RT-qPCR

Total RNA content was extracted from the cells and tissue samples with the help of TRIzol reagents (Invitrogen Inc., Carlsbad, CA, USA). The RNA mass and concentration were detected using UV-vis spectrophotometry (ND-1000, Nanodrop Technologies Inc., Wilmington, USA). For miRNA, complementary DNA (cDNA) was obtained using miRNA First Strand cDNA Synthesis (Tailing Reaction) kits (B532453-0020, Sangon Biotech, Shanghai, China), and for mRNA, cDNA was obtained with reverse transcription kits (RR047A, Takara Bio Inc., Shiga, Japan). The fluorescence quantitative PCR was subsequently performed using cDNA as a template with reference to SYBR® Premix Ex Taq^TM II (perfect real time) kit (DRR081, Takara) instructions. The RT-qPCR reaction was carried out using a real-time fluorescence quantitative PCR instrument (ABI 7500, Applied Biosystems, Foster City, CA, USA) instrument. U6 and GAPDH mRNA levels were normalized as the internal parameters for the results. The primers are shown in Table 1, and 2-ΔΔCt represents the doubling relationship between the target gene expression of the experimental group and the control group.

Primer sequences used for RT-qPCR