Real time system

Total RNA was extracted using the Total RNA Miniprep kit (Axygen, China) according to the manufacturer's instruction. First‐strand cDNA and the miRNA first‐strand cDNA were synthesized using the ReverTra Ace qPCR‐RT kit (Toyobo, Japan) and miRNA 1st Strand cDNA Synthesis Kit (by stem‐loop) (Vazyme, https://www.vazyme.com/Home.html) according to the user's manual respectively. Quantitative real‐time PCR (qRT‐PCR) analyses were performed using SYBR Premix Ex Taq (Takara, Japan) and gene‐specific primers (Table S5) on a CFX96TM Real‐Time System with snRNA U6 or Ubiquitin (UBQ) as an internal control.

Total RNAs from both clinical tissues and cultured cells were extracted by a QuickPrep mRNA Purification Kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s protocols. The first-strand cDNA was produced by a synthesis kit (TaKaRa, Dalian, P.R. China) for reverse transcriptions. Real-time PCR was performed with the SYBR Premix Ex Taq Kit (TaKaRa) in an ABI PRISM 7900 Real-Time System. The protocol is briefly listed here: denaturation (95°C for 5 min), annealing (35 repetition of 95°C for 30 s, 60°C for 45 s, and 72°C for 60 s), and extension (72°C for 10 min). The primers were synthesized by JieLi Co. (Shanghai, P.R. China). TRIM59: 5′-TACGAGAGCAGCAGCTTGAA-3′ (forward) and 5′-ACGGGTTGAACCTCAGGAAG-3′ (reverse). GAPDH: 5′-GTGGACATCCGCAAAGAC-3′ (forward) and 5′-AAAGGGTGTAACGCAACTA-3′ (reverse). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. All experiments were performed at least three times in triplicate.