Reverse transcription-quantitative PCR (RT-Qpcr)

To determine the Mrna expression levels, RT-Qpcr was performed. Total RNA was extracted from VSMC using the RNA simple Total RNA kit (Tiangen Biotech Co., Ltd.), according to the manufacturer’s instructions. Subsequently, 1 μg total RNA was reverse transcribed into Cdna using Cdna Reverse Transcription kits (Thermo Fisher. Scientific, Inc.) The mRNA expression levels of WWP2, UTX, SIRT1, α-SMA, SM22a and OPN were detected by qPCR using a SYBR green PCR Kit (DBI Bioscience) on the LightCycler System (Roche Applied Science). The primer sequences used are listed in Table I in S1 Table. The thermocycler conditions were as follows: 37˚C for 30 sec; 95˚C for 5 min; followed by 45 cycles of 95˚C for 10 sec; 55˚C for 30 sec; and 72˚C for 30 sec. The obtained Ct values were analyzed based on the amplification curves and the relative expression levels of the target genes were calculated using the 2^-ΔΔCq method [19]. PCR reactions were performed in triplicate and normalized using GAPDH as a reference gene.