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@lsu.edu.cn (W.Z.); dxie@sibs.ac.cn (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