Transcriptome analysis after PPARγ activation in human meibomian gland epithelial cells (hMGEC).

PURPOSE: 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.

METHODS: 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.

RESULTS: 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.

CONCLUSIONS: 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.