Isolation of total RNA and quantitative real-time PCR
Total RNA was extracted by TRIzol TM Reagent (Ambion, Texas, USA). Concentration and purity of RNA were determined using NanoDrop Microvolume spectrophotometer (Thermo ScientificTM, USA). For miRNA analysis, 500 ng of total RNA was performed with miRNA 1st Strand cDNA Synthesis Kits (by stem-loop) (Vazyme, Nanjing, P.R. China) to generate cDNA. For other mRNA analysis, cDNA was generated by reverse transcription kit HiScript Q RT SuperMix (Vazyme, Nanjing, P.R. China). Quantification of mRNA was done by by real-time PCR performed on resulting cDNA via miRNA Universal SYBR qPCR Master Mix or ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and an ABI 7500 thermocycler (Applied Biosystems Life Technologies, Foster City, CA, USA). The housekeeping gene U6 and GAPDH were used as standards for the normalization of mRNA expression values. The primers (Sangon Biotech Co., Ltd., Shanghai, P.R. China) used in this study were listed inTable 2 :