Supplementary Material Legends Table S1. IHC characterization of the HER2+ BC subtype. Expression of estrogen receptor (ER), progesterone receptors (Pr), Ki-67 and C-erbB-2 in breast cancer tumor tissues. Table S2 Sequences of the primers for RT-PCR miRNA amplification (in white) or miRNA modulation (in gray). Table S3 miR-429, miR-190 and miR-584 putative targets in HER2+ BC samples. For each miRNA (1st column) we indicated the pairwise pathways (2nd column), the number (3rd column) and the name of the target genes (4-5th column) within these pathways. Figure S1 miRNA-429 is upregulated, while miR-190 and miR-584 are downregulated in HER2+ BC cell lines. The expression levels of miR-429 (A), miR-190-5p (B) and miR-584 (C) were evaluated in SKBR3 HER2+ cell line compared to MCF10A ‘normal-like’ cell line, by RT-PCR analysis. The results are the average of three independent experiments in triplicate (t test, p value <0.05, *; <0.01, **, <0.001, ***). Figure S2 RT-PCR analysis revealed that miR-429 is specific for HER2+ BC. RT-PCR analysis of miR-429 expression level in Luminal A, Luminal B and HER2+ BC compared to normal-like cell lines. The results are the average of three independent experiments in triplicate. T test, p value<0.05,*; <0.01, **; <0.001, ***. Figure S3. miR-429 is significantly overexpressed in each of the 3 considered stages compared to normal samples (t test p value<0.001, ***) (3A). Each stage composition is variable, containing either Luminal A Luminal B, Her2+ and basal. We observed that miR-429 expression is not associated with different stages of BC. Figure S4 miRNA-429 controls HER2+ BC cell proliferation migration and invasion. In order to verify if miR-429 is involve in cell proliferation, the SKBR-3 cells were treated with 200nM of As miR-429. The expression level of miR-429 was evaluated by RT-PCR analysis (t test compared to scramble treated cells, p value <0.01, **; <0.001, ***) (A). Wound healing test was performed on SKBR-3 treated with 200nM scramble oligonucleotide or As miR-429 in culture for 9 days. In each sample, the wound area was quantified by Image J analysis and normalized on the same area at time 0 [31] (t test compared to scramble-treated cells; n=3 experiments in triplicate, p value <0.01, **) (B). Cell count was performed on HER2+ MDA-MB-453 cell line at 0, 24, 48, 72, 96 hours of 200nM scramble or As miR-429 oligonucleotide treatment. The results are the average of three independent experiments in triplicate (t test performed on scramble treated cells, p value<0.05, *)(C).

Figure S5 As miR-429 is counteracts the growth of HER2+ BC tumors xenografted in mouse. Schematic representation of tumor injection and As miR-429 treatment workflow (A). The pictures represent the tumors, treated with As miR-429 (on the left) or with scramble oligonucleotides (on the right) after explanting from the mouse (n=5 tumors from SKBR3 cells, T test, p value<0.05, *; p value <0.01, **) (B). Figure S6 Analysis of the mRNA sequence of human VHL revealed a seed for miR-429 pairing in the 3’ UTR of the target. Figure S7 RT-PCR analysis on tumors formed by scramble- or As miR-429 treated SKBR3 cells, explanted from mouse. The tumors, in which we succeeded in miR-429 reduction (A), have a higher VHL expression (B), reduction in HIF1a-target SLUG (C), VEGF (D) and SNAIL (E) expression in respect to Scramble-treated tumors. T test, p value <0.05, *; < 0.01, **.

#sample % ER % Pr %Ki67 C-erbB-2

1 0 0 30 2+

2 0 0 15 3+

3 0 0 15 3+

4 0 0 15 3+

5 0 0 30 3+

6 0 0 15 3+

7 0 0 30 3+

8 0 0 15 3+

9 0 0 20 3+

10 0 0 15 3+

11 0 0 20 3+

Table S1.

Supplementary material Cava et al., 2019

miRNA ID Sequence (5’-3’)

miR-429 (MIMAT0001536)

TCTAATACTGTCTGGTAAAACCGT

miR-190a-5p (MIMAT0000458)

GTGTGATATGTTTGATATATTAGGTTGTT

miR-584-5p (MIMAT0003249)

TTATGGTTTGCCTGGGACTGAG

miR-103-3p AGCAGCATTGTACAGGGCTATGA

Scramble Sense (S) ATGATGTCCTTCTAGTACGCATC

miR-429 AntiSense(As)

ACGGTTTTACCAGACAGTATTA

Table S2Sequences of the primers for RT-PCR miRNA amplification (in white) or miRNA modulation (in gray)

miRNA Pairwise pathways n. ofgenes/tot genes

Genes in pathway A Genes in pathway B

Hsa-miR-429 A) Acute phase response signaling;B) HIF1 signaling

33/484 CP, IL18, MAP2K7, NRAS, SERPINA1, SERPING1

ATM, CREBBP, NRAS, PIK3C3, VEGFB

A) HIF1 signalingB) Glioblastoma multiforme signaling

ATM, CREBBP, NRAS, PIK3C3, VEGFB ATM, CDK2, E2F5, NRAS, PIK3C3, RHOC

A) HIF1 signalingB) Growth hormone signaling

ATM, CREBBP, NRAS, PIK3C3, VEGFB ATM, PIK3C3, PRKCB, RPS6KA2,SOCS6

Hsa-miR-190 A) Axonal guidance signalingB) CXCR4 Signaling

30/693 ADAM8, BMP1, FZD9, GNB2L1, HRAS, NGEF, PIK3C3, PIK3CB, ROCK1, SEMA3E

GNB2L1,HRAS, PIK3C3, PIK3CB, ROCK1

A) Axonal guidance signalingB) P2Y Purinergic Receptor signaling pathway

ADAM8, BMP1, FZD9, GNB2L1, HRAS, NGEF, PIK3C3, PIK3CB, ROCK1, SEMA3E

GNB2L1, HRAS, NFKB1, PIK3C37, PIK3CB

A) HIF1 signalingB) Glioblastoma multiforme signaling

COPS5, CREBBP, MMP15, MMP24, NOS3, NRAS, PIK3CA

FOXO1, FZD8, NRAS, PDGFRB, PIK3CA, PLCL1, RHOH, RHOQ

Hsa-miR-584 A) Axonal guidance signalingB) CXCR4 Signaling

49/693 ARHGEF12, ARPC3, ARHGEF15, ATM, FARP2, GNAI1, GNB2, MAP2K1, NRAS, PLCB2, PLCD3, PXN, TUBA4A, UNC5B, WAS, