Quantitative Analysis of miRNA and mRNA Levels

Total RNA was isolated using TRIzol Reagent (ZYMO Research, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was generated using the TransScript First-Strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). Exon-exon qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Mannheim, Germany). U6 small nuclear RNA was used as an internal control for miR663a; the level of miR663AHG was normalized to that of GAPDH (for cultured cells) or Alu (for tissues). Similarly, the levels of TP53, JUNB, JUND, PIK3CD, P21, and TGFB1 mRNAs in cell lines were normalized to that of GAPDH. The relative mRNA levels further normalized to the control were further calculated using the typical 2^-ΔΔCT method [7 (link)]. The relative level of pre-miR663a was calculated according to the formula [41 (link)]: pre-miR663a = 2^{-CT(pri-miR663a+pre-miR663a)} − 2^{-CT(pri-miR663a)}. Sequences of these PCR primers are listed in Table S2.