Lightcycle 96 apparatus

Total RNA was extracted using TRIzol (Beyotime, China) and then converted to cDNA using BeyoRT II First Strand cDNA synthesis kit (RNase H minus). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed using the SYBR Green mix (Qiagen, Shanghai, China) in the LightCycle 96 apparatus (Roche, Basel, Switzerland) (19 (link)). The primers used are shown in Table 1.
The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 72°C for 20 s, and 56°C for 15 s. Data were normalized by the level of GAPDH expression in each of the samples described above. Results were obtained in triplicates.

Total RNA was extracted using Trizol reagent (Invitrogen) following reagent specifications. Total RNA and reverse transcription reagent (Tiangen) were prepared to obtain cDNA. The expression of ZEB1-AS1 and MAP4K4 were calculated by the 2^−ΔΔCt method using SuperReal PreMix Plus kit (Tiangen), and 18S rRNA acted as an internal control. The expression of miR-1224-5p was calculated by the 2^−ΔΔCt method using miRcute miRNA kit (Tiangen), and U6 served as an internal control. qRT-PCR was performed on a LightCycle 96 apparatus (Roche, Basel, Switzerland). The amplification procedure was a three-step process consisting of 40 cycles, and three parallel replicates were performed for all results. The specific qRT-PCR primer sequences were listed in Supplementary Table 1.