qRT-PCR

Total RNA from the cells was prepared by TRIzol reagent (Invitrogen, Gaithersburg, MD, USA) and quantified by UV-Vis spectroscopy. Then, 2 μg of total RNA was subjected to reverse transcription, and 1 μL of the product was used for PCR to detect specific gene mRNA level. GAPDH was used as the internal reference gene. For miRNA quantification, the Taqman advanced miRNA cDNA synthesis kit was used to reverse transcription according to the manufacturer’s protocols (Applied Biosystems, Carlsbad, CA, USA). Taqman microRNA reverse transcription kit was performed for U6 small nuclear RNA (snRNA) reverse transcription following manufacturer’s protocol (Applied Biosystem).

All probes were purchased from Thermofisher Scientific. The comparative 2^−ΔΔCT method was used to analyze the relative expression of the target genes and miR-26b.¹⁹