LncRNA CEBPA-AS1对瘢痕疙瘩成纤维细胞增殖、迁移、侵袭的影响及机制

Effects of LncRNA CEBPA-AS1 on proliferation,migration and invasion of keloid fibroblasts

目的探讨LncRNA CEBPA-AS1对瘢痕疙瘩成纤维细胞增殖、迁移、侵袭的影响及机制。方法原代分离培养人正常皮肤成纤维细胞与瘢痕疙瘩成纤维细胞,采用RT-qPCR法检测CEBPA-AS1与miR-194-3p表达;siNC、si-CEBPA-AS1、si-CEBPA-AS1与anti-miR-NC、si-CEBPA-AS1与anti-miR-194-3p分别转染瘢痕疙瘩成纤维细胞(si-NC组、si-CEBPA-AS1组、si-CEBPA-AS1+anti-miR-NC组、si-CEBPA-AS1+anti-miR-194-3p组);采用CCK-8实验、划痕实验、Transwell小室实验分别检测细胞增殖、迁移及侵袭;双荧光素酶报告基因实验检测CEBPA-AS1与miR-194-3p的靶向关系;采用Western blotting法检测ProcollagenⅠ、α-SMA、Col1、N-cadherin、E-cadherin蛋白表达。结果与正常皮肤成纤维细胞比较,瘢痕疙瘩成纤维细胞中CEBPA-AS1表达升高(P<0.05),miR-194-3p表达降低(P<0.05)。与si-NC组比较,si-CEBPA-AS1组细胞存活率降低(P<0.05),迁移距离减小(P<0.05),侵袭细胞数减少(P<0.05),ProcollagenⅠ、α-SMA、Col1、N-cadherin蛋白水平降低(P均<0.05),E-cadherin蛋白水平升高(P<0.05)。CEBPA-AS1可靶向调控miR-194-3p。与si-CEBPA-AS1+anti-miR-NC组比较,si-CEBPA-AS1+anti-miR-194-3p组miR-194-3p表达降低(P<0.05),细胞存活率升高(P<0.05),迁移距离增加(P<0.05),侵袭细胞数增多(P<0.05),ProcollagenⅠ、α-SMA、Col1、N-cadherin蛋白水平升高(P均<0.05),E-cadherin蛋白水平降低(P<0.05)。结论干扰LncRNA CEBPA-AS1表达可通过靶向调控miR-194-3p而抑制瘢痕疙瘩成纤维细胞增殖、迁移、侵袭。

Objective To explore the effects and mechanism of LncRNA CEBPA-AS1 on the proliferation,migra⁃tion and invasion of keloid fibroblasts and its regulatory effect on miR-194-3p.Methods Human normal skin fibroblasts and keloid fibroblasts underwent primary isolation and culture.The RT-qPCR method was used to detect the expression of CEBPA-AS1 and miR-194-3p.The si-NC,si-CEBPA-AS1,si-CEBPA-AS1 and anti-miR-NC,si-CEBPA-AS1 and an⁃ti-miR-194-3p were transfected into keloid fibroblasts(which were taken as the si-NC group,si-CEBPA-AS1 group,siCEBPA-AS1+anti-miR-NC group,si-CEBPA-AS1+anti-miR-194-3p group).CCK-8 experiment,Scratch experiment,and Transwell chamber experiment were used to detect cell proliferation,migration and invasion.The dual luciferase re⁃porter gene experiment was used to detect the targeting relationship between CEBPA-AS1 and miR-194-3p.Western blot⁃ting was used to detect the protein expression levels of ProcollagenⅠ,α-SMA,Col1,N-cadherin,and E-cadherin.Re⁃sults Compared with the normal skin fibroblasts,the expression level of CEBPA-AS1 in the keloid fibroblasts increased(P<0.05),while the expression level of miR-194-3p decreased(P<0.05).Compared with the si-NC group,the cell sur⁃vival rate of the si-CEBPA-AS1 group was reduced(P<0.05),the migration distance was reduced(P<0.05),and the number of invasive cells was reduced(P<0.05),and the protein levels of ProcollagenⅠ,α-SMA,Col1 and N-cadherin decreased(all P<0.05),while the protein level of E-cadherin increased(P<0.05).CEBPA-AS1 could target and regu⁃late miR-194-3p.Compared with the si-CEBPA-AS1+anti-miR-NC group,the expression level of miR-194-3p in the siCEBPA-AS1+anti-miR-194-3p group decreased(P<0.05),and the cell survival rate increased(P<0.05),the migration distance increased(P<0.05),the number of invasive cells increased(P<0.05),and the protein levels of ProcollagenⅠ,α-SMA,Col1,and N-cadherin increased(all P<0.05),while the protein level of E-cadherin decreased(P<0.05).Conclusion Interference with LncRNA CEBPA-AS1 expression could inhibit the proliferation,migration,invasion and collagen synthesis of keloid fibroblasts by targeting miR-194-3p.