Table2_Construction of a HOXA11-AS-Interact Ed Network in Keloid Fibroblasts Using Integrated Bioinformatic Analysis and in Vitro Validation.XLSX

Background: Expression of the long noncoding RNA (lncRNA) HOXA11-AS significantly increased in keloids by unclarified molecular regulation mechanisms.
Methods: Using successfully primary cultured keloid-derived fibroblasts from central region of chronic keloid tissues (sample 0), small interfering RNAs were designed and transfected into two keloid fibroblast samples (samples 1 and 2) to knockdown HOXA11-AS. One nonspecific transfection control (sample 3) and one blank control (sample 4) were used to remove nonspecific overlap from the studied group. The lncRNAs, messenger RNAs (mRNAs), and microRNAs (miRNAs) of five samples were sequenced to identify differentially expressed (DE) profiles in HOXA11-AS-knockdown keloid fibroblasts in samples 1 and 2 (by intersection), which facilitated removal of overlap with the nonspecific controls (samples 3 and 4, by union). Using stepwise bioinformatic analysis, a HOXA11-AS-interacted competing endogenous network (ceRNA) was screened based on three DE profiles.
Results: Keloid fibroblasts with or without HOXA11-AS as well as with or without nonspecific interferences were successfully constructed respectively. A total of 1,396 mRNAs and 39 lncRNAs were significantly changed in keloid fibroblast with HOXA11-AS knockdown. Simultaneously, 1,626 mRNAs and 99 lncRNAs were significantly changed in keloid fibroblast with nonspecific interference. With removal of nonspecific overlap, a lncRNA–mRNA interactive network characterized by close natural/intronic antisense relationship was initially constructed in keloid fibroblast with HOXA11-AS knockdown. Based on this network, a lncRNA–mRNA–protein interaction network was extended by integration of the human protein–protein interaction network. Significant functional genes were screened using PageRank algorithm in the extended network. Three genes, including SNED1, NIPAL3, and VTN, were validated by real-time PCR in HOXA11-AS-knockdown keloid fibroblasts. Only NIPAL3 was predicted to be a target gene for HOXA11-AS via three competing endogenous miRNAs (hsa-miRNA-19a-3p, hsa-miR-141-3p, and hsa-miR-140-5p).
Conclusion: An interactive network of HOXA11-AS–three miRNAs–NIPAL3 was predicted in keloid fibroblasts by integrative bioinformatic analysis and in vitro validation.

背景：长链非编码RNA（long noncoding RNA, lncRNA）HOXA11-AS在瘢痕疙瘩中表达显著上调，但其分子调控机制尚未阐明。
方法：从慢性瘢痕疙瘩组织中央区域成功原代培养瘢痕疙瘩来源成纤维细胞（样本0），设计小干扰RNA（small interfering RNAs, siRNA）并转染至两株瘢痕疙瘩成纤维细胞样本（样本1、2）以敲低HOXA11-AS的表达；同时设置非特异性转染对照（样本3）与空白对照（样本4），以排除研究组中的非特异性干扰。对5个样本的长链非编码RNA、信使RNA（messenger RNA, mRNA）及微小RNA（microRNA, miRNA）进行测序，通过取样本1和2的交集，鉴定HOXA11-AS敲低的瘢痕疙瘩成纤维细胞中的差异表达（differentially expressed, DE）谱；再通过取样本3和4的并集，剔除与非特异性对照存在重叠的差异表达谱。通过逐步生物信息学分析，基于三类差异表达谱筛选得到HOXA11-AS相互作用的竞争性内源RNA（competing endogenous RNA, ceRNA）调控网络。
结果：成功分别构建了敲低或不敲低HOXA11-AS，以及存在或不存在非特异性干扰的瘢痕疙瘩成纤维细胞模型。在HOXA11-AS敲低的瘢痕疙瘩成纤维细胞中，共鉴定到1396个信使RNA与39个长链非编码RNA发生显著表达变化；同时在非特异性干扰组的瘢痕疙瘩成纤维细胞中，共鉴定到1626个信使RNA与99个长链非编码RNA发生显著表达变化。剔除非特异性重叠的差异表达谱后，初步构建了以紧密天然/内含子反义关系为特征的长链非编码RNA-信使RNA互作网络。基于该网络，整合人类蛋白质-蛋白质互作网络，进一步扩展得到长链非编码RNA-信使RNA-蛋白质互作网络。利用PageRank算法在扩展网络中筛选得到核心功能基因，并通过实时荧光定量PCR（real-time PCR）验证了HOXA11-AS敲低的瘢痕疙瘩成纤维细胞中的SNED1、NIPAL3与VTN三个基因。其中仅NIPAL3被预测为HOXA11-AS的靶基因，可通过三种竞争性内源微小RNA（hsa-miRNA-19a-3p、hsa-miR-141-3p及hsa-miR-140-5p）发挥调控作用。
结论：本研究通过整合生物信息学分析与体外验证，在瘢痕疙瘩成纤维细胞中预测得到HOXA11-AS-三种微小RNA-NIPAL3互作调控网络。