METTL3-mediated methylation of CYP2C19 mRNA may aggravate clopidogrel resistance in ischemic stroke patients

Figures

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  Figure 1. PRU of human blood samples. CR was defined as having a test result >208 P2Y12 Reaction Units (PU). Values are mean ± std. N = 5. Unpaired t-test, ****P < 0.0001.

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  Figure 2. METTL3 levels in peripheral blood were elevated in humans with CR. (a) qPCR test results of m6A regulators expression levels in mRNA. (b) WB analysis results of METTL3 expression levels. Values are mean ± std. N = 3. Unpaired t-test, *P < 0.05.

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  Figure 3. METTL3 modifies CYP2C19 RNA m6A in the peripheral blood of human subjects with CR, which is associated with decreased CYP2C19 RNA and CYP2C19 expression. (a) Prediction of the possible m6A methylation site of CYP2C19 mRNA. (b) MeRIP results. M6A methylation levels of CYP2C19 mRNA in the peripheral blood of human clopidogrel resistant is higher than that of non-clopidogrel resistant. (c and d) The levels of CYP2C19 in human peripheral blood were determined by qPCR and WB analysis. Values are mean ± std. N = 3. Unpaired t-test, *P < 0.05, **P < 0.01. R, resistance; N, non-resistance.

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  Figure 4. Relative decline rate of MAR in the peripheral blood of rats. CR is defined as platelet aggregation inhibition rate = (unadministered MAR – MAR after administration)/unadministered MAR <30%. Values are mean ± std. N = 3. Unpaired t-test, **P < 0.001.

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  Figure 5. METTL3 levels in peripheral blood and liver were elevated in rats with CR. (a) qPCR results of METTL3 expression levels in mRNA in peripheral blood of rats. (b) qPCR results of METTL3 expression levels in mRNA in the liver of rats. (c) WB results of METTL3 protein expression in peripheral blood of rats. (d) WB results of METTL3 protein expression in the liver of rats. (e) Dot blot analysis results of global m6A levels in the liver of rats. Values are mean ± std. N = 3. Unpaired t-test, *P < 0.05.

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  Figure 6. METTL3 modifies CYP2C6v1 RNA in the peripheral blood and liver of rats with CR and results in decreased CYP2C6v1 RNA expression. (a) Prediction of the possible m6A methylation site of CYP2C6v1 mRNA in rats. (b) MeRIP-PCR results. m6A methylation level of CYP2C6v1 mRNA in the peripheral blood of rats of the clopidogrel-resistant group was significantly higher than the non-clopidogrel-resistant group. (c) qPCR results of CYP2C6v1 expression levels in mRNA in peripheral blood of rats. (d) MeRIP results. M6A methylation level of CYP2C6v1 mRNA in liver of rats in clopidogrel-resistant group was higher than that in non-clopidogrel-resistant group. (e) qPCR results of CYP2C6v1 expression levels in mRNA in the liver of rats. Values are mean ± std. N = 3. Unpaired t-test, *P < 0.05. ****P < 0.0001. R, resistance; N, non-resistance.

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  Figure 7. Mechanism by which METTL3 promotes CR in IS by targeting CYP2C19 mRNA N6-methyladenosine.