The Effects of avb3 Integrin Signaling on the Transcriptome of an Immortalized Line of Human Trabecular Meshwork Cells (TM-1)

Purpose: To study the effects of overexpressing either a wild type (WT) or a constitutively active (CA) avb3 integrin on the transcriptome of an immortalied line of human trabecular meshwork cells. Methods: RNA was collected from four samples each of control cells expressing an empty vector (EV), cells overexpressing a WT avb3 integrin (WTb3) or a CA avb3 (CAb3) integrin. All cells had been cultured at confluence for 24 hr in 1%FBS-containing medium. RNA libraries were prepared using 1µg of RNA/sample with the TruSeq RNA sample preparation kit and sequenced on four lanes of an Illumina HiSeq 2500 System. Quality control was performed on the sequencing reads using fastq and skewer trimming software was used to preprocess raw fastq files. A quality control pipeline was then applied and each RNA-Seq sample was subsequently normalized by the method of trimmed mean M-values prior to DGE analysis using EdgeR. Results: 5,186 genes had significantly altered expression (p<0.05) in WTb3 cells compared to EV cells (2,645 upregulated vs 2541 downregulated); 8,249 genes had significantly altered expression (p<0.05) in CAb3 cells compared to EV cells (4,094 upregulated vs 4,255 downregulated). However, 6,999 genes had significantly altered expression (p<0.05) in WTb3 cells vs CAb3 cells (3,524 upregulated vs 3,475 downregulated). Conclusions: Overexpressing of both a WT avb3 and a constitutively active avb3 integrin caused signficant alterations in the transcriptome of immortalized TM-1 cells, however constitutive activation of avb3 signaling had a more pronounced effect on the TM-1 transcriptome than did mere overexpression of the integrin. Overall design: Transcriptomes of immortalized trabecular meshwork cells overexpressing a wild type avb3 integrin and a constitutivelyl active avb3 integrin were compared using RNA-seq analysis.