Research ArticleCancer

The lncRNA H19 mediates breast cancer cell plasticity during EMT and MET plasticity by differentially sponging miR-200b/c and let-7b

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Science Signaling  13 Jun 2017:
Vol. 10, Issue 483, eaak9557
DOI: 10.1126/scisignal.aak9557
  • Fig. 1 H19 was essential for cancer metastasis.

    (A) The classification for pair comparison between cell clones with differently metastatic potency (group N, nonmetastatic clones; group W, weakly metastatic clones; group H, highly metastatic clones). (B) qRT-PCR analysis of H19 abundance in 16 heterogeneous subclones isolated from the primary tumor of one TA2 mice with spontaneous breast cancer and normalized to that in clone 1. (C) qRT-PCR assessment of H19 abundance in metastatic 168FARN, 4TO7, and 4T1 cells and nonmetastatic 67NR cells and normalized to that in mouse mammary epithelial Scp2 cells. (D) qRT-PCR assessment of H19 abundance in TA2-C13 and TA2-C47 cells infected with lentiviral short hairpin RNA (shRNA) control or H19 shRNA and normalized to U6 small nuclear RNA (snRNA). (E) IVIS bioluminescence imaging of mice (n = 3 each) or extracted tissue from syngeneic TA1 mice 4 weeks after orthotopic injection of TA2-C13 and TA2-C47 cells transfected with luciferase and H19 shRNA or control shRNA. (F) The circulating tumor cells (CTCs) and metastatic lesions were calculated from syngeneic TA1 mice 4 weeks after orthotopic injection of TA2-C13 and TA2-C47 cells transfected with luciferase and H19 shRNA or control shRNA. (G) qRT-PCR assessment of H19 abundance of primary tumors from 48 primary breast cancer (PBC) patients and of metastases from 60 metastatic breast cancer (MBC) patients and normalized to U6 snRNA (Sh-Ctr, shRNA control; Sh-H19, H19 shRNA). Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001.

  • Fig. 2 Git2 expression was conditionally targeted by miR-200b/c.

    (A) Schematic outlining the origin of C13-PT, C13-PB, and C13-LM cells. (B) Gene cluster by microarray analysis of C13-PT, C13-PB, and C13-LM cells using the genes encoding GIT2, CYTH3, E-cadherin, N-cadherin, and vimentin. For each gene, the average signal among all cell samples was used as the baseline (onefold). Changes from baseline are represented by color intensity, with up-regulation shown as red and down-regulation shown as green. (C and D) qRT-PCR analysis of miR-200b (C) and miR-200c (D) expression in nonmetastatic TA2-C7 cells, weakly metastatic TA2-C47 cells, and highly metastatic TA2-C13 cells and in TA2-C13–derived cells and normalized to U6 snRNA. (E and F) qRT-PCR assessment of miR-200b (E) or miR-200c (F) abundance in C13-PT, C13-PB, or C13-LM cells transfected with the antagomirs of miR-200b, miR-200c, or their antagomir controls and normalized to U6 snRNA. (G and H) Western blotting analysis of GIT2 in C13-PT, C13-PB, or C13-LM cells transfected with the antagomirs of miR-200b (G), miR-200c (H), or their antagomir controls. Densitometry was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). E-cad, E-cadherin; Vim, vimentin; PT, primary tumor; PB, peripheral blood; LM, lung metastasis. Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001.

  • Fig. 3 H19 sponged miR-200b/c.

    (A) Schematic outlining the Ago2 RIP strategy to validate endogenous miRNA:H19 binding. (B) qRT-PCR assessment of H19 in C13-PT, C13-PB, and C13-LM cells that were pulled down by AGO2 or negative control IgG and normalized to U6 snRNA. (C) qRT-PCR detection of miR-200b/c endogenously associated with H19 in C13-PT, C13-PB, and C13-LM cells that were pulled down by AGO2 or negative control IgG and normalized to U6 snRNA. (D) The luciferase activity assessment in TA2-C7 cells transfected with sensor (miR-200b/c 4×, psiCHECK2-miR-200b/c 4×), together with 0, 20, 40, 80, or 160 ng of sponge plasmid pH19 or pH19mut1. (E) Western blotting detection of GIT2 in C13-PT, C13-PB, and C13-LM cells transfected with #2 H19 small interfering RNA (siRNA) or siRNA control and normalized to GAPDH. (F) Western blotting detection of guanosine 5′-triphosphate (GTP)–bound ARF6 that were pulled down by glutathione S-transferase (GST)–GGA3–protein binding domain (PBD) beads in C13-PB, C13-PT, and C13-LM cells and normalized to total ARF6. (G) Endogenous CYTH3 in C13-PT, C13-PB, and C13-LM cells was detected by Western blotting and quantified relative to GAPDH as a loading control (siCtr, siRNA control; siH19, H19 siRNA). Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001.

  • Fig. 4 H19 sponged let-7b.

