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Roles of miR-16 in vascular endothelial injury in patients with coronary heart disease

Objective: This study is to detect miR-16 expression in peripheral blood of patients with Coronary Heart Disease (CHD), and explore the role of miR-16 in vascular endothelial injury.

Methods: Peripheral blood was collected from coronary patients. QRT-PCR was applied to detect miR-16 expression and autophagy related 14 (AGT14) mRNA expressions. After transfection with miR-16 inhibitor in vitro, the biological functions of miR-16 were detected in endothelial cells by CCK-8, apoptosis assay, and cell cycle analysis. Bioinformatics methods predicted that ATG14 was one of target genes regulated by miR-16. Dual luciferase assay was used to validate whether miR-16 directly regulated ATG14. Expression changes of ATG14, Beclin1 and microtubule-associated protein 1 light chain 3 beta (LC3B) were detected by Western blot.

Results: miR-16 expression was significantly increased in blood in CHD patients, and miR-16 expression was higher in patients with unstable angina than patients with stable angina (P<0.05), which indicated that miR-16 was related to the development of CHD. In vitro experimental results showed that miR-16 inhibited the proliferation and promoted apoptosis of endothelial cells significantly (P<0.05). Compared with control group, the percentage of cells in G1 phase in miR-16 inhibitor group significantly decreased (58.8 ± 3.51 vs. 40.7 ± 3.32) whereas the percentage of cells in S phase significantly increased (21.8 ± 1.62 vs. 32.7 ± 2.47) (P<0.05), indicating that miR-16 inhibited G1/S conversion of endothelial cells. The dual luciferase assay validated that miR-16 directly targeted ATG14. Western blot and qRT-PCR results demonstrated that expressions of Beclin1 and LC3B were repressed when miR-16 was over-expressed while were significantly increased after down-regulated miR-16. And, electronic microscopy showed that miR-16 inhibited the formation of autophagy bodies in endothelial cells.

Conclusion: MiR-16 expression was up-regulated in peripheral blood of patients with CHD. MiR-16 regulated ATG14 expression in endothelial cells. Thus, miR-16 may play a role in the development of vascular endothelial injury and CHD.

Author(s): Zheng Cao, Li Qian, Shen Wu