Quantitative Real-Time Polymerase Chain Reaction

Total RNA of PAECs was extracted in accordance with the instructions of the Trizol reagent (Invitrogen), and reverse transcription was performed using reverse transcriptase (Thermo Fisher Scientific, Inc.) as reported previously (Zhao et al., 2017). The primers were designed and obtained from Sangon Biotech Co. (Shanghai, China) as follows: Dec1, forward primer 5′-GAAAGGATCGGCGCAATTAA-3′, reverse primer 5′-CATCATCCGAAAGCTGCATC-3′; and β-actin, forward primer 5′-TGGCACCACACCTTCTACAA-3′, reverse primer 5′-GCAGCTCGTAGCTCTTCTCC-3′. The relative level of mRNA was calculated by the 2^–ΔΔCT method (Teng et al., 2012). Fold change of the relative mRNA expression was evaluated as the average fold change of the hypoxic PAECs compared with that of the normoxic PAECs. Measurements were repeated three times.