Long non-coding RNA histone deacetylase 4 antisense RNA 1 (HDAC4-AS1) inhibits HDAC4 expression in human ARPE-19 cells with hypoxic stress

Age-related macular degeneration (AMD) is resulted from choroidal neovascularization (CNV)-mediated cicatrization and vision loss. The sustained retinal hypoxia in retinal pigment epithelium (RPE) cells was reported to contribute to CNV. However, the underlying genetic regulatory network of hypoxia response in RPE is not fully understood. In this study, human ARPE-19 RPE cells were cultured under the anoxia for 24 h and later re-oxygenated in normoxia. Then the transcriptome was investigated via high throughput sequencing. We observed that long non-coding RNA (lncRNA) histone deacetylase 4 antisense RNA 1 (HDAC4-AS1) was increased in hypoxic condition compared to normal control and decreased after re-oxygenation addition, while the change of HDAC4 expression was reduced in hypoxic condition compared to normal control and up-regulated after re-oxygenation addition in ARPE-19 cells. Furthermore, HDAC4-AS1 knockdown could suppress the transcription activity of HDAC4 only in hypoxia condition, and fluorescence in situ hybridization and pull down assay indicated that transcripts of HDAC4-AS1 could substantially bind to the promoter of HDAC4 and facilitate the recruitment of HIF-1α. Finally, we also determined the specific regions of HDAC4-AS1 that contribute to the interaction with HIF-1α and the promoter of HDAC4. Taken together, these outcomes declared that HDAC4-AS1 could inhibit HDAC4 expression through regulating HIF-1α in human ARPE-19 cells with hypoxic stress.年龄相关性黄斑变性（Age-related macular degeneration, AMD）是由脉络膜新生血管（choroidal neovascularization, CNV）介导的瘢痕形成及视力丧失所引发的疾病。已有研究证实，视网膜色素上皮（retinal pigment epithelium, RPE）细胞持续的视网膜缺氧会促进CNV的发生，但目前对于RPE细胞中缺氧应答的潜在遗传调控网络仍未完全阐明。本研究将人源ARPE-19 RPE细胞置于缺氧环境中培养24小时，随后将其转移至常氧条件下进行复氧处理。通过高通量测序对细胞转录组进行分析后，我们观察到：与正常对照组相比，长链非编码RNA（long non-coding RNA, lncRNA）组蛋白去乙酰化酶4反义RNA1（histone deacetylase 4 antisense RNA 1, HDAC4-AS1）在缺氧条件下表达上调，而复氧后其表达水平显著下降；而ARPE-19细胞中HDAC4的表达变化在缺氧条件下较对照组有所减弱，复氧后则出现上调。进一步实验表明，仅在缺氧条件下，HDAC4-AS1敲低可抑制HDAC4的转录活性；荧光原位杂交与下拉实验结果显示，HDAC4-AS1转录本可显著结合HDAC4的启动子区域，并促进缺氧诱导因子-1α（HIF-1α）的招募。此外，本研究还明确了HDAC4-AS1中参与与HIF-1α及HDAC4启动子相互作用的特定功能区域。综上，本研究结果证实，在遭受缺氧应激的人源ARPE-19 RPE细胞中，HDAC4-AS1可通过调控HIF-1α的活性抑制HDAC4的表达。