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EZH2 Drives Cholangiocarcinogenesis Through Downregulation of Tumor Suppressors Genes via Histone Methylation And microRNAs
Author(s) -
Zhang Jinqiang,
Chen Weina,
Han Chang,
Song Kyoungsub,
Ma Wenbo,
Wu Tong
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02196
Subject(s) - ezh2 , small hairpin rna , biology , gene knockdown , microrna , cancer research , rna interference , epigenetics , downregulation and upregulation , microbiology and biotechnology , cell culture , gene , rna , genetics
EZH2 regulates gene expression through epigenetic machinery. Increased expression of EZH2 is present in cholangiocarcinoma (CCA). Importantly, EZH2 level in CCA is closely correlated with patient prognosis. The objective of this study is to further delineate the effect of EZH2 in CCA. Alb‐CRE/EZH2flox/flox mice were subjected to hydrodynamic tail vein injection of transposase‐based plasmids expressing AKT and Notch intracellular domain (NICD). This model was employed, along with xenograft and CCA cells, to assess the effect and mechanism of EZH2. ChIP‐seq, small RNA‐seq and bioinformatics analyses were performed to identify the targets of EZH2. In CCA induction by HDTV injection of NICD/AKT, we observed that the Alb‐CRE/EZH2 flox/flox mice exhibited significantly decreased tumor burden and prolonged overall survival. These findings reveal an important role of EZH2 in CCA development. In a CCA xenograft model with intrahepatic inoculation of human CCA cell line (CCLP1), we observed that EZH2 knockdown by shRNA significantly decreased CCA progression. Further in vitro studies showed that EZH2 depletion by shRNA significantly reduced the growth and colony formation capability of human CCA cells (CCLP1) and mouse CCA cells. RNA‐seq and RT‐PCR analysis revealed that depletion of EZH2 by shRNA in CCA cells restored the expression of 12 putative tumor suppressor genes (including Osgin1, Pax3, Itga7, Il1rn, Gas1, Cdkn1a, Bbc3, Wnt11, Wt1, Sulf, Sfrp1, and Ptprv). By analyzing the human CCA database GSE76297 (containing 91 CCA and 92 paired non‐tumor tissues), we found that the expression levels of 12 tumor suppressor genes in CCA tissues were decreased by an average of 1.7 ‐folds in comparison to paired non‐tumorous liver tissues. The tumor inhibitory effects of the identified genes were verified in CCA cells with altered EZH2 expression. Furthermore, we observed that intraperitoneal injection of the EZH2 inhibitor, GSK126, decreased CCA tumor burden in mice receiving HDTV injection of NICD/AKT. Treatment of human and mouse CCA cells with GSK126 restored the expression of the 12 tumor suppressor genes. These results indicate that EZH2 downregulates multiple tumor suppressor genes in CCA cells. We next carried out ChIP‐seq (using anti‐H3K9me2 or anit‐EZH2) and small RNA‐seq to determine the mechanisms underlying EZH2‐mediated downregulation of tumor suppressor genes. The ChIP‐seq results showed that both anti‐H3K9me2 and anti‐EZH2 IP led to significant enrichments of the DNA fragments of 5 out of 12 genes. The small RNA‐seq analyses identified a group of EZH2‐regulated microRNAs which target the 12 tumor suppressor genes. Our findings suggest that EZH2 downregulates the expression of tumor suppressor genes either directly through histone methylation or indirectly through specific microRNAs. Taken together, our findings disclose a novel mechanism by which EZH2 drives cholangiocarcinogenesis through downregulation of tumor suppressors genes via histone methylation and microRNAs. Our results provided further evidence supporting EZH2 inhibition for CCA treatment.