外泌体环状RNA 30741在小鼠血管平滑肌细胞增殖中的作用

目的 探讨环状RNA在血管平滑肌细胞增殖过程中的作用。方法 在体外原代培养小鼠血管平滑肌细胞,采用不同浓度（10~50 ng/mL）的血小板源性生长因子-BB（PDGF-BB）诱导血管平滑肌细胞（VSMC）增殖。在给予PDGF-BB诱导48 h后,采用细胞计数和BrdU法检测血管平滑肌细胞增殖;同时采用高速离心法分离培养液中外泌体,提取外泌体中总RNA,利用Real-time PCR法检测外泌体中6种环状RNA（环状RNA 30741、1853、723、3069、1776、1092）的表达情况,在Circbase等数据库对环状RNA的靶小分子RNA进行生物信息学分析。结果 与对照组（Vehicle）相比,给予10 ng/mL PDGF-BB刺激后血管平滑肌细胞的数目和BrdU OD值没有明显变化（vs Vehicle, P>0.05）,20~50 ng/mL的PDGF-BB刺激后血管平滑肌细胞数目和BrdU OD值呈浓度依赖性增加（vs Vehicle, P<0.05）。在血管平滑肌细胞增殖过程中,环状RNA 1853、723、3069、1776、1092的表达水平无明显变化（P>0.05）,环状RNA30741的表达水平上调（P<0.05）,且环状RNA 30741的mRNA水平与VSMC的细胞数呈显著正相关性 (P<0.05)。生物信息学分析提示环状RNA 30741的靶小分子RNA包括小分子RNA-21等。结论 环状RNA 30741可能参与了血管平滑肌细胞的增殖过程。

The exosome circular RNA 30741 promoted the proliferation of vascular smooth muscle cells in mice

Objective To detect the effect of circular RNA (circRNA) extracted from exosome on the proliferation of vascular smooth muscle cells (VSMCs). Methods Primary mouse VSMCs were cultured in vitro and were induced to proliferate by platelet-derived growth factor-BB (10-50 ng/mL, PDGF-BB). The proliferation property of VSMCs were investigated by cell number counting as well as 5-bromodeoxyuridine (BrdU) OD test 48 h post stimulation of PDGF-BB. The exosome was isolated from supernatant using high speed centrifugation. Total RNA was extracted form exosome and 6 circRNAs (circRNA 30741, 1853, 723, 3069, 1776, 1092) were explored by real time PCR. The target microRNA (miR) of circRNA was predicted by bioinformatics analysis in database such as Circbase. Results Compared with Vehicle, VSMCs induced by 10 ng/mL PDGF-BB did not show significant differences in cell number as well as BrdU OD (vs Vehicle, P>0.05). Nevertheless, the cell number as well as BrdU OD significantly increased in VSMCs induced by 20 -50 ng/mL PDGF-BB in dose dependent manner (vs Vehicle, P<0.05). Except circRNA 1853, 723, 3069, 1776, 1092, the mRNA level of circRNA 30741 significantly increased during the proliferation progress of VSMCs (P<0.05). Bioinformatics analysis indicated that miR-21 was the target microRNA of circRNA 30741. Conclusion Our data indicated that circRNA 30741 promoted the proliferation of vascular smooth muscle cells in mice.