Superscript 3 rt

Using TRIzol reagent (Vazyme, Nanjing, China), total RNA was extracted from liver cancer cell lines according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA by superscript III RT (Vazyme, Nanjing, China) and random primers. Then, quantitative PCR with SYBR PreMix Ex Taq (Vazyme, Nanjing, China) was carried out by using an iQ5 quantitative RT–qPCR detection system (Bio-Rad, Richmond, CA, USA). The relative expression of genes was analyzed by the 2^−ΔΔCT method. The primer sequence was as follows: NAP1L5 forward primer ATGGCCGACTCGGAAAACC; reverse primer TAGGCAGGCTCTCGATAAAGT; GAPDH forward primer GGAGCGAGATCCCTCCAAAAT; reverse primer GGCTGTTGTCATACTTCTCATGG.

Cervical spinal cord, the skin of neck and HaCat cells were collected in RNase-free condition sand isolated total RNAs using TRIzol reagent (Sigma-Aldrich). RNA (1 mg) was reverse transcribed for each sample using SuperScript III RT (Vazyme). The sequences of the forward and reverse primers for:

IL-6: Forward: TCCATCCAGTTGCCTTCTTGG; Reverse: CCACGATTTCCCAGAGAACATG;

IL-17α: Forward: AGTGTTTCCTCTACCCAGCAC; Reverse: GCCACTGCCTCGTATTGAGT;

IL-33: Forward: TCCTTGCTTGGCAGTATCCA; Reverse: TGCTCAATGTGTCAACAGACG;

IL-1β: Forward: TGTCTTGGCCGAGGACTAAG; Reverse: TGGGCTGGACTGTTTCTAATG;

Gapdh: Forward: TCCATGACAACTTTGGCATTG; Reverse: CAGTCTTCTGGGTGGCAGTGA;

The qPCR analysis was performed in the StepOnePlus real-time PCR system by SYBR green I dye detection (Vazyme). The final volume of the quantitative PCR amplification reaction is 10 μl, The thermal cycling conditions include pre-denaturation at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 10 s, and annealing and extension at 60 °C for 30 s. Gapdh is used as an endogenous control to standardize the difference. The melting curve is constructed after the cycle to ensure that no non-specific products are present. Quantification was performed by using the normalized Ct (cycle threshold) value of Gapdh Ct and analysis using the 2^−ΔΔCt method.