2.4. Ribonuclease R (RNase R) and actinomycin D (ActD) treatment

RNase R is an exoribonuclease that degraded linear RNA but left circular transcripts intact [27]. The extracted RNA in 100 μg/mL ox-LDL-treated VSMCs was treated with RNase R (Geneseed, Guangzhou, China) with a ratio of 3 U enzyme/μg RNA for 30 min at 37°C or treated with the 1× Reaction Buffer (as Mock group). Ethanol precipitation was carried out to remove the enzyme and salts. After that, RT-qPCR was used to examine circ_UBR4 and UBR4 expression. ActD was used to block transcription, and 100 μg/mL ox-LDL-treated VSMCs in 24-well plate were added to 2 μg/mL ActD (Amyjet, Wuhan, China) for 0, 4, 8, 16, and 24 h before total RNA isolation and RT-qPCR for the detection of circ_UBR4 and UBR4 expression.