Total RNA was extracted from cHCECs by use of the miRNeasy Mini Kit (QIAGEN, Hilden, Germany). The cDNA was synthesized by use of the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). Polymerase chain reaction was performed via TaqMan Fast Advanced Master Mix (Applied Biosystems) and TaqMan Gene Expression Assays (inventoried) (Applied Biosystems) under the following conditions: activation of enzyme at 95°C for 20 seconds, 40 cycles of denaturation at 95°C for 1 second, and annealing/elongation at 60°C for 20 seconds. The StepOnePlus Real-Time PCR system (Applied Biosystems) was used for PCR amplification and analysis.
The levels of MMP1, MMP2, MMP4, TIMP1, BMP2, SPARC, TGFβ1, TGFβ2, FN1, SERPINB2, CD44, CD166, CD105, CD24, Col3A1, Col4A1, Col4A2, Col8A2, CDH2, VIM, CDKN1B, CDKN1C, IGFBP3, SNAIL1, SNAIL2, and IL1B were normalized to that of 18S rRNA. The results were presented as ΔΔCt (relative units of expression).