Cell Lines, Clinical Samples Collection, RNA Extraction, and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

The human KIRC cell lines,786-O, caki-1, and human kidney cell (HK-2 cell, proximal tubule epithelial cell) were originally purchased from cell repository of Shanghai Institute of Life Sciences, Chinese Academy of Sciences. RPMI 1640 medium, containing 10% fetal bovine serum (FBS), penicillin (25 U/ml), and streptomycin (25 mg/ml), was used to culture these KIRC cells at 37°C in a humidified 5% CO2 environment. In addition, a total of 25 fresh samples from patients who underwent laparoscopic radical or partial nephrectomy for KIRC were collected in Southeast University Zhongda Hospital from 2019 to 2020, including tumor tissue and matched adjacent normal kidney tissue and stored at −80°C. All patients were diagnosed with KIRC and did not undergo any antitumor therapy before surgery. The research was authorized by the Medical Ethics Committee of the Southeast University Zhongda Hospital (ZDKYSB077), and each patient gave informed consent.

Total RNA was isolated from KIRC cells and clinical tissues using Total RNA Kit I (50) (OMEGAbiotec, China). Then cDNA was synthesized using the HiScript II Q RT SuperMix (R223-01) reagent kit (vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was performed using the SYBR green PCR mix (vazyme, Nanjing, China) according to the manufacturer’s instructions. The 2^−ΔΔCT calculation method (21, 22), a relative quantification to calculate the proportion of transcripts in a sample, was applied to determine the relative expression levels of the five m6A-related lncRNAs in the prognostic signature. It described the expression levels of the target genes relative to the reference genes. The detailed calculation method of ΔΔCT was as follows: ΔΔCT= (CT_lncRNA -CT_GAPDH) _sample- (CT_lncRNA -CT_GAPDH) _control (The control group in this study was HK-2 cell or normal kidney tissue). GAPDH was employed as the endogenous control. The final results obtained from the 2^−ΔΔCT calculation were the relative expression of the target genes. The primer sequences used in the present study were listed in Table S1 .