长非编码RNA NEAT1通过miR-141-3p/EGFR参与瘢痕疙瘩形成的调控机制

Involvement of Long Non-coding RNA NEAT1 in the Regulatory Mechanism of Keloid Formation through miR-141-3p/EGFR

目的探讨长非编码RNA NEAT1在瘢痕疙瘩中表达情况及参与瘢痕疙瘩形成的调控机制。方法收集27例瘢痕疙瘩组织作为观察组,27例瘢痕疙瘩旁的正常皮肤组织作为对照组,采用实时荧光定量(RT-qPCR)检测其NEAT1、miR-141-3p的表达水平。取对数生长期的瘢痕疙瘩成纤维细胞(HKF)或293T细胞,随机分为空白对照组(Black组)、阴性对照组(NC组)、si-NEAT1组、miR-141-3p mimic组、si-NEAT1+OE-EGFR组、miR-141-3p mimic+OE-EGFR组。si-NEAT1组、miR-141-3p mimic组、si-NEAT1+OE-EGFR组、miR-141-3p mimic+OE-EGFR组细胞应用脂质体转染法分别进行si-NEAT1、miR-141-3p mimic、si-NEAT1+OE-EGFR、miR-141-3p mimic+OE-EGFR、阴性对照质粒转染,Black组细胞不作处理。RT-qPCR检测各组细胞中NEAT1和miR-141-3p的相对表达量。CCK-8法检测细胞增殖。使用生物信息学分析预测NEAT1与miR-141-3p及miR-141-3p与EGFR的结合位点,通过双荧光素酶报告基因实验确认NEAT1与miR-141-3p及miR-141-3p与EGFR的靶向作用。过表达EGFR的拯救实验,检测沉默NEAT1和上调miR-141-3p的细胞增殖抑制效应的变化。结果与对照组比较,观察组中NEAT1表达升高(P<0.05),而miR-141-3p的表达异常下调(P<0.05);沉默NEAT1表达可以显著抑制瘢痕疙瘩成纤维细胞的增殖,升高miR-141-3p的表达;NEAT1靶向负调控miR-141-3p;miR-141-3p靶向负调控EGFR;过表达EGFR可拯救沉默NEAT1和上调miR-141-3p表达对瘢痕疙瘩成纤维细胞增殖的抑制作用。结论NEAT1的表达在瘢痕疙瘩组织中异常升高,沉默NEAT1可通过miR-141-3p/EGFR轴降低瘢痕疙瘩成纤维细胞的增殖,抑制瘢痕疙瘩的形成和发展。

Objective To investigate the expression of long non-coding RNA NEAT1 in keloids and the regulatory mechanisms involved in keloid formation.Methods 27 cases of keloid tissues were collected as the experimental group and 27 cases of normal skin tissues next to the keloid as the control group,and the expression levels of NEAT1 and miR-141-3p in the experimental and control groups were detected by real-time fluorescence quantitative PCR(RT-qPCR).Keloidal fibroblasts(HKF)or 293T cells at logarithmic growth stage were randomly divided into blank control group(Black group),negative control group(NC group),si-NEAT1 group,miR-141-3p mimic group,si-NEAT1+OE-EGFR group and miR-141-3p mimic+OE-EGFR group.Cells in the si-NEAT1 group,miR-141-3p mimic group,si-NEAT1+OE-EGFR group and miR-141-3p mimic+OE-EGFR group were transfected by applying liposome transfection for si-NEAT1,miR-141-3p mimic,si-NEAT1+OE-EGFR,miR-141-3p mimic+OE-EGFR,respectively,with negative control plasmid transfection,and cells in the Black group were left untreated.The relative expression of NEAT1 and miR-141-3p in each group of cells was detected by RT-qPCR.CCK-8 assay was used to detect cell proliferation.The binding sites of NEAT1 to miR-141-3p and miR-141-3p to EGFR were predicted using bioinformatics analysis,and the targeting of NEAT1 to miR-141-3p and miR-141-3p to EGFR was confirmed by dual luciferase reporter gene assay.Rescue assays of overexpression of EGFR were performed to detect changes in the proliferation inhibitory effects of cell proliferation by silencing NEAT1 and upregulating miR-141-3p.Results Compared with normal tissues,the expression of NEAT1 was increased in keloid tissues(P<0.05),while the expression of Mir-141-3p was abnormally down-regulated(P<0.05).Silencing NEAT1 expression significantly inhibited the proliferation of keloid fibroblasts and elevated miR-141-3p expression;NEAT1 targeted negative regulation of miR-141-3p;miR-141-3p targeted negative regulation of EGFR.Overexpression of EGFR rescued the inhibitory effect of silencing NEAT1 and up-regulated miR-141-3p expression on HKF proliferation in keloids.Conclusion NEAT1 expression is abnormally elevated in keloid tissues and silencing NEAT1 reduces the proliferation of keloid fibroblasts through the miR-141-3p/EGFR axis,inhibiting keloid formation and progression.