Department of Ophthalmology, Yonsei University Wonju College of Medicine, Wonju, South Korea; Gavin Herbert Eye Institute, University of California, Irvine, CA, United States.
Gavin Herbert Eye Institute, University of California, Irvine, CA, United States.
Ocul Surf. 2019 Oct;17(4):809-816. doi: 10.1016/j.jtos.2019.02.003. Epub 2019 Feb 8.
PPARγ plays a critical role in the maturation of immortalized human meibomian gland epithelial cells (hMGEC). To further understand the molecular changes associated with meibocyte differentiation, we analyzed transcriptome profiles from hMGEC after PPARγ activation.
Three sets of cultivated hMGEC with or without exposure to PPARγ agonist, rosiglitazone were used for RNA-seq analysis. RNA was isolated and processed to generate 6 libraries. The libraries were then sequenced and mapped to the human reference genome, and the expression results were gathered as reads per length of transcript in kilobases per million mapped reads (RPKM) values. Differential gene expression analyses were performed using DESeq2 and NOISeq. Gene ontology enrichment analysis (GOEA) was performed on gene sets that were upregulated or downregulated after rosiglitazone treatment. Five genes were selected for validation and differential expression was confirmed using quantitative PCR. The Differential expression of CK5 was evaluated using Western blotting.
Expression data indicated that about 58,000 genes are expressed in hMGEC. DESeq2 and NOISeq indicated that 296 and 3436 genes were upregulated and 258 and 3592 genes were down regulated after rosiglitazone treatment, respectively. Of genes showing significant differences > 2 fold, GOEA indicated that cellular and metabolic processes were highly represented. Expression of ANGPTL4, PLIN2, SQSTM1, and DDIT3 were significantly upregulated and HHIP was downregulated by rosiglitazone. CK5 was downregulated by rosiglitazone.
The RNA-seq data suggested that PPARγ activation induced alterations in cell differentiation and metabolic process and affected multiple signaling pathways such as PPAR, autophagy, WNT, and Hedgehog.
过氧化物酶体增殖物激活受体 γ（PPARγ）在永生化人眼睫毛囊上皮细胞（hMGEC）的成熟过程中起着关键作用。为了进一步了解与类脂细胞分化相关的分子变化，我们分析了 PPARγ 激活后 hMGEC 的转录组谱。
三组培养的 hMGEC 分别用或不用 PPARγ 激动剂罗格列酮处理，用于 RNA-seq 分析。分离 RNA 并进行处理，生成 6 个文库。然后将文库测序并映射到人类参考基因组，将表达结果以每转录长度每百万映射读数的读长数（RPKM）值收集。使用 DESeq2 和 NOISeq 进行差异基因表达分析。对罗格列酮处理后上调或下调的基因集进行基因本体富集分析（GOEA）。选择 5 个基因进行验证，使用定量 PCR 确认差异表达。使用 Western 印迹评估 CK5 的差异表达。
表达数据表明，hMGEC 中约有 58000 个基因表达。DESeq2 和 NOISeq 分别表明，罗格列酮处理后分别有 296 和 3436 个基因上调，258 和 3592 个基因下调。在差异倍数 > 2 的基因中，GOEA 表明细胞和代谢过程高度代表。罗格列酮显著上调 ANGPTL4、PLIN2、SQSTM1 和 DDIT3 的表达，下调 HHIP 的表达。罗格列酮下调 CK5 的表达。
RNA-seq 数据表明，PPARγ 激活诱导细胞分化和代谢过程的改变，并影响多个信号通路，如 PPAR、自噬、WNT 和 Hedgehog。
