Expression of miRNA-26a in platelets is associated with clopidogrel resistance following coronary stenting

Abstract

The present study aimed to evaluate the association between platelet microRNA (miRNA)-26a expression and clopidogrel resistance in patients who underwent coronary stenting. Between September 2013 and August 2014, 43 patients with coronary heart disease underwent percutaneous coronary intervention at Heibei General Hospital (Shijiazhuang, China). In the same period, 20 healthy volunteers without any history of cardiovascular disease were enrolled in the present study as the control group. Flow cytometry was used to measure the phosphorylation levels of vasodilator-stimulated phosphoprotein (VASP), and to calculate the platelet reactivity index (PRI). Low response to clopidogrel was defined as PRI ≥50% on day 7 following clopidogrel administration. Western blotting was used to measure protein expression of VASP and reverse transcription-quantitative polymerase chain reaction analysis was performed to determine the expression levels of mRNA and miRNAs. Bioinformatics tools were employed to predict that miR-26a, miR-199 and miR-23a may target VASP mRNA. The results of the present study demonstrated that the activity of platelets in patients with low or high clopidogrel response was increased, as compared with healthy subjects. No differences in platelet VASP protein expression levels were detected between patients with high clopidogrel response and healthy subjects; whereas VASP protein expression was elevated in patients with low clopidogrel response. Furthermore VASP gene transcription was maintained at low levels in healthy subjects and patients with high clopidogrel response, whereas patients with low clopidogrel response exhibited increased VASP mRNA expression levels. Platelet expression of miRNA-26a, but not miRNA-199 or miRNA-23a, was associated with high platelet reactivity. Serum miRNA-26a, miRNA-199 and miRNA-23a were not demonstrated to be involved in clopidogrel resistance. Therefore, the present study demonstrated that platelet miRNA-26a has an important role in clopidogrel resistance. Combined miRNA and VASP PRI tests may aid the early diagnosis and prediction of clopidogrel resistance.

Keywords: clopidogrel resistance; microRNA; percutaneous coronary intervention; platelet; vasodilator-stimulated phosphoprotein.

Figure 1.

Platelet reactivity index (PRI). Flow cytometry was used to measure the phosphorylation levels of vasodilator-stimulated phosphoprotein following stimulation with vasodilators, and to calculate the PRI. Low response to clopidogrel was defined as PRI ≥50% on day 7 post-clopidogrel administration. Data are expressed as the mean ± standard deviation. *P<0.05, vs. the control group; ^#P<0.05, vs. the low clopidogrel response group.

Figure 2.

Relative expression of VASP protein in platelets, as detected by western blotting. The relative content of VASP protein was expressed as VASP/GAPDH ratio. Data are expressed as the mean ± standard deviation. *P<0.05, vs. the control group; ^#P<0.05, vs. the high clopidogrel response group. VASP, vasodilator-stimulated phosphoprotein.

Figure 3.

Relative expression of VASP mRNA in platelets, as detected by reverse transcription-quantitative polymerase chain reaction. GAPDH was used as an internal reference for quantification analysis. Data are expressed as the mean ± standard deviation. *P<0.05, vs. the control group; ^#P<0.05, vs. the high clopidogrel response group. VASP, vasodilator-stimulated phosphoprotein.

Figure 4.

Relative expression levels of (A) miR-26a, (B) miR-199 and (C) miR-23a in platelets. miRNA expression was detected by reverse transcription-quantitative polymerase chain reaction with SYBR Green. U6 gene was used as an internal reference for relative quantification. Data are expressed as the mean ± standard deviation. *P<0.05, vs. the control group; ^#P<0.05, vs. the high clopidogrel response group.

Figure 5.

Relative expression levels of (A) miR-26a, (B) miR-199 and (C) miR-23a in serum. miRNA expression was detected by reverse transcription-quantitative polymerase chain reaction with SYBR Green. U6 gene was used as an internal reference for relative quantification. Data are expressed as the mean ± standard deviation.