    (A) Western blotting detection of CYTH3 protein expression of C13-PT, C13-PB, and C13-LM cells transfected with let-7b antagomir or antagomir control and normalized to GAPDH. (B) qRT-PCR detection of let-7b endogenously associated with H19 in C13-PT, C13-PB, and C13-LM cells that were pulled down by AGO2 or negative control IgG and normalized to U6 snRNA. (C) The luciferase activity assessment in TA2-C7 cells transfected with sensor (let-7b 4×, psiCHECK2-let-7b 4×), together with 0, 20, 40, 80, or 160 ng of sponge plasmid pH19 or pH19mut2. (D) Western blotting detection of CYTH3 in C13-PT, C13-PB, and C13-LM cells transfected with #2 H19 siRNA or siRNA control and normalized to GAPDH. (E) Number of CTCs in syngeneic TA1 mice 4 weeks after orthotopic injection of C13-PT, C13-PB, and C13-LM cells infected by CYTH3 shRNA or control shRNA. (F) Bioluminescence (Biolum), hematoxylin and eosin (H&E) staining, and immunohistochemistry staining with luciferase antibody (Anti-Luc) of lung metastatic lesions of TA1 mice (n = 3) 4 weeks after orthotopic injection of C13-PT cells infected with Git2 shRNA or control shRNA. (G) Quantification of bioluminescence of region of interest (ROI) for lung metastatic lesions of TA1 mice (n = 3) 4 weeks after orthotopic or tail vein injection of C13-PT cells infected with Git2 shRNA or control shRNA. (H) Immunostaining for CTCs in peripheral blood samples from MBC patients. CTCs are defined as CK+/CD45/DAPI+ cells with cytokeratin 8 (CK8) (red), CD45 (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 10 μm. NC, negative control; BF, before filtrating; AF, after filtrating; siCtr, siRNA control; siH19, H19 siRNA. Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001.

  • Fig. 5 ArfGAP GIT2 and ArfGEF CYTH3 are involved in metastatic initiation or colonization.

    (A and B) Western blotting for GIT2 (A) and CYTH3 (B) in the primary breast tumor specimens, peripheral blood samples, and metastatic samples from the same patient of 13 MBC patients. (C and D) A possible mechanism explains the sequential metastatic capability of nonmetastatic TA2-C7, weakly metastatic TA2-C47, and highly metastatic TA2-C13 cells. (E) Schemes describing the mechanism of H19 regulating the step acquirement of the competence to flexibly shift EMT and MET during metastasis.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/483/eaak9557/DC1

    Fig. S1. Git2 transcript is a predicted target of miR-200b/c.

    Fig. S2. Cyth3 transcript is a predicted target of let-7b.

    Fig. S3. The functional effect of let-7 assessed with a targeted antagomir.

    Fig. S4. The role of GIT2 and CYTH3 as analyzed by RIP and in vitro assays.

    Fig. S5. Luciferase assay results.

    Table S1. Metastases resulting from orthotopic injection of cells from distinct clones.

    Table S2. Clinical characteristics of MBC patients.

    Data file S1. Microarray results.

    Data file S2. miRNA and H19 sequences.

    Data file S3. miRNA target information.

    Data file S4. ArfGEF target information.

    Data file S5. siRNA, shRNA, and PCR primer sequences.

  • Supplementary Materials for:

    The lncRNA H19 mediates breast cancer cell plasticity during EMT and MET plasticity by differentially sponging miR-200b/c and let-7b

    Wu Zhou,* Xiao-lei Ye, Jun Xu, Ming-Guo Cao, Zheng-Yu Fang, Ling-Yun Li, Guang-Hui Guan, Qiong Liu, Yue-Hui Qian, Dong Xie*

    *Corresponding author. Email: zhouwu{at}lsu.edu.cn (W.Z.); dxie{at}sibs.ac.cn (D.X.)

    This PDF file includes:

    • Fig. S1. Git2 transcript is a predicted target of miR-200b/c.
    • Fig. S2. Cyth3 transcript is a predicted target of let-7b.
    • Fig. S3. The functional effect of let-7 assessed with a targeted antagomir.
    • Fig. S4. The role of GIT2 and CYTH3 as analyzed by RIP and in vitro assays.
    • Fig. S5. Luciferase assay results.
    • Table S1. Metastases resulting from orthotopic injection of cells from distinct clones.
    • Table S2. Clinical characteristics of MBC patients.
    • Legend for data file S1
    • Data file S2. miRNA and H19 sequences.
    • Data file S3. miRNA target information.
    • Data file S4. ArfGEF target information.
    • Data file S5. siRNA, shRNA, and PCR primer sequences.

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    Other Supplementary Material for this manuscript includes the following:


    Citation: W. Zhou, X.-l. Ye, J. Xu, M.-G. Cao, Z.-Y. Fang, L.-Y. Li, G.-H. Guan, Q. Liu, Y.-H. Qian, D. Xie, The lncRNA H19 mediates breast cancer cell plasticity during EMT and MET plasticity by differentially sponging miR-200b/c and let-7b. Sci. Signal. 10, eaak9557 (2017).

    © 2017 American Association for the Advancement of Science

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