Identification of differentially expressed genes by microarray
To investigate RGC death and survival in the transcriptional level, the rAION-inducted rats were treated with phosphate buffered saline (PBS) or GCSF. The transcriptome profiles were analyzed using oligonucleotide microarrays. Microarray data were analyzed using the Gene Expression Pattern Analysis Suite to identify the differentially expressed genes. In a total of 24,358 analyzed genes, 3101 and 3332 transcripts were regulated by GCSF treatment on days 3 and 7 post rAION, respectively. In addition, 702 and 179 transcripts were regulated by PBS treatment on days 3 and 7 post rAION, respectively (Figure 1A–D). Unsupervised hierarchical clustering analysis of differentially expressed genes from all groups was conducted to investigate the similarity of the whole gene expression between the experimental samples. The result indicated that the profile of gene expression in the GCSF-treated group was similar (Figure 1E). Additionally, the PBS-treated rats on days 3 and 7 post rAION also exhibited similar gene expression. As stated above, the trend of gene expression is consistent between the PBS- and GCSF-treated groups.
TAFs involved in regulation of cell death and proliferation
To classify the biological function of differentially expressed genes, we employed a gene ontology (GO) analysis. After GO analysis, there were many TBP-associated proteins that are classified into the category of regulation of cell death and proliferation. We found that 18 TAFs, including TAF1, TAF1a, TAF1b, TAF1c, TAF1d, TAF2, TAF5, TAF6l, TAF7, TAF7l, TAF8, TAF9, TAF9b, TAF10, TAF11, TAF12, TAF13, and TAF15, were upregulated by GCSF treatment. Additionally, 17 TAFs, including TAF1, TAF1a, TAF1b, TAF1c, TAF1d, TAF3, TAF5, TAF6, TAF6l, TAF7, TAF7l, TAF8, TAF9, TAF10, TAF11, TAF12, and TAF13, were downregulated by PBS treatment (Figure 2). It indicated that the expression level of many TAFs was suppressed by ON ischemic injury and induced by GCSF treatment.
Network analysis revealed that TAFs directly interact with TP53 and TBP
STRING network analysis exhibited that many TAFs interact directly with TP53, including TAF1, TAF1L, TAF2, TAF3, TAF4, TAF5, TAF6, TAF7, TAF7L, TAF9, TAF9b, TAF10, TAF11, TAF12, and TAF13 (Figure 3). Additionally, TAFs have direct interaction with TBP, including TAF1, TAF1a, TAF1b, TAF1c, TAF1d, TAF1L, TAF2, TAF3, TAF4, TAF5, TAF6, TAF7, TAF7L, TAF9, TAF9b, TAF10, TAF11, TAF12, and TAF13. Among these TAFs, TAF9 was predicted to bind with TP53, and the expression level of TAF9 was dramatically elevated by GCSF treatment and suppressed by PBS treatment. The biological function of TAF9 is involved in gene regulation associated with apoptosis [32]. Thus, we selected TAF9 as a candidate gene to evaluate its function in RGC apoptosis and survival after ON ischemia.
Taf9 knockdown impaired the protective effect of GCSF on the visual function
To evaluate the role of TAF9 in the protection of visual function in rAION, flash visually evoked potentials (FVEPs) were measured at day 28 post infarct (Figure 4A). On TAF9 knockdown, we found no improvement in visual function despite GCSF treatment. The P1-N2 amplitudes in the sham-operated, scramble siRNA-treated, GCSF plus scramble siRNA-treated, and GCSF plus TAF9 siRNA-treated groups were 65.8 ± 12.7 μV, 23.4 μV ± 4.2 μV, 49.5 ± 6.6 μV, and 28.9 ± 8.4 μV, respectively (Figure 4B). Treatment with GCSF plus TAF9 siRNA reduced the P1-N2 amplitude by 1.71-fold compared to treatment with GCSF plus scramble siRNA (Figure 4B, p < 0.05).
Taf9 knockdown impaired the protective effect of GCSF on RGC density
In the central retina, the RGC density in the sham-operated, scramble siRNA-treated, GCSF plus scramble siRNA-treated, and GCSF plus TAF9 siRNA-treated groups were 1402.5 ± 99.1, 655.2 ± 199.6, 1265.1 ± 352.5, and 649.7 ± 227.6 cells/mm2, respectively (Figure 5A). In the midperipheral retina, the RGC densities in the sham-operated, scramble siRNA-treated, GCSF plus scramble siRNA-treated, and GCSF plus TAF9 siRNA-treated groups were 1219.4 ± 201.3, 319.2 ± 195.8, 863.3 ± 161.3, and 492.9 ± 250.1 cells/mm2, respectively (Figure 5A). The RGC density in the GCSF plus TAF9 siRNA-treated group was significantly reduced by 1.94- and 1.75-fold in the central and midperipheral retinas, respectively, compared with that in the GCSF plus scramble siRNA-treated group (Figure 5B, p < 0.05).
TAF9 knockdown impaired the anti-apoptotic ability of GCSF
The numbers of TUNEL+ cells in the sham-operated, scramble siRNA-treated, GCSF plus scramble siRNA-treated, and GCSF plus TAF9 siRNA-treated groups were 0.2 ± 0.4/HPF, 7.4 ± 2.7/HPF, 2.1 ± 1.3/HPF, and 6.3 ± 2.2/HPF, respectively. The number of TUNEL+ cell in the GCSF plus TAF9 siRNA-treated group significantly increased by threefold compared to that in the GCSF plus scramble siRNA-treated group (p < 0.05), but there was no significant difference between the scramble siRNA-treated and GCSF plus TAF9 siRNA-treated groups (Figure 6), further suggesting a survival pathway dependent on TAF9.
TAF9 knockdown suppressed GCSF-induced TP53 and TRIAP1 expression
Western blotting confirmed that the GCSF plus scramble siRNA-treated group exhibited the highest protein level of TAF9 compared with other groups (Figure 7, p < 0.05). GCSF plus TAF9 siRNA treatment significantly repressed TAF9 protein expression by 6.9-fold compared to GCSF plus scramble siRNA treatment (p < 0.05). In the GCSF plus TAF9 siRNA-treated group, the TP53 level was reduced by 2.4-fold compared to that in the GCSF plus scramble siRNA-treated group (p < 0.05). One of TP53 regulated genes, TP53-regulated inhibitor of apoptosis gene 1 (TRIAP1), can inhibit apoptosis through interaction with APAF1 and heat shock protein 70 (HSP70) complex [33]. Our Western blotting data demonstrated that the TRIAP1 level was reduced by 4.7-fold in the GCSF plus TAF9 siRNA-treated group compared with that in the GCSF plus scramble siRNA-treated group (p < 0.05).
Overexpression of TAF9 inhibited RGC death by modulating TP53-TRIAP1-CASP3 axis
To explore the role of TAF9 in the regulation of RGC death and survival, the AAV2-rTAF9 was used to overexpress the TAF9 level in the rAION model. Four weeks after rAION, the numbers of TUNEL positive cells in the PBS-treated and AAV2-r-TAF9-treated groups were 7.4 ± 2.7/HPF and 2.4 ± 1.7/HPF, respectively (Figure 8A). The number of TUNEL positive cell was 3.1-fold lower in the AAV2-r-TAF9-treated group than that in the PBS-treated group (p < 0.05). Western blotting confirmed that the TP53 and TRIAP1 levels in the AAV2-r-TAF9-treated group were significantly increased by 2.04- and 2.71-fold, respectively, compared to those in the PBS-treated group (Figure 8B, p < 0.05). Moreover, the cleaved-caspase 3 (Cl-casp3) level was reduced by 2.33-fold in the AAV2-rTAF9-treated group compared to that in the PBS-treated group (Figure 8B, p < 0.05).