Regulatory long non-coding RNAs and their potential roles in aortic diseases

Rationale:

Aortic valve stenosis (AVS) and coronary artery disease (CAD) are among the most common subtypes of cardiovascular disease, which are significantly shown as a comorbidity in the patients undergoing transcatheter aortic valve implantation (TAVI). In human transcriptome there are >80% of non-coding RNAs, that cannot be translated into protein, however, play crucial roles in the regulation of genes expression and epigenetic modification. However, a specific, in-depth study of lncRNAs on AVS needs to be performed to investigate novel diagnostic biomarkers and to potentially develop therapeutic strategies. Herein, we explored the biological functions of differently expressed lncRNAs; SENCR, MIAT, MEG3 in patients with AVS.

Methods and Results:

RNA sequencing was performed on the tissue of aortic valve explant from patients with AVS for lncRNAs screening. Loss-of-function experiment of SENCR, MIAT, MEG3 was performed in vitro by lncRNAs knockdown via siRNA transient transfection (48h) in different cell types involved in CAD and AVS; human coronary artery endothelial cells, and smooth muscle cells, valvular endothelial cells and interstitial cells, followed by various functional assays. qPCR validated the effectivity of lncRNAs knockdown. In addition, tube formation assay was performed to investigate how siSENCR, siMIAT, siMEG3-conditioned HCAEC affect the angiogenesis, which altogether showed direct proportional results to cell proliferation and migration. Set of experiments were performed in VEC to study endothelial-to-mesenchymal transition (EndMT) and simultaneously in VIC to study calcification involved in AVS. After lncRNAs knockdown, VEC was treated with TNFα (30 ng/ml) and TGFβ (5 ng/ml) + IL1 (1 ng/ml) separately for 5 days to induce EndMT. In parallel, after lncRNAs knockdown, VIC was treated with osteogenic medium (supplemented with dexamethasone, L-ascorbic acid, glycerol-2-phosphate) and pro-calcifying medium (supplemented with NaH₂PO₄, L-ascorbic acid) separately for 7 days to induce calcification. qPCR preliminary data showed that knockdown of SENCR, MIAT, MEG3 in VEC exerts different genes expression (ACTA2, PECAM1, NOS3, VIM, VWF), suggesting that downregulated lncRNAs may affect different cellular mechanisms upon EndMT process in AVS. Furthermore, knockdown of SENCR, MIAT, MEG3 in VIC also exerts different genes expression (BGLAP, RUNX2) as shown in qPCR preliminary data, indicating that downregulated lncRNAs may affect the pathophysiology in different manners upon progressive calcification in AVS.  

Outlook:

Further investigation of intercellular communication among different cell populations in AVS is to be performed via incorporating lncRNA into extracellular vesicle (EV), which act as a cargo and can be transported from donor cells to recipient cells to exert the biological functions of target lncRNAs on paracrine signaling, followed by further analysis of proteomics and transcriptomics to decipher lncRNAs binding partners and RNA-protein interactions in the underlying molecular mechanisms of the disease. A required to elaborate deeper insights into lncRNA roles in AVS and CAD. For a more advanced in vitro experimental study, 3D models of human induced pluripotent stem cells (hiPSC)-derived vascular organoids are also to be established to investigate the translational relevance of lncRNAs as potential diagnostic biomarkers and novel therapeutic approach to treat patients with AVS and CAD.