Figure 3. Effects of silencing the glucocorticoid receptor gene in nontransformed human trabecular meshwork cells

Cultures of human trabecular meshwork cells at passage 5 were transfected by nucleofector electroporation with either GR siRNA or scramble control as indicated in Methods. Twenty-four h later, cells were treated with 100 nM DEX or vehicle for 72 h. Total RNA extraction was followed by real-time TaqMan PCR using GR, MYOC, and 18S TaqMan probes. Normalization of expression in each sample was achieved using the 18S TaqMan probe hybridized to its own 10^-4 diluted RT. A: There is significant reduction of GR cDNA in HTM-55 and HTM-69 cells transfected with GR siRNA as compared with those transfected with scramble control in the presence of vehicle or DEX. Values of 0 and 100 indicate null and full reduction of the GR gene expression by GR siRNA. Scramble negative control samples were normalized to a value of 0% reduction and GR percent silencing values from scramble are expressed as the mean±SEM. Vehicle: HTM-55, n=6, p=0.001; HTM-69, n=6, p=0.015. DEX: HTM-55, n=6, p=0.003; HTM-69, n=6, p=0.015. B: Induction of the MYOC gene by DEX after transfection with scramble and/or GR siRNA compared to vehicle. Vehicle samples were normalized to a value of 1.0 and MYOC fold change values versus vehicle are expressed as the mean±SEM. Scramble-transfected: HTM-55, n=6, p=0.014; HTM-69, n=6, p=0.001. GR siRNA-transfected: HTM-55, n=6, p=0.02; HTM-69, n=6, p=0.009. C: Expression of the MYOC gene in the presence of DEX after transfection with GR siRNA compared to expression after transfection with scramble control. Values of 0 and 100 indicate null and full reduction of the MYOC gene expression by the GR siRNA. Scramble control samples were normalized to a value of 0% reduction and MYOC percent reduction from scramble is expressed as the mean±SEM. GR siRNA transfected: HTM-55, n=6, p0.002; HTM-69, n=6, p=0.01. Silencing the GR receptor with siRNA significantly reduces the expression of the glucocorticoid-induced gene MYOC in HTM cells.