Targeting High‐Mobility Group Box2 by miR‐127 Modulates Pluripotency of Mouse Embryonic Stem Cells and Contributes to Aggressiveness of Hepatocellular Carcinoma

Abstract

BackgroundCancer progenitor cells possess some characteristics associated with normal stem cells. MiR‐127 is associated with germ layer differentiation and its promoter is hypermethylated in cancer cells. High‐mobility‐group protein 2 (HMGB2) is highly expressed in embryonic stage and hepatocellular carcinoma (HCC), but low in adult liver. The current study aimed at elucidating a novel regulation of HMGB2 by miR‐127 in mouse stem cells and liver cancer disease.MethodsMouse ESCs (CGR8, Sigma) were maintained on 0.1% gelatin coated dish in Knockout DMEM containing 15% ES‐FBS, 2 mM L‐glutamin, 0.1 mM NEAA, 0.1 mM β‐mercaptoethanol 50 μg/ml penicillin‐streptomycin and 10 ng/ml Leukemia inhibitory factor (LIF). For embryonic body (EB) formation, ESCs were trypsinized into single cells and cultured by seeding 900 ESCs in 30 μl hanging droplets of ES medium devoid of LIF. Liver tumor initiating cells (TICs) spheroid assay: 1000 cells were suspended in serum free TIC medium with 1% methylcellulose supplemented with B27, 20 ng/ml EGF and 20 ng/ml FGF in each well of 96‐well ultralow attachment plate for 7 days. Other methods: luciferase reporter assay, qPCR, Western blots, Co‐immunoprecipitation (Co‐IP), cell invasion, shRNA knockdown and adenovirus overexpression.ResultsThe level of miR‐127 was markedly elevated in CGR8 cells during EB differentiation, which correlated with the significant down‐regulation of HMGB2, OCT4 and SOX2. A conserved seed region for miR‐127 exists in the 3′UTR of human, mouse and rat HMBG2 genes. Ectopic expression of miR‐127 inhibited HMGB2 3′UTR activity in a dose dependent manner, which was abolished by mutation of the miR‐127 seed region and miR‐127 antagomir (anti‐miR‐127). Ectopic expression of miR‐127 reduced HMGB2 protein levels in a variety of human HCC cells. HMGB2 interacted with OCT4 and increased protein levels of OCT4 and SOX2, which further enhanced their transcriptional activity. On the other hand, HMGB2 inhibitor Inflachromene (ICM) induced differentiation of mouse stem cells and alleviated proliferation of TIC cells. Ectopic expression of miR‐127 in mouse stem cell reduced mRNA levels of Oct4, Nanog and Sox2, but increased mesendoderm markers during EB formation. Ectopic expression of miR‐127 in TIC cells lead to smaller spheroid formation, which was rescued by HMGB2. This was associated with decreased RhoA, CDC42, and Rac1/2/3 expression by miR‐127.ConclusionmiR‐127 is a novel regulator of mouse stem cell differentiation and liver cancer progenitor cell proliferation by targeting HMGB2. Our work provides a therapeutic perspective for future translational studies to inhibit HCC metastasis by blocking HMGB2.Support or Funding InformationGrant support: NIH R01DK104656, R01DK080440, R01ES025909, R21AA022482, R21AA024935, VA Merit Award 1I01BX002634.