High Mobility Group Box-1Promote Hepatocellular Carcinoma Progression Via MiR-21-dependent Pathway

Background&Aims:Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is associated with a very poor prognosis. The discovery of microRNA (miRNA) has significantly altered traditional concepts of gene regulation and miRNAs have emerged as novel modulators of gene expression in cancer. As an oncomir, the over-expression of miR-21has been reported in various cancers. However, the role and regulation of miR-21in HCC are not elucidated. High mobility group box1(HMGB1) is a prototypical Damage-Associated Molecular Pattern molecule (DAMP) with multiple extracellular functions, including modulation of inflammation and immunity. We hypothesize that HMGB1induces the expression of miR-21in HCC to promote tumor progression.Methods:Human HCC cell lines (Huh7, HepG2) and normal human hepatocytes (HHC) were stimulated with varying doses of Human recombinant HMGB1(rHMGB1) or treated with neutralizing antibody to HMGB1. IL6, IL6R and Signal transducer and activator of transcription3(STAT3) inhibitor were used to examine their role in HMGB1-mediated miR-21induction. MRNA expression was quantitated by SYBR Green quantitative reverse transcription-PCR (SYBR Green qRT-PCR) and TaqMan real-time polymerase chain reaction (Taqman qRT-PCR). Protein expressions were assessed by Western blot analysis and immunofluorescent staining. A luciferase reporter assay was conducted to confirm target association. In vivo studies using xenograft tumors in nude mice and in vivo image system (IVIS) were performed to verify our in vitro findings.Results:HMGB1and miR-21, both upregulated in HCC, showed a clear relationship between them in HCC cell lines. The modulation of miR-21by HMGB1is time-and dose-dependent. The induction of IL6, IL6R neutralizing antibodies and STAT3inhibitor demonstrated the important role of them in HMGB1-mediated miR-21induction. We used gain-and loss-of-function methods to evaluate the effect of miR-21in human HCC cell lines (Huh7and HepG2). And a luciferase reporter assay was used to confirm the associations between miR-21and its targets RECK and TIMP3. We further demonstrate that RECK and TIMP3are not only miR-21’s targets but also the down-streams of HMGB1-miR-21pathway and inhibition of miR-21could suppress HCC cells migration and invasion induced by HMGB1. Moreover, a xenograft tumor modle in nude mice was conducted to analyzed the role of miR-21in HMGB1-inducing HCC progression.Conclusion:These results suggest that in Human HCC cells, extracellular HMGB1activates IL6/stat3signaling pathways to induce miR-21expression with the subsequent suppression of multiple tumor suppressors, which, in turn, promote tumor invasion and metastasis. This study provides new insights into the interaction of inflammatory mediators and miRNA regulation of oncogenic pathways in HCC progression.