In the FUSCC cohort, total RNA of tumor tissue was isolated from all 123 patients' samples using TRIzol reagent (15596-026, Invitrogen). A PrimeScript RT reagent kit (K1622, Thermo Scientific) was used to synthesize first-strand cDNA from total RNA. After that, we performed SYBR Green real-time PCR analysis using ABI 7900HT machine (Applied Biosystems, USA). We used β-actin mRNA expression level as a reference. NR1H3 and NR2F6 mRNA expression level was normalized to β-actin 21 (link). The primers for qRT-PCR analysis were synthesized by Sangon (Shanghai, P.R. China), sequences of which are shown as following:
NR1H3: Forward primer: 5'-CCTTCAGAACCCACAGAGATCC-3'; Reverse primer: 5'-ACGCTGCATAGCTCGTTCC-3'
NR2F6: Forward primer: 5'-GAGCGGCAAGCATTACGGT-3'; Reverse primer: 5'-GGCAGGTGTAGCTGAGGTT-3'
β-actin: Forward primer: 5'-CATGTACGTTGCTATCCAGGC-3'; Reverse primer: 5'-CTCCTTAATGTCACGCACGAT-3'
NR1H3: Forward primer: 5'-CCTTCAGAACCCACAGAGATCC-3'; Reverse primer: 5'-ACGCTGCATAGCTCGTTCC-3'
NR2F6: Forward primer: 5'-GAGCGGCAAGCATTACGGT-3'; Reverse primer: 5'-GGCAGGTGTAGCTGAGGTT-3'
β-actin: Forward primer: 5'-CATGTACGTTGCTATCCAGGC-3'; Reverse primer: 5'-CTCCTTAATGTCACGCACGAT-3'
