Mir 182 5p

EV RNA was purified using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, and RNA was eluted in 10 μL RNase-free water. The RT primers and Taqman probes were ordered from Thermofisher Scientific (miR-182-5p, Assay ID 002599, Catalog number 4427975; miR-183-5p, Assay ID 002269, Catalog number 4427975) (Waltham, MA, USA). cDNA was synthesized using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) and qPCR was performed using TaqMan Universal Master Mix II (Thermo Fisher Scientific, Waltham, MA, USA). We used the same number of EVs when comparing HR and N EVs from the same batch. Three independent batches of paired HR and N EVs were included, and we used 1.37 × 10¹¹ EVs, 1.38 × 10¹¹ EVs, and 2.74 × 10¹¹ EVs from each batch, respectively. Each EV sample was analyzed in triplicates. The fold change of the miRNA expression was calculated using 2^–ΔCT (HR EVs relative to N EVs of the same batch). The p-value was calculated using unpaired Student’s t-test.

Total RNA was isolated from tissues and cells using Trizol reagent (Invitrogen, USA) based on the manufacturer’s recommendations. RNA quality and concentration were measured using NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). For mRNA, All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgene, China) was used for RT-qPCR, while for miRNA, the miRNA RT-qPCR Starter kit (RiboBio, China) was used. The CMTM7 and GAPDH primers were synthesized by Genewiz (Tianjin, China), and miR-182-5p (ID: 002334) and U6 (ID: 001093) probes were purchased from Thermo Fisher Scientific (USA). All specific sequences are listed in Additional file 1: Table S2.