Identification of novel retinal target genes of thyroid hormone in the human WERI cells by expression microarray analysis

Abstract

Using the human WERI-Rb1 cell line as a model system, we performed a genome-wide search for retinal target genes of thyroid hormone (TH) via expression microarray analysis followed by quantitative real-time RT-PCR verification. We identified 12 novel retinal targets of TH, including 10 up-regulated genes (OPN1MW, OPN1LW, TIMP3, RP1L1, GNGT2, CRX, ARR3, GCAP1, IMPDH1, and PDE6C) and 2 down-regulated genes (GNGT1 and GNB3). In addition, we found a number of novel TH-targets that are not currently known to be retinal genes. This is the first report of human retinal targets regulated by thyroid hormone.

Figure 1. WERI cells express high levels of TRβ2 and RXRγ

RNA extracted from WERI cells was used to perform RT-PCR analysis using isoform-specific primers as described in Table 1. The predicted product size for TRα1 = 346 bp, TRα2 = 467 bp, TRβ1= 484 bp, TRβ2 = 307 bp, RXRα = 201 bp, RXRβ = 239 bp, RXRγ = 228 bp. * = TRβ1 PCR product size is smaller than predicted (484 bp). See text for explanation.

Figure 2. T3 induces the transcription of the L/M opsin gene in WERI cells

WERI cells were either treated with vehicle (mock), 10 uM all-trans retinoic acid (ATRA), 10 uM 9-cis retinoic acid (9c-RA), or 10 nM T3 (T3) for 48 hours. RNA extracted from these samples was used to perform RT-PCR analyses using primer pairs that amplify the L/M opsin gene (top panel), COUPTF1 (middle panel), or the housekeeping gene GAPDH (bottom panel).

Figure 3. Microarray Expression profile of WERI cells treated with T3

(A). Scatter plot of hybridization signals of RNA from WERI cells treated with 100 nM T3 (TH, Y-axis) versus signals from a mock treated sample (Mock). Up-regulated transcripts are indicated by red + sign; down-regulated transcripts are indicated by green + sign; yellow + sign indicate transcripts with similar expression levels in the TH and Mock samples. (B). Scatter plot of p values (Y-axis) versus fold change (X-axis, log2 scale). The red + sign on the bottom left are transcripts down regulated by 4 fold or more with p value of ≤0.01 ; red + signs on the bottom right represent transcripts that are up-regulated by 4 fold or more with p values of ≤0.01. (C). Heatmap of probe sets on ±4 fold, p≤0.01 list generated by cluster analysis. Note the three biological replicates show similar levels of expression.

Figure 4. Verification of microarray data by qRT-PCR analysis

RNA samples used for microarray analysis mock (Mock) and 100 nM T3 (TH), were subjected to qRT-PCR to measure the expression levels of the 37 target genes identified by microarray analysis. The data for all 37 genes can be found in Table 4. This figure shows the amplification curves of 8 genes: GAPDH (used for normalization), LMOD1 (an up-regulated non-retinal gene), 5 up-regulated retinal genes (OPN1LW/MW, CRX, RP1L1, TIMP3 and GNGT2), and 1 down-regulated retinal gene (GNGT1). Each sample contains three biological replicates and was assayed in duplicate.

Figure 5. Dose-response analyses on identified retinal TH-targets

RNA samples from WERI cells treated for 48 hours with either vehicle control (No T3) or different concentrations of T3 (1nM, 10 nM, 100 nM) were used to perform qRT-PCR to measure the expression level of each gene as indicated. Fold inductions was calculated using the ddCt method which show the relative amount of each sample compared to the control (No T3). The data shown are normalized mean values ± SD of three biological replicates done in duplicate. Statistical analyses were performed using the Student’s t-test and compared the 1 nM group to the control group (No T3). The * sign indicates p≤0.05, ** sign indicates p≤0.01.

Figure 6. Time course analyses on identified retinal TH-targets

RNA samples from WERI cells treated with 5 nM T3 at different time points were used to perform qRT-PCR to measure the expression levels of each gene as indicated. GAPDH was also measured for normalization. Fold induction was calculated using the ddCt method which shows the relative amount of samples from each time point compared to the 0 time point. The data shown are normalized mean values ± SD of three biological replicates done in duplicate. F=fast, M=medium, S=slow.