miR-128-3p靶向沉默HO-1促进角质形成细胞炎症介质分泌

miR-128-3p Promotes the Secretion of Inflammatory Mediators from Keratinocytes via Silencing HO-1

目的探讨miR-128-3p对角质形成细胞HaCaT促炎介质分泌的影响及机制。方法收集银屑病患者的皮损组织和正常皮肤组织,荧光实时定量PCR(qRT-PCR)检测miR-128-3p和血红素氧合酶-1(HO-1)mRNA表达,并对两者的相关性进行分析。生物信息学预测和荧光素酶报告基因分析miR-128-3p与HO-1之间的靶向作用关系。培养HaCaT细胞,在脂多糖处理后,转染miR-128-3p mimic/inhibitor,qRT-PCR检测白介素(IL)-1β和IL-6 mRNA表达,qRT-PCR和Western blot检测HO-1表达,酶联免疫吸附法(ELISA)检测细胞培养上清液中IL-1β和IL-6水平。以HO-1抑制剂锌原卟啉Ⅸ(ZnPPⅨ)处理HaCaT细胞后再用miR-128-3p inhibitor转染,qRT-PCR检测IL-1β和IL-6 mRNA表达,ELISA检测上清液中IL-1β和IL-6水平。结果银屑病患者皮损组织中miR-128-3p表达上调,HO-1表达下调,两者呈负相关。HO-1为miR-128-3p的靶基因,miR-128-3p在转录后水平降低HO-1表达,促进IL-1β和IL-6表达与分泌,并且HO-1抑制剂ZnPPⅨ预处理消除了miR-128-3p inhibitor对IL-1β和IL-6表达与分泌的影响。结论miR-128-3p促进HaCaT细胞分泌IL-1β和IL-6,其机制与靶向沉默HO-1有关。

Objective To explore the effects and underlying mechanisms of miR-128-3 p on the secretion of pro-inflammatory mediators from keratinocytes.Methods Both normal skin tissue and lesion tissue were collected from psoriasis patients.The expression of miR-128-3 p and heme oxygenase-1(HO-1)was detected by quantitative real-time PCR(qRT-PCR)and their correlation was analyzed.The targeted relationship between miR-128-3 p and HO-1 was analyzed by bioinformatic prediction and luciferase reporter assay.Lipopolysaccharide(LPS)-treated HaCaT cells were transfected with miR-128-3 p mimic or its inhibitor.The mRNA expression of interleukin(IL)-1βand IL-6 was measured using qRT-PCR.Both qRT-PCR and Western blot were employed to determine HO-1 expression.The levels of IL-1βand IL-6 in the cell culture supernatant were examined by enzyme-linked immunosorbent assay(ELISA).HaCaT cells were treated with HO-1 inhibitor protoporphyrin zincⅨ(ZnPPⅨ)and then transfected with miR-128-3 p inhibitor,followed by assessing IL-1βand IL-6 expression and secretion by qRT-PCR and ELISA,respectively.Results Increased miR-128-3 p expression and decreased HO-1 mRNA levels were observed in lesion tissue and showed a negative correlation.Using bioinformatic prediction and luciferase reporter assay,HO-1 was identified as a putative target gene of miR-128-3 p.Importantly,miR-128-3 p decreased HO-1 expression at the post-transcriptional level and promoted the expression and secretion of IL-1βand IL-6 from LPS-treated HaCaT cells.Further,pretreatment with HO-1 inhibitor,ZnPPⅨabrogated the effects of miR-128-3 p inhibitor on IL-1βand IL-6 expression and secretion in LPS-treated HaCaT cells.Conclusion miR-128-3 p promotes the secretion of IL-1βand IL-6 from HaCaT cells by targeting HO-1.