A double-negative feedback loop between EZH2 and miR-26a regulates tumor cell growth in hepatocellular carcinoma.

Accumulating evidence demonstrates the important roles of microRNAs (miRNAs) in tumor development and progression. miR-26a has been reported to be downregulated in several types of cancers including hepatocellular carcinoma, but the underlying mechanism of how miR-26a is repressed remains largely unknown. In the present study, we performed western blot analysis, qRT-PCR, luciferase reporter assay and chromatin immunoprecipitation assay to investigate the relationship between miR-26a and the enhancer of zest homologue 2 (EZH2). CCK-8 assay and colony formation assay were carried out to explore the effect of miR-26a on HCC cells proliferation. We demonstrated that miR-26a was epigenetically repressed by EZH2-mediated H3K27 trimethylation within the miR-26a promoter. Moreover, we confirmed that EZH2 was also a direct target of miR-26a in HCC cells, thus, creating a double-negative feedback loop. Furthermore, miR-26a restoration increased the expressions of its host genes (CTDSPL and CTDSP2). Overexpression of EZH2 abrogated miR-26a induction of CTDSPL and CTDSP2. Restoring the balance of the double-negative feedback loop by miR-26a overpression or EZH2 silence significantly inhibited HCC cell growth. Overexpression of EZH2 rescued the growth inhibition effect of miR-26a. These findings suggest that an imbalanced double-negative feedback loop between EZH2 and miR-26a exists in HCC cells, which contributes to miR-26a deregulation and regulates tumor cells proliferation.

[1]  D. Hu,et al.  MicroRNA-124a is epigenetically regulated and acts as a tumor suppressor by controlling multiple targets in uveal melanoma. , 2013, Investigative ophthalmology & visual science.

[2]  D. Tindall,et al.  Polycomb protein EZH2 suppresses apoptosis by silencing the proapoptotic miR-31 , 2014, Cell Death and Disease.

[3]  Jian-Rong Yang,et al.  MicroRNA-26a/b and their host genes cooperate to inhibit the G1/S transition by activating the pRb protein , 2011, Nucleic acids research.

[4]  Yushun Wan,et al.  MicroRNA-548 down-regulates host antiviral response via direct targeting of IFN-λ1 , 2012, Protein & Cell.

[5]  M. Lerman,et al.  NotI Microarrays: Novel Epigenetic Markers for Early Detection and Prognosis of High Grade Serous Ovarian Cancer , 2012, International journal of molecular sciences.

[6]  G. Wang,et al.  miR-26 a Promoted by Interferon-Alpha Inhibits Hepatocellular Carcinoma Proliferation and Migration by Blocking EZH 2 , 2014 .

[7]  Anke Sparmann,et al.  Polycomb silencers control cell fate, development and cancer , 2006, Nature Reviews Cancer.

[8]  Gösta Winberg,et al.  RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  V. Cicinnati,et al.  MicroRNA-101 inhibits human hepatocellular carcinoma progression through EZH2 downregulation and increased cytostatic drug sensitivity. , 2014, Journal of hepatology.

[10]  R. Jove,et al.  miR-26a enhances miRNA biogenesis by targeting Lin28B and Zcchc11 to suppress tumor growth and metastasis , 2014, Oncogene.

[11]  Pei Ma,et al.  Long non-coding RNA TUG1 is up-regulated in hepatocellular carcinoma and promotes cell growth and apoptosis by epigenetically silencing of KLF2 , 2015, Molecular Cancer.

[12]  M. Lerman,et al.  Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer , 2010, BMC Cancer.

[13]  A. Covey,et al.  Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma , 2012, CA: a cancer journal for clinicians.

[14]  Pierre-Olivier Angrand,et al.  Diverse involvement of EZH2 in cancer epigenetics. , 2015, American journal of translational research.

[15]  Hui Li,et al.  Long non-coding RNA HOTAIR promotes tumor cell invasion and metastasis by recruiting EZH2 and repressing E-cadherin in oral squamous cell carcinoma. , 2015, International journal of oncology.

[16]  C. Kang,et al.  DNMT1 and EZH2 mediated methylation silences the microRNA-200b/a/429 gene and promotes tumor progression. , 2015, Cancer letters.

[17]  M. Rubin,et al.  Genome-wide DNA methylation events in TMPRSS2-ERG fusion-negative prostate cancers implicate an EZH2-dependent mechanism with miR-26a hypermethylation. , 2012, Cancer discovery.

[18]  Swathi Chinaranagari,et al.  EZH2 dependent H3K27me3 is involved in epigenetic silencing of ID4 in prostate cancer , 2014, Oncotarget.

[19]  Lu Wang,et al.  MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. , 2011, Cancer research.

[20]  Z. Yin,et al.  The functional and mechanistic relatedness of EZH2 and menin in hepatocellular carcinoma. , 2014, Journal of hepatology.

[21]  A. D. De Marzo,et al.  Myc Enforces Overexpression of EZH2 in Early Prostatic Neoplasia via Transcriptional and Post-transcriptional Mechanisms , 2011, Oncotarget.

[22]  Epigenetic silencing of microRNA-218 via EZH2-mediated H3K27 trimethylation is involved in malignant transformation of HBE cells induced by cigarette smoke extract , 2016, Archives of Toxicology.

[23]  Jun Yu,et al.  EZH2-mediated concordant repression of Wnt antagonists promotes β-catenin-dependent hepatocarcinogenesis. , 2011, Cancer research.

[24]  Wanggang,et al.  miR-26a Promoted by Interferon-Alpha Inhibits Hepatocellular Carcinoma Proliferation and Migration by Blocking EZH2 , 2015 .

[25]  Kathryn A. O’Donnell,et al.  Therapeutic microRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model , 2009, Cell.

[26]  Tomohiko Ichikawa,et al.  MicroRNA-26a/b directly regulate La-related protein 1 and inhibit cancer cell invasion in prostate cancer. , 2015, International journal of oncology.

[27]  X. Zhao,et al.  Disruption of the MYC-miRNA-EZH2 loop to suppress aggressive B-cell lymphoma survival and clonogenicity , 2013, Leukemia.

[28]  G. Krasnov,et al.  Epigenetic Alterations of Chromosome 3 Revealed by NotI-Microarrays in Clear Cell Renal Cell Carcinoma , 2014, BioMed research international.

[29]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[30]  J. Simon,et al.  Roles of the EZH2 histone methyltransferase in cancer epigenetics. , 2008, Mutation research.

[31]  R. Young,et al.  Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells. , 2009, Molecular cell.

[32]  Li-xiang Xue,et al.  EZH2 Represses Target Genes through H3K27-Dependent and H3K27-Independent Mechanisms in Hepatocellular Carcinoma , 2014, Molecular Cancer Research.

[33]  Jiejie Xu,et al.  EZH2-mediated loss of miR-622 determines CXCR4 activation in hepatocellular carcinoma , 2015, Nature Communications.

[34]  N. Gammoh,et al.  MicroRNA-26a promotes anoikis in human hepatocellular carcinoma cells by targeting alpha5 integrin , 2014, Oncotarget.

[35]  A. Ma,et al.  MicroRNA-26a regulates tumorigenic properties of EZH2 in human lung carcinoma cells. , 2012, Cancer genetics.

[36]  M. Fraga,et al.  Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. , 2007, Cancer research.

[37]  R. Gregory,et al.  MicroRNA biogenesis pathways in cancer , 2015, Nature Reviews Cancer.

[38]  Zhaohui S. Qin,et al.  Coordinated regulation of polycomb group complexes through microRNAs in cancer. , 2011, Cancer cell.

[39]  Wei Zhao,et al.  Honokiol inhibits bladder tumor growth by suppressing EZH2/miR-143 axis , 2015, Oncotarget.

[40]  Yu Yin,et al.  Autoregulatory feedback loop of EZH2/miR-200c/E2F3 as a driving force for prostate cancer development. , 2014, Biochimica et biophysica acta.

[41]  Jing Zhao,et al.  c‐Myc‐mediated epigenetic silencing of MicroRNA‐101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma , 2014, Hepatology.

[42]  Q. Ye,et al.  MicroRNA‐26a suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting interleukin‐6‐Stat3 pathway , 2013, Hepatology.