Data Sheet 2_Identification and histological validation of autophagy-related core genes ADRB2 and PLK2 in keloids, with integrated immune infiltration analysis.pdf

IntroductionKeloids are pathological fibroproliferative scars characterized by excessive collagen deposition and a lack of effective targeted therapies. Autophagy dysregulation has been linked to keloid pathogenesis, but the underlying molecular mechanisms remain unclear.
MethodsTranscriptomic datasets were integrated and analyzed using differential expression analysis and weighted gene co-expression network analysis. Three machine learning algorithms—least absolute shrinkage and selection operator (LASSO), support vector machine–recursive feature elimination (SVM-RFE), and random forest—were applied to identify autophagy-related hub gene candidates in keloids. Immune infiltration and functional analyses were conducted to explore immune microenvironment alterations. Histological staining (H&E and Masson), immunohistochemistry, and Western blotting were used for tissue-level validation, while cellular experiments were performed in keloid fibroblasts with autophagy modulation.
ResultsADRB2 and PLK2 were consistently identified as key autophagy-related candidate genes. Immune-related analyses suggested that these genes may be involved in remodeling the keloid immune microenvironment by influencing the abundance and functional status of multiple immune cell subsets. Histological and protein-level assays demonstrated significantly higher expression of ADRB2 and PLK2 in keloid tissues compared with adjacent normal skin. In keloid fibroblasts, fibrotic markers (COL1/COL3) and autophagy-related markers (LC3-II/LC3-I and p62) were upregulated concomitantly with ADRB2 and PLK2 at baseline. Autophagy modulation altered ADRB2 expression (decreased with EBSS and increased with chloroquine), whereas PLK2 expression remained largely unchanged.
DiscussionThese findings identify ADRB2 and PLK2 as under-recognized autophagy- andimmunity-related candidate biomarkers in keloids, highlighting their potential relevance asdiagnostic indicators and future therapeutic research targets.

引言：瘢痕疙瘩（Keloids）是一类病理性纤维增生性瘢痕，以胶原过度沉积、缺乏有效靶向治疗方案为典型特征。自噬失调与瘢痕疙瘩的发病机制密切相关，但其潜在分子机制仍未阐明。
方法：本研究通过差异表达分析与加权基因共表达网络分析，对转录组数据集进行整合与分析。采用最小绝对收缩和选择算子（LASSO）、支持向量机-递归特征消除（SVM-RFE）以及随机森林三种机器学习算法，筛选瘢痕疙瘩中与自噬相关的核心候选基因。开展免疫浸润与功能分析，以探究瘢痕疙瘩免疫微环境的改变情况。采用组织学染色（苏木精-伊红（H&E）染色、马松（Masson）染色）、免疫组化及蛋白质印迹（Western blotting）进行组织层面验证；同时通过调控自噬水平，在瘢痕疙瘩成纤维细胞中开展细胞实验。
结果：肾上腺素能受体β2（ADRB2）与Polo样激酶2（PLK2）被一致鉴定为关键的自噬相关候选基因。免疫相关分析显示，这两个基因可通过调控多种免疫细胞亚群的丰度与功能状态，参与瘢痕疙瘩免疫微环境的重塑过程。组织学与蛋白质水平实验结果表明，相较于瘢痕旁正常皮肤，瘢痕疙瘩组织中ADRB2与PLK2的表达水平显著升高。在瘢痕疙瘩成纤维细胞中，纤维化标志物（I型胶原COL1/III型胶原COL3）与自噬相关标志物（LC3-II/LC3-I及p62）的基础表达水平与ADRB2、PLK2呈协同上调趋势。调控自噬水平可改变ADRB2的表达（Earle's平衡盐溶液（EBSS）处理后表达下调，氯喹处理后表达上调），而PLK2的表达水平基本无变化。
讨论：本研究结果证实，ADRB2与PLK2是瘢痕疙瘩中尚未被充分关注的自噬与免疫相关候选生物标志物，凸显了二者作为诊断指标及未来治疗研究靶点的潜在应用价值。