Figure 4. piRNA-6426 increases the methylation of SOAT1 CpG island. (A) The online prediction software MethPrimer was used to predict the location of the CpG island in the SOAT1 promoter region. (B) In a 50 μL system, the extracted DNA (2~5 μg) was denatured with NaOH (final concentration value 0.2 mol/L) at 37° C for 10 min, and 30 μL of just prepared 10 mmol/L hydroquinone and 520/1 40.5% sodium bisulfite were added and mixed, and then paraffin oil was added to isolate from the air, and incubated for 16 h in the dark. The modified DNA was passed through a Wizard DNA purification column (Chemicon) and eluted at room temperature, and then modified by using NaOH (The final concentration is 0.3 mol/L) for 5 min and precipitated by using ethanol, the DNA is dissolved in 20 μL of water, and specific primers were used for methylation-specific PCR (MSP) detection. (C) RT-qPCR assay was used to detect the expression of SOAT1 mRNA in cardiomyocytes incubated with LV-piRNA-6426 or empty vector. (D, E) Western blotting was used to detect the expression level of SOAT1 protein after piRNA-6426 overexpressed. (F) RT-qPCR assay was used to detect the expression of SOAT1 mRNA in cardiomyocytes incubated with sh-piRNA-6426 or scrambled shRNA. (G, H) Western blotting was used to detect the expression level of SOAT1 protein after piRNA-6426 interfered. Values were expressed as mean ± SEM. *P<0.05, n=6.