Cytokines and hepatocellular carcinoma: Potential progression markers and cell typespecific modulators of the mirnome (Interleukin-6 and JAK/STAT activating cytokines)

HCC is the fifth most common and the third deadliest cancer worldwide. There is an urgent need to find sensitive markers for early diagnosis and to monitor postoperative recurrence in order to provide treatment for HCC. Cytokines play an important role in liver physiology, i.e., IL-6-type cytokines as mediators of the acute phase response. In addition, they contribute to cancer development and progression. Thus, HCC-associated cytokines as well as miRNAs are candidates for diagnostic and prognostic biomarkers of HCC.

Methods: So far, serum samples of 8 healthy controls, 23 nonalcoholic steatohepatitis (NASH) patients and 18 HCC patients have been analyzed regarding the levels of up to 48 cytokines with the Luminex immunoassay technology (Bio-Plex 2200, Bio-Rad). The effect of four cytokines: Hyper-IL-6 (H-IL-6), OSM, IFN-γ, and IL-27 on the miRNome and on the transcriptome of nine healthy and cancerous cell lines derived from human liver, colon and skin cells (PH5CH8, Hep3B, Huh-7; NCM460, HCT15, HCT116; NHEM, A375, MelJuso) has been analyzed by microarray (Affymetrix GeneChip miRNA Array 3.0, Human Transcriptome Array 2.0) and validated by qPCR.

Results: In a first step, we analyzed and correlated the expression levels of inflammatory cytokines in HCC and NASH patients, being at high risk to develop HCC. Herein, serum levels of IL-6 and of HGF were higher in HCC patients than in healthy controls and in advanced NASH patients, as confirmed by Bio-Plex cytokine immunoassays. Next, we wanted to study a possible correlation between cytokine levels and the presence of the PNPLA3 p. 148 M risk variant in patients with NASH, thereby contributing to biomarker research in liver diseases.

Next, we have studied the effect of IL-6 on the miRNome in hepatoma cell lines and primary hepatocytes. Surprisingly, IL-6 caused thousands of mRNAs to be differentially regulated in HepG2 and HuH-7 hepatoma cell lines, whereas levels of only few miRNAs were significantly changed (FDR < 0.05, logFC 0.5). In contrast to HCC cell lines, a stronger response of the miRNome following IL-6 stimulation was observed in primary hepatocytes (68 and 27 differentially expressed miRNAs in two samples, respectively). To further address this possible cell type and tissue specificity, we tested the effect of STAT-1 or STAT-3- activating cytokines (IL-27, IFN-γ, OSM and H-IL-6) on both the miRNome and the mRNA transcriptome of healthy and cancerous cell lines, derived from human liver, colon and skin cells. Moreover, the regulation and roles of selected IL-6/Jak/STAT3-related miRNAs with possible relevance for HCC are currently further analyzed.

Conclusions: Our work may on one hand further help to correlate cytokine levels and the presence of a risk variant of pnpla3 in NASH patients, thereby contributing to biomarker research in liver diseases. On the other hand, we could participate to improve the knowledge on the question of the effect of cytokine stimulation on the miRNome and the transcriptome, in various cells and tissues cancerous or not.

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Lammert, F.

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