supplementary materials for€¦ · fig. s2. cyth3 transcript is a predicted target of let-7b....
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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: [email protected] (W.Z.); [email protected] (D.X.)
Published 13 June 2017, Sci. Signal. 10, eaak9557 (2017)
DOI: 10.1126/scisignal.aak9557
The 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.
Other Supplementary Material for this manuscript includes the following:
(available at www.sciencesignaling.org/cgi/content/full/10/483/eaak9557/DC1)
Data file S1 (Microsoft Excel format). Microarray results.
www.sciencesignaling.org/cgi/content/full/10/483/eaak9557/DC1
Fig. S1. Git2 transcript is a predicted target of miR-200b/c. (A and B) Western
blotting and quantification assessing the abundance of epithelial and mesenchymal
markers in C13-PT, C13-PB and C13-LM cells. (C) The endogenous protein level of
GIT2 in C13-PT, C13-PB and C13-LM cells was detected by Western blotting. (D)
The miRNA target prediction tool revealed that mouse Git2 transcript was a predicted
target of miR-200b/c. (E) Conserved miR-200b/c target sites in Git2. PT, primary
tumor; PB, peripheral blood; LM, lung metastasis; E-cad, E-cadherin; N-cad, N-
cadherin; Vim, Vimentin. Data are means ± SD from 3 experiments; blots are
representative. *P<0.05, **P<0.01, ***P<0.001, by Student’s t-test.
Fig. S2. Cyth3 transcript is a predicted target of let-7b. (A)The H19 LncRNA of
nonmetastatic TA2-C7 cells, weakly metastatic TA2-C47 cells, highly metastatic
TA2-C13 cells and TA2-C13-derived sublines was analyzed by qRT-PCR, and
normalized by TA2-C7 cells. (B. The Ago2 antibody precipitated the Ago2 protein
from cell lysates from C13-PT, C13-PB and C13-LM cells, but the control non-
immune IgG did not. The input lane indicates lysate samples used as a positive
control for Ago2. (C and D) Knockdown efficiency of three H19-targeted siRNAs in
TA2-C13 cells, analyzed by Western blotting (C) and quantified from RT-PCR assays
(D). Ctr, Control; Si, siRNA. (E and F) Targetscan results for predicted miRNA
binding sites in the mouse Cyth3 transcript. Red arrows and frames indicate the
predicted eraction region and conserved target site sequences for let-7 miRNA. Data
are means ± SD from 3 experiments; blots are representative. *P<0.05, **P<0.01,
***P<0.001, by Student’s t-test.
.
Fig. S3. The functional effect of let-7 assessed with a targeted antagomir. (A) let-7
miRNA expression in non-metastatic TA2-C7 cells, weakly metastatic TA2-C47 cells,
highly metastatic TA2-C55 and TA2-C13 cells, and TA2-C13-derived sublines was
analyzed by qRT-PCR and normalized to U6 snRNA. (B) Abundance of let-7
isoforms in TA2-C13 cells transfected with an antagomir of let-7, assessed with qRT-
PCR. (C) Quantified results of Western blotting for CYTH3, in C13-PT, C13-PB or
C13-LM cells transfected with a control or let-7 antagomir. (D)
Immunohistochemistry staining for E-cadherin in the indicated cell line-derived
primary tumors. Negative control is stained without E-cadherin secondary antibody.
Tumors were each grown in 3 mice. (E and F) Quantified results of Western blotting
for GIT2 (E) and CYTH3 (F) in the indicated cell lines or cell line-derived primary
tumors (“-PT”). GAPDH served as a loading control. Data are means ± SD from 3
experiments; blots are representative. ***P<0.001; *, not significant; analyzed by
Student’s t-test.
Fig. S4. The role of GIT2 and CYTH3 as analyzed by RIP and in vitro assays. (A
to D) Western blotting assessment of the effect of GIT2 knockdown (A and B) or
CYTH3 knockdown (C and D) in the indicated cell lines on the amount of GTP-bound
ARF6 (A, assessed by pulled down with immobilized GST-GGA3-PBD beads) and E-
cadherin (B). (E and F) Western blotting assessment of the effect of miR-200b/c
antagomir (E) or let-7b antagomir (F) on the abundance of E-cadherin in the indicated
cells. (G and H) Western blotting assessment of the effect of H19 knockdown (with
each of three targeted siRNAs: #1, #2 and #3) on the abundance of E-cadherin (G)
and vimentin (H) in C13-PT, C13-PB or C13-LM cells. GAPDH served as a loading
control. (I to L) Cell invasion in cells transfected with GIT2 shRNA (I and K) or
CYTH3 shRNA (J and L), as assessed by Transwell invasion assays. (M to R)
Proliferation and colony forming ability in cells transfected with GIT2 shRNA (M, O
and Q) or CYTH3 shRNA (N, P and R), as assessed by BrdU incorporation and soft
agar assays, respectively. Data are means ± SD from 3 experiments; blots are
representative. N.S, not significant; *P<0.05, **P<0.01, analyzed by Student’s t-test.
Fig. S5. Luciferase assay results. (A) Activity of Git2 pMirTarget luciferase reporter
plasmids in TA2-C7 cells co-transfected with miR-200b/c mimics, miR-200b/c
mimics and full-length H19 plasmids (pH19), or miR-200b/c mimics and a miR-
200b/c binding site-mutant H19 plasmids (pH19mut1) were. (B and C) Abundance of
miR-200b/c (B) or let-7b (C) in C13-PT, C13-PB and C13-LM cells transfected with
H19 siRNA (#2 siRNA) or a control. (D to G) Relative amount of GTP-bound ARF6
(D and F; assessed by GST-GGA3 pulls down analysis) or E-cadherin (E and G) in
C13-PT, C13-PB and C13-LM cells transfected with Git2 cDNA mutated at miR-
200b/c binding sites (D and E), Cyth3cDNA mutated at let-7b binding sites (F and G),
or the respective control plasmids. Data are means ± SD (n=3); N.S, not significant;
**P<0.01, ***P<0.001.
Table S1. Metastases resulting from orthotopic injection of cells from distinct
clones. The numbers of circulating tumor cells, metastatic lesions of TA1 mice 4
weeks after orthotopic injection of cells isolated from single mouse mammary tumor
that arose spontaneously without chemical stimulus in a wild-type TA2 mouse.
Clone ID Cells in blood Pulmonary tumor lesions Extra-pulmonary tumor lesions
Clone 1 0 0 0
Clone 3 0 0 0
Clone 7 0 0 0
Clone 13 13.71±7.29 3.12±1.37 10.31±2.40
Clone 18 11.34±7.41 0 0
Clone 22 0 0 0
Clone 41 12.64±6.38 0 0
Clone 44 0 0 0
Clone 47 14.92±8.23 0 0
Clone 51 12.09±7.21 2.65±2.35 9.36±2.45
Clone 53 0 0 0
Clone 55 11.76±9.28 3.73±1.86 11.37±1.97
Clone 60 0 0 0
Clone 63 0 0 0
Clone 73 0 0 0
Clone 82 10.56±9.23 0 0
Table S2. Clinical characteristics of MBC patients. The clinical stages and
subtypes of 60 patients with metastatic breast cancer were evaluated by TNM system
and pathologic feature. IDC, invasive ductal carcinoma; ILC, invasive lobular
carcinoma; ER, estrogen receptor; PR, progesterone receptor; HER, human epidermal
growth factor receptor.
T T1 21(35.0%)
T2-T4 39(65.0%)
N N1 19(31.7%)
N2-N3 41(68.3%)
Histology IDC 47(78.3%)
ILC 11(18.3%)
other 2(3.3%)
Grading G1 2(3.3%)
G2 26(43.3%)
G3 32(53.3%)
ER ER- 18(30%)
ER+ 42(70%)
PR PR- 21(35.0%)
PR+ 39(65.0%)
HER HER2- 48(80%)
HER2+ 12(20%)
Data file S1. Microarray results. The raw microarray data is available on Dryad.
Here, the normalized microarray data were further sorted by pair comparison between
non-metastatic clones (Group N), weakly metastatic clones (Group W) and highly
metastatic clones (Group H). Data are provided in an Excel (.xls) file in the auxiliary
files online.
Data file S2. miRNA and H19 sequences. Online searching for the mouse miR-
200b/c, Let-7b and H19 sequences that were presented 5’-3’.
>mmu-miR-200b-3p MIMAT0000233: UAAUACUGCCUGGUAAUGAUGA
>mmu-miR-200c-3p MIMAT0000657: UAAUACUGCCGGGUAAUGAUGGA
>mmu-let-7b-5p MIMAT0000522: UGAGGUAGUAGGUUGUGUGGUU
>mmu-H19, Accession ID AK145379:
GGTTGGGGGGTATCGGGGAAACTGGGGAAGATGGGAGAGCTGGAGGAGAGTCGTGGGGT
CCGAGGAGCACCTCGGCATCTGGAGTCTGGCAGGAATGTTGAAGGACTGAGGGGCTAGCT
CAGGCAGAGCAAAGGCATCGCAAAGGCTGGAAAACATCGGAGTGAAGCTGAAGGGCCTG
AGCTAGGGTTGGAGAGGAATGGGGAGCCAGACATTCATCCCGGTTACTTTTGGTTACAGG
ACGTGGCGGCTGGTCGGATAAAGGGGAGCTGCTGGGAAGGGTTCGACCCCAGACCTGGG
CAGTGAAGGTATAGCTGGCAGCAGTGGGCAGGTGAGGACCGCCGTCTGCTGGGCAGGTG
AGTCTCCTTCTTCTCTCTTGGCCTCGCTCCACTGACCTTCTAAACGAAGGTTTAGAGAGGG
GGCCTGGTGAGAAGAAGCGGCTGGCCTCGCAGCAGAATGGCACATAGAAAGGCAGGATA
GTTAGCAAAGGAGACATCGTCTCGGGGGGAGCCGAGACAGAAGGAGGCTGGGGGACCAT
TGGCGACCCCAGGTGGAAAGAGCTCTTAGAGAGAAGAAAGAAGAGGTGCAGGGTTGCCA
GTAAAGACTGAGGCCGCTGCCTCCAGGGAGGTGATAGGAGTCCTTGGAGACAGTGGCAG
AGACCATGGGATCCAGCAAGAACAGAAGCATTCTAGGCTGGGGTCAAACAGGGCAAGAT
GGGGTCACAAGACACAGATGGGTCCCCAGCCGCCACAACATCCCACCCACCGTAATTCAC
TTAGAAGAAGGTTCAAGAGTGGCTCTGGCAAAGTCCCAAGTTTGCCAGAGCCTCAATAAC
TGGAGAATGGAAAAGAAGGGCAGTGCAGGGTGTCACCAGAAGGGGAGTGGGGGCTGCAG
GTATCGGACTCCAGAGGGATTTTACAGCAAGGAGGCTGCAGTGGGTCCAGCCTGCAGACA
CACCATTCCCATGAGGCACTGCGGCCCAGGGACTGGTGCGGAAAGGGCCCACAGTGGACT
TGGTACACTGTATGCCCTAACCGCTCAGTCCCTGGGTCTGGCATGACAGACAGAACATTTC
CAGGGGAGTCAAGGGCACAGGATGAAGCCAGACGAGGCGAGGCAGGCGGGGCAGAATG
AATGAGTTTCTAGGGAGGGAGGTTGGGTGCAGGTAGAGCGAGTAGCTGGGGTGGTGAGC
CAGGGAGGCACTGGCCTCCAGAGTCCGTGGCCAAGGAGGGCCTTGCGGGCGGCGACGGA
GCAGTGATCGGTGTCTCGAAGAGCTCGGACTGGAGACTAGGCCAGGTCTCCAGCAGAGGT
GGATGTGCCTGCCAGTCACTGAAGGCGAGGATGACAGGTGTGGTCAATGTGACAGAAAG
ACATGACATGGTCCGGTGTGATGGAGAGGACAGAAGGGCAGTCATCCAGCCTTCTTGAAC
ACCATGGGCTGGCGCCTTGTCGTAGAAGCCGTCTGTTCTTTCACTTTTCCCAAAGAGCTAA
CACTTCTCTGCTGCTCTCTGGATCCTCCTCCCCCTACCTTGAACCCTCAAGATGAAAGAAA
TGGTGCTACCCAGCTCATGTCTGGGCCTTTGAATCCGGGGACTTCTTTAAGTCCGTCTCGTT
CTGAATCAAGAAGATGCTGCAATCAGAACCACTACACTACCTGCCTCAGGAATCTGCTCC
AAGGTGAAGCTGAAAGAACAGATGGTGTCAACATTTTGAAAGAGCAGACTCATAGCACCC
ACCCACCCCTGAGAATCCATCTTCATGGCCAACTCTGCCTGACCCGGGAGACCACCACCC
ACATCATCCTGGAGCCAAGCCTCTACCCCGGGATGACTTCATCATCTCCCTCCTGTCTTTTT
CTTCTTCCTCCTTTCCTGTAATTCTGTTTCTTTCCTTTTGTTCCTTCCTTGCTTGAGAGACTC
AAAGCACCCGTGACTCTGTTTCCCCATTTACCCCCTTTTGAATTTGCACTAAGTCGATTGCA
CTGGTTTGGAGTCCCGGAGATAGCTTTGAGTCTCTCCGTATGAATGTATACAGCGAGTGTG
TAAACCTCTTTGGCAATGCTGCCCCAGTACCCACCTGTCGTCCATCTCCGTCTGAGGGCAA
CTGGGTGTGGCCGTGTGCTTGAGGCCTCGCCTTCCCCTCGCCTAGTCTGGAAGCAGTTCCA
TCATAAAGTGTTCAACATGCCCTACTTCATCCTTTGCCCCTCCTCACCAGGGCCTCACCAG
AGGTCCTGGGTCCATCAATAAATACAGTTACAGCC
Data file S3. miRNA target information. The predict target sites and exact positions
of mouse H19 for miR-200b/c, let-7b were analyzed by online RNAhybrid tool.
target : mmu-H19
length: 2257
miRNA : mmu-miR-200b-3p
length: 22
mfe: -23.9 kcal/mol
position 339
target 5' C G 3'
CGUC UGCUGGGCAG
GUAG AUGGUCCGUC
miRNA 3' A UA AUAAU 5'
target: mmu-H19
length: 2257
miRNA : mmu-miR-200c-3p
length: 23
mfe: -26.3 kcal/mol
position 338
target 5' G G G 3'
CCGUC UGCU GGCAG
GGUAG AUGG CCGUC
miRNA 3' A UA G AUAAU 5'
target: mmu-H19
length: 2257
miRNA : mmu-let-7b-5p
length: 22
mfe: -29.3 kcal/mol
position 1637
target 5' G U A C G 3'
AACCAC AC CUA CUGCCUCA
UUGGUG UG GAU GAUGGAGU
miRNA 3' UGU 5'
Data file S4. ArfGEF target information. The screen shot of let-7b target sites on
10 ArfGEF-encoding genes.
1. BIG1 (ARFGEF1)
2. BIG2 (ARFGEF2)
3. GBF1
4. BRAG1 ( IQSEC2)
5. BRAG2 (IQSEC1)
6. CYTH1
7. EFA6A (PSD)
8. PSD2
9. FBXO8
10. CYTH3
Data file S5. siRNA, shRNA, and PCR primer sequences. The sequences for
commercially provided or synthesized oligonucleotides.
SiRNAs:
27mer H19 siRNA duplexes:
ShRNAs:
H19 shRNA:
#1. V3SM11247-245861005 GACGGCTTCTACGACAAGG
#2. V3SM11247-245995018 TTCAGAACGAGACGGACTT
#3. V3SM11247-246067486 TTATCCGACCAGCCGCCAC
Git2 shRNA:
#1. V3SM11241-232760563 GTCAACTTCGTCGTACACA
#2. V3SM11241-233375008 TGGACGAACGCTAACATCT
#3. V3SM11241-235331065 TCATGACCTAAGCACTTGG
CYTH3 shRNA:
#1. V3SM11241-235505932 CAGCAGGTCGTTCTCAATT
#2. V3SM11241-236387017 GAACGTGTGTGTCAGGTCA
#3. V3SM11241-237030037 CTGACATTTACCTATTTCT
Primers:
pH19mut1:
Forward: AAGCTTCAGCAGAATGGCACATA
Reverse: CTCGAGGGAGGCTGGGGGACCAT
pH19mut2:
Forward: AAGCTTCGTTCTGAATCAAGA
Reverse: CTCGAGCCACTACACTACCTGCCT
Git2 miR-200b/c mutant:
Forward: GCGGATCCCTGGACCCTGCCGC
Reverse: GACCATGGAGG CGGAAAGCTATT
CYTH3 let-7b mutant:
Forward: GCGGATCCTATGAAAGTCAAGCG
Reverse: GACCATGGTAAGATCCCAAGCTTAC
Oligonucleotides:
pSiCHECK2-miR-200b 4X:
GCAAGCTTAGGCCGTCGTGCTGGGCAGGCCGTCGTGCTGGGCAGGCCGTCGTGCTGGGCA
GGCCGTCGTGCTGGGCAGGCGGCCGCAAGAGCTC
pSiCHECK2-miR-200b 4X:
GCAAGCTTGGCCGTCGTGCTGGGCGGCCGTCGTGCTGGGCGGCCGTCGTGCTGGGCGGCC
GTCGTGCTGGGCGAGCTC
pSiCHECK2-let-7b 4X:
GAACCACTACACTACCTGCCTCAGGCGGCTCGAGGAACCACTACACTACCTGCCTCAGGA
ACCACTACACTACCTGCCTCAGGAACCACTACACTACCTGCCTCAGGAACCACTACACTAC
CTGCCTCAG GCGGCCGCAA