IL-35 is highly expressed in peripheral blood and tumor tissues of HCC patients
The transcriptome dataset of GSE14520 has 249 HCC tumor and 239 non-tumor samples. We detected IL-35, represented by the mean expression of subunits IL-12p35 and Ebi3, in the HCC tumor and paired non-tumor samples. The results showed that level of IL-35 expression in tumor tissues were significantly higher than paired non-tumor tissues (P < 0.0001, Fig.1A). A similar trend was detected in the IL-35R gene expression examination, which was calculated by the mean expression of IL-12Rβ2 and gp130 (P < 0.0001, Fig.1B). To further validate the bioinformatics analysis results, we detected the level of serum IL-35 expression by AimPlex bead-based immunoassays and performed IHC analyses of IL-35 by the level of p35 and Ebi3 co-expression. Our results showed that the level of serum IL-35 expression was significantly higher in HCC patients compared with health control volunteers (HC) (P=0.0006, Fig.1C). IHC staining results showed that p35 and Ebi3 co-expressed in the hepatic cytoplasm and both subunits were highly expressed in the tumor tissues compared with the paired non-tumor samples (Fig.2A). The same results were observed through western blot analysis (Fig.2B and 2C).