外泌体环状RNA 30741在小鼠血管平滑肌细胞增殖中的作用

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

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Brd U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Brd U 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Brd U 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m RNA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。

Objective To detect the effect of circular RNA（circ RNA） extracted from exosome on the proliferation ofvascular smooth muscle cells（VSMCs）. Methods Primary mouse VSMCs were cultured in vitro and were induced toproliferate by platelet-derived growth factor-BB（10-50 ng/m L, PDGF-BB）. The proliferation property of VSMCs wereinvestigated by cell number counting as well as 5-bromodeoxyuridine（Brd U） OD test 48 h post stimulation of PDGF-BB. Theexosome was isolated from supernatant using high speed centrifugation. Total RNA was extracted form exosome and 6 circ RNAs（circ RNA 30741, 1853, 723, 3069, 1776, 1092） were explored by real time PCR. The target micro RNA（miR） ofcirc RNA was predicted by bioinformatics analysis in database such as Circbase. Results Compared with Vehicle, VSMCsinduced by 10 ng/m L PDGF-BB did not show significant differences in cell number as well as Brd U OD（vs Vehicle, P〉0.05）.Nevertheless, the cell number as well as Brd U OD significantly increased in VSMCs induced by 20-50 ng/m L PDGF-BB indose dependent manner（vs Vehicle, P〈0.05）. Except circ RNA 1853, 723, 3069, 1776, 1092, the m RNA level of circ RNA30741 significantly increased during the proliferation progress of VSMCs（P〈0.05）. Bioinformatics analysis indicated that miR-21 was the target micro RNA of circ RNA 30741. Conclusion Our data indicated that circ RNA 30741 promoted theproliferation of vascular smooth muscle cells in mice.