Abstract：
Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence in situ hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.
摘要：
瘢痕疙瘩是由皮肤创伤或自发形成的病理性瘢痕组织，常伴有瘙痒和疼痛。尽管GNAS反义RNA1(GNAS-AS1)在瘢痕疙瘩中显示异常上调,潜在的分子机制尚不清楚。使用定量逆转录PCR测量瘢痕疙瘩患者和人瘢痕疙瘩成纤维细胞（HKFs）的临床组织中的基因和蛋白质水平，蛋白质印迹和酶联免疫吸附测定。HKFs的特点，包括扩散和迁移，使用细胞计数试剂盒8和伤口愈合测定进行评估。使用荧光原位杂交测量HKF中GNAS-AS1和miR-196a-5p的共定位。采用Pearson相关分析方法分析瘢痕疙瘩患者样本中GNAS-AS1、miR-196a-5p和C-X-C基序趋化因子配体12(CXCL12)之间的关系。通过染色质免疫沉淀和荧光素酶报告基因测定验证基因相互作用。在瘢痕疙瘩患者的临床组织中，GNAS-AS1和CXCL12表达上调，miR-196a-5p表达下调。GNAS-AS1敲低抑制增殖,迁移,和HKF的细胞外基质（ECM）积累，所有这些都被miR-196a-5p下调逆转。信号转导和转录激活因子3(STAT3)通过GNAS-AS1启动子相互作用诱导GNAS-AS1转录,还有氯硝柳胺,STAT3抑制剂，GNAS-AS1表达降低。GNAS-AS1通过构建miR-196-5p正调控CXCL12。此外，CXCL12敲低克制HKFs中STAT3的磷酸化。我们的发现揭示了促进HKF增殖的STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3的反馈回路，迁移和ECM积累并影响瘢痕疙瘩的进展。
