EED regulates epithelial-mesenchymal transition of cancer cells induced by TGF-β

13301甲第4285号博士（医学）金沢大学博士論文本文Full 以下に掲載：Biochemical and Biophysical Research Communications 453(1) pp.124-130 2014.Elsevier. 共著者：Dulamsuren Oktyabri, Shoichiro Tange, Minoru Terashima, Akihiko Ishimura, Takeshi Suzuk

Poster Session: Abstracts

The 3rd International Symposium on Carcinogenic Spiral & International Symposium on Tumor Biology in Kanazawa, [DATE]: January 24(Thu)-25(Fri),2013, [Place]:Kanazawa Excel Hotel Tpkyu, Kanazawa, Japan, [Organizers]:Infection/Inflammation-Assisted Acceleration of the Carcinogenic Spiral and its Alteration through Vector Conversion of the Host Response to Tumors / Scientific Research on Innovative Areas, a MEXT Grant-in Aid Projec

Knockdown of <i>JARID2</i> antagonized TGF-ß-induced morphological changes of HT29 cells.

<p>(A) Cell morphological changes of HT29 cells after TGF-ß treatment. HT29 cells were infected with retroviruses expressing control shRNA or <i>JARID2</i> shRNA (described as <i>JARID2</i> KD) without or with the treatment of 5 ng/ml of TGF-ß for 72 hours. (B) Immunofluorescence images of cells showing the localization of E-cadherin. The panels of HT29 cells with the same arrangement with (A) were stained with anti-E-cadherin antibody and with DAPI. (C) Fluorescence images of cells showing reorganization of actin cytoskeleton by staining with TRITC-phalloidin (Actin) and with DAPI. Scale bars: 20 µm.</p

Over-expression of <i>JARID2</i> enhanced the TGF-ß-induced repression of <i>CDH1</i>, <i>miR-200a</i> and <i>miR-200c</i> through the regulation of histone H3 methylation in A549 cells.

<p>ChIP analyses of H3K27me3, H3K4me3, EZH2 and FLAG-tagged JARID2 on the regulatory regions of <i>CDH1</i> (A), <i>miR-200b/200a/429</i> (B) and <i>miR-200c/141</i> genes (C) in A549 cells are shown. The occupancies of methylated histones, EZH2 or FLAG-JARID2 protein on the regions were analyzed by quantitative PCR (*, <i>P</i><0.01 comparing to control; **, <i>P</i><0.05 comparing to control; ***, <i>P</i><0.05 comparing to control plus TGF-ß; ****, <i>P</i><0.01 comparing to control plus TGF-ß).</p

JARID2 Is Involved in Transforming Growth Factor-Beta-Induced Epithelial-Mesenchymal Transition of Lung and Colon Cancer Cell Lines

<div><p>Histone methylation plays a crucial role in various biological and pathological processes including cancer development. In this study, we discovered that JARID2, an interacting component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in Transforming Growth Factor-beta (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 lung cancer cell line and HT29 colon cancer cell line. The expression of <i>JARID2</i> was increased during TGF-ß-induced EMT of these cell lines and knockdown of <i>JARID2</i> inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. <i>JARID2</i> knockdown itself had no effect in the expression of EMT-related genes but antagonized TGF-ß-dependent expression changes of EMT-related genes such as <i>CDH1</i>, <i>ZEB</i> family and <i>microRNA-200</i> family. Chromatin immunoprecipitation assays showed that JARID2 was implicated in TGF-ß-induced transcriptional repression of <i>CDH1</i> and <i>microRNA-200</i> family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of JARID2 protein, which may control PRC2 recruitment and histone methylation during TGF-ß-induced EMT of lung and colon cancer cell lines.</p></div

The expression of <i>JARID</i> genes during TGF-ß-induced EMT of HT29 colon cancer cells.

<p>QRT-PCR analysis was performed to detect the expression of <i>JARID1A</i> (A), <i>JARID1B</i> (B), <i>JARID1C</i> (C) and <i>JARID2</i> (D) in HT29 cells before and after the treatment of 5 ng/ml of TGF-ß (12 h, 24 h, 48 h and 72 h) (*, <i>P</i><0.01 comparing to control; **, <i>P</i><0.05 comparing to control). (E) Western blot was performed to detect the expression of JARID2 proteins during TGF-ß-induced EMT. As a control, anti-GAPDH antibody was used.</p

Knockdown of <i>JARID2</i> antagonized TGF-ß-induced morphological changes of A549 cells.

<p>(A) Cell morphological changes of A549 cells after TGF-ß treatment. A549 cells were infected with retroviruses expressing control shRNA or <i>JARID2</i> shRNA (described as <i>JARID2</i> KD) without or with the treatment of 1 ng/ml of TGF-ß for 48 hours. (B) Immunofluorescence images of cells showing the localization of E-cadherin. The panels of A549 cells with the same arrangement with (A) were stained with anti-E-cadherin antibody and with DAPI. (C) Fluorescence images of cells showing reorganization of actin cytoskeleton by staining with TRITC-phalloidin (Actin) and with DAPI. Scale bars: 20 µm.</p

Knockdown of <i>JARID2</i> affected the TGF-ß-dependent changes in expression of EMT-related genes in A549 cells.

<p>QRT-PCR analysis was performed to detect the expression of <i>CDH1/E-cadherin</i> (A), <i>FN1/Fibronectin</i> (B), <i>Vimentin</i> (C), <i>ZEB1</i> (D), <i>ZEB2</i> (E), <i>miR-200a</i> (F), <i>miR-200c</i> (G) and <i>JARID1B</i> (H) in A549 cells infected with retroviruses expressing control shRNA or <i>JARID2</i> shRNA with or without the treatment of 1 ng/ml of TGF-ß for 24 hours (*, <i>P</i><0.01 comparing to control). (I) Western blot was performed to detect the expression of E-cadherin, Fibronectin, Vimentin, ZEB1, ZEB2, phosphorylated SMAD3 (P-SMAD3) and GAPDH proteins using the corresponding antibodies.</p

Over-expression of <i>JARID2</i> enhanced the TGF-ß-dependent changes in expression of EMT-related genes in A549 cells.

<p>QRT-PCR analysis was performed to detect the expression of <i>CDH1/E-cadherin</i> (A), <i>FN1/Fibronectin</i> (B), <i>Vimentin</i> (C), <i>ZEB1</i> (D), <i>ZEB2</i> (E), <i>miR-200a</i> (F) and <i>miR-200c</i> (G) in A549 cells infected with the control retrovirus or the retrovirus expressing <i>JARID2</i> with or without treatment of TGF-ß for 24 hours (*, <i>P</i><0.01 comparing to control; **, <i>P</i><0.05 comparing to control; ***, <i>P</i><0.05 comparing to control plus TGF-ß). (H) Western blot was performed to detect the expression of E-cadherin, Fibronectin, Vimentin, ZEB1, ZEB2 and GAPDH proteins using the corresponding antibodies.</p