The endothelial-enriched lncRNA LINC01235 regulates hypoxia response via HIF-3a

Abstract

Background Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Hypoxia induces significant changes in cardiovascular control mechanisms potentially resulting in pathophysiology. Recently, an increasing number of long non-coding RNAs (lncRNAs) was reported to participate in the regulation of Hypoxia-inducible factors (HIF). Analysis of single-cell RNA-sequencing of human Abdominal Aortic Aneurysms pinpointed the endothelial-enriched lncRNA LINC01235. LINC01235 was previously correlated with tumour progression in gastric cancer and worse patient prognosis in breast cancer. Globally, the role of LINC01235 in the cardiovascular system remains unknown. Purpose The objective of this study is to unravel the function of LINC01235 in endothelial cells (ECs). Methods and results LINC01235 levels were elevated in human umbilical vein ECs (7.66 fold, p<0.05), human aortic ECs (16.84 fold, p<0.05) and human dermal microvascular ECs (639.73 fold, p<0.05) over other human cardiovascular cells like vascular smooth muscle cells, aortic fibroblasts and cardiomyocytes. Severe hypoxia (0.2% O2 for 24h) reduced LINC01235 expression significantly (0.33 fold, p<0.05). SiRNA-mediated LINC01235 silencing in HUVECs (0.12, p<0.05) resulted in decreased proliferation (0.76 fold, p<0.05) and vascular endothelial growth factor A (VEGFA)-stimulated angiogenic sprouting (0.39 fold, p<0.05). Loss of LINC01235 did not affect apoptosis, metabolism or barrier function. Analysis of RNA-sequencing data revealed that many hypoxia-responsive genes were downregulated after knockdown of LINC01235 (siCtrl vs. siLINC01235). These included HIF-3α (0.24 fold, p<5.86e-28) as a potential key regulator of the cellular feedback to hypoxia. Phenotypically, knockdown of HIF3A using siRNAs (0.07 fold, p<0.05) resulted in decreased proliferation (0.82 fold, p<0.05) and VEGFA-stimulated angiogenic sprouting (0.50 fold, p<0.05). Accordingly, hypoxia response and LINC01235 knockdown exhibit a negative correlation based on transcriptomics data (R=−0.157, p<2.2e-16), further emphasizing a role of LINC01235 in hypoxia response. Conclusion In summary, the EC-enriched lncRNA LINC01235 is likely required for the suppression of hypoxia-induced gene expression under normoxic conditions potentially mediated by HIF-3α. Functionally, loss of LINC01235 decreased proliferation and VEGFA-stimulated angiogenic sprouting without an effect on cell death, metabolism or barrier integrity. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG - TRR267