Department of Plastic and Aesthetic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430000, P.R. China.
Mol Med Rep. 2021 Jan;23(1). doi: 10.3892/mmr.2020.11693. Epub 2020 Nov 17.
Keloids are a skin fibroproliferative condition characterized by the hyperproliferation of fibroblasts and the excessive deposition of extracellular matrix (ECM) components. Previous studies have determined that Caveolin‑1 controlled hyperresponsiveness to mechanical stimuli through Runt‑related transcription factor 2 (Runx2) activation in keloids. However, the molecular mechanism of Runx2 regulating the pathological progression of keloids has not been elucidated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that most of the differentially expressed genes (DEGs), including Runx2, were significantly enriched in the biological processes 'Positive regulation of cell proliferation', in the cellular components 'Extracellular matrix', in the molecular functions 'Extracellular matrix structural constituents' and in the KEGG 'PI3K‑Akt signaling pathway'. The aim of the present study was to investigate the expression levels of the Runx2 in human keloid tissues and primary human keloid fibroblasts (HKFs), and to determine the underlying molecular mechanisms involved in the fibrotic roles of Runx2 in keloid formation. Runx2 expression levels were analyzed in patient keloid tissues and HKFs using western blotting, reverse transcription‑quantitative PCR (RT‑qPCR) and immunofluorescence microscopy. Primary HKFs were transfected with a small interfering RNA (si) specifically targeting Runx2 (si‑Runx2). Subsequently, Cell Counting Kit‑8, wound healing and Transwell assays, flow cytometry, RT‑qPCR and western blotting were applied to evaluate the proliferation, migration, apoptosis, ECM deposition and PI3K/AKT signaling pathway of HKFs, respectively. In addition, western blotting was also used to determine the expression levels of phosphorylated AKT and PI3K in HKFs. The results revealed that Runx2 expression levels were upregulated in keloid tissues and primary HKFs compared with the normal skin tissues and human normal fibroblasts. Following the transfection with si‑Runx2, the proliferative and migratory abilities of HKFs were significantly reduced and the apoptotic rate was increased. The expression levels of type I, type III collagen, fibronectin, and α‑smooth muscle actin were downregulated in si‑Runx2‑transfected cells, which was hypothesized to occur through following the downregulation of the phosphorylation levels of PI3K and AKT. In conclusion, the findings of the present study indicated that Runx2 silencing in HKFs might significantly inhibit the cell proliferation, migration and the expression levels of ECM‑related proteins, and promote apoptosis via suppressing the PI3K/AKT signaling pathway. Thus, Runx2 siRNA treatment may reverse the pathological phenotype of keloids through the inhibition of PI3K/AKT signaling in patients.
瘢痕疙瘩是一种皮肤纤维增生性疾病，其特征是成纤维细胞过度增殖和细胞外基质（ECM）成分的过度沉积。先前的研究已经确定，Caveolin-1 通过激活 Runt 相关转录因子 2（Runx2）控制对机械刺激的超敏反应。然而，Runx2 调节瘢痕疙瘩病理进展的分子机制尚未阐明。基因本体论和京都基因与基因组百科全书（KEGG）分析表明，大多数差异表达基因（DEGs），包括 Runx2，在生物过程“细胞增殖的正调控”、细胞成分“细胞外基质”、分子功能“细胞外基质结构成分”和 KEGG“PI3K-Akt 信号通路”中显著富集。本研究的目的是检测 Runx2 在人瘢痕疙瘩组织和原代人瘢痕疙瘩成纤维细胞（HKFs）中的表达水平，并确定其在瘢痕疙瘩形成中纤维化作用的潜在分子机制。通过 Western blot、逆转录-定量 PCR（RT-qPCR）和免疫荧光显微镜检测患者瘢痕疙瘩组织和 HKFs 中的 Runx2 表达水平。用特异性靶向 Runx2 的小干扰 RNA（siRNA）转染原代 HKFs（si-Runx2）。随后，通过细胞计数试剂盒-8 法、划痕愈合实验和 Transwell 实验、流式细胞术、RT-qPCR 和 Western blot 分别评估 HKFs 的增殖、迁移、凋亡、ECM 沉积和 PI3K/AKT 信号通路。此外，Western blot 还用于检测 HKFs 中磷酸化 AKT 和 PI3K 的表达水平。结果显示，与正常皮肤组织和人正常成纤维细胞相比，Runx2 在瘢痕疙瘩组织和原代 HKFs 中的表达水平上调。转染 si-Runx2 后，HKFs 的增殖和迁移能力明显降低，凋亡率增加。si-Runx2 转染细胞中 I 型、III 型胶原、纤连蛋白和α-平滑肌肌动蛋白的表达水平下调，推测这是通过下调 PI3K 和 AKT 的磷酸化水平实现的。综上所述，本研究结果表明，HKFs 中 Runx2 的沉默可能通过抑制 PI3K/AKT 信号通路显著抑制细胞增殖、迁移和 ECM 相关蛋白的表达水平，并促进细胞凋亡。因此，通过抑制患者的 PI3K/AKT 信号通路，Runx2 siRNA 治疗可能通过抑制 PI3K/AKT 信号通路逆转瘢痕疙瘩的病理表型。
