Taqman mirna assay system

The levels of U6 and miR-455-3p were tested using the TaqMan miRNA assay system (Life Technologies Corporation, Shanghai, China). To detect mRNA expression, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA). Quantitative real-time PCR was performed using SYBR green (Life Technologies, Grand Island, NY, USA). The expression levels of target genes and the results were normalized against β-actin expression. The primers used in this study were as follows: Has-miR-455-3p, forward: 5′-GCAGTCCATGGGCATATACAC-3′; U6, forward: 5′-CTCGCTTCGGCAGCACA-3′; HSF1, forward: 5′-CCTGGTCAAGCCAGAGAGAG-3′, reverse: 5′-CTGCACCAGTGAGATCAGGA-3′; α-SMA, forward: 5′-GGCTCTGGGCTCTGTAAGG-3′, reverse: 5′-CTCTTGCTCTGGGCTTCAT; C-3′; Collagen-I, forward: 5′-GCTCCTCTTAGGGGCCACT-3′, reverse: 5′-CCACGTCTCACCATT; GGGG-3′; TIMP-1, forward: 5′-GCAACTCGGACCTGGTCATAA-3′, reverse: 5′-CGGCCCGTGAT; GAGAAACT-3′; β-actin, forward: 5′-CTAAGGCCAACCGTGAAAAG-3′, reverse: 5′-ACCAGAGGCATACAGGGACA-3′.