Quantitative real-time PCR assay
Peripheral blood mononuclear cells were isolated from peripheral blood using density gradient centrifugation. Then, total RNA were extracted and purified from peripheral blood mononuclear cells by TRIzolTM LS reagent. Total RNA was reverse translated as complementary DNA after the degradation of mixed genomic DNA using PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara Bio Inc., Japan). SYBR Green kit (Takara Bio Inc., Japan) was used in quantitative real-time PCR process based on QuantStudioTM 7 Flex (Life Technologies, USA). The expression data of ERAP1 was normalized to internal reference β-actin, and the relative expression level of ERAP1 was calculated by comparative 2-ΔΔCt method. Primers sequence of ERAP1 and β-actin were listed as following: β-actin, forward: 5’-CTCCATCCTGGCCTCGCTGT-, reverse: 5’-GCTGTCACCTTCACCGTTCC-; ERAP1, forward: 5’-TTTGAACTTGGCTCATCTTCC-, reverse: 5’-AATTGTCTGTTGGACACAACG .