Synthetically designed circular RNAs inhibit miR-21-5p and miR-146a-5p to improve senescent vascular cell functions

Vascular remodeling, endothelial dysfunction, and cellular senescence are complex, connected processes influencing cellular function and further contributing to the pathogenesis of cardiovascular diseases (CVDs). Numerous studies have confirmed that microRNAs (miRs), including miR-21-5p and miR-146a-5p, are associated with CVDs. Inhibiting miRs through sponges, like anti-miRs and circular RNAs (circRNAs), might represent a therapeutic approach.

This project studies miR-21-5p and miR-146a-5p in senescent human vascular smooth muscle and endothelial cells (VSMCs and ECs) to establish a therapeutic approach using specifically designed circular RNAs. Cells were analyzed regarding their miR-21 and miR-146a expression and their senescent marker expression via qPCR using a replicative senescent model. CircRNAs were designed for the respective miRNA and evaluated by northern blotting. Methods to characterize the effect of miR inhibition through circRNA transfection included quantification of target gene and protein expression through PCR, western blots, and functional analysis of proliferation and migration.

We could show that ECs and VSMCs have higher levels of miR-21 and miR-146a expression in senescent cells than in non-senescent cells (miR-21 p<0.05, miR-146a p<0.01 in ECs). CircRNAs were designed, expressing either four binding sites for miR-21 or miR-146a. The northern blot analysis confirmed the successful design and transfection of circRNAs into VSMC and ECs. After transfection, circRNAs could be detected within the cells for several days. In VSMCs, a significant upregulation of miR-146a target genes TRAF6 (p<0.05) and KLF4 (p<0.0001) was shown after a 48-hour transfection with circRNA-146a. CircRNA inhibition of miRNA-146a was more efficient in target regulation than the respective inhibition through anti-miRs. Protein analysis confirmed target regulation. Comparable results were observable for ECs. Migration and proliferation assays depicted an improvement of both parameters in ECs (p<0.05) and a decrease in migration capacity in VSMCs (p<0.05). Senescence marker analysis was performed to evaluate the impact of circRNAs on the senescence status of vascular cells. Results showed downregulation of senescence markers after either transfecting circRNA-21 or circRNA-146a.

In summary, we could demonstrate the efficiency of circRNAs by inhibiting miR-21 and miR-146a functions in senescent vascular cells, especially in VSMCs. Due to their circular structure, which protects against exonuclease activity, circRNAs are more stable and efficient when transfected compared to anti-miRs. These properties of circRNAs might present a better alternative and a realistic approach for future clinical studies.