosorbent assay was used to measure the content of tumor necrosis factorα(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)in the culture medium.MTT assay was used to evaluate cell activity and EdU assay was to used to test cell proliferation.The binding site between miR-128-3p and LEP was determined by using a dual luciferase reporter gene assay.Results·Compared with mice in the control group,mice in the model group showed downregulated expression of miR-128-3p and upregulated expression of LEP at both RNA and protein levels(all P<0.05).The dual luciferase reporter gene assay confirmed that LEP was a downstream target of miR-128-3p.Compared with the negative control mimic(NC mimic)group,expression of miR-128-3p was up-regulated in the miR-128-3p mimic group,and expression of LEP was reduced.The levles of TNF-α,IL-6,and IL-1βwere significantly lower in the miR-128-3p mimic group than in the NC mimic group.The relative cell viability and EdU-positive cell rate were also reduced after miR-128-3p up-regulation(all P<0.05).Compared with the negative control inhibitor(NC inhibitor)group,expression of miR-128-3p was down-regulated in the miR-128-3p inhibitor group,and expression of LEP was increased.The levles of TNF-α,IL-1βand IL-6 were increased after miR-128-3p downregulation.miR-128-3p down-regulation led to an increase in relative cell viability and EdU-positive cell rate(all P<0.05).Further experimental results showed that LEP expression was up-regulated in the miR-128-3p inhibitor+LEP inhibitor group compared with that in the LEP inhibitor group,whereas the levels of TNF-α,IL-6,and IL-1βwere elevated,and the relative viability of the cells and the rate of EdU-positive cells were increased(all P<0.05).Conclusion·miR-128-3p downregulates LEP to inhibit the proliferation and inflammatory response of keratinocytes,thereby inhibiting the occurrence and development of psoriasis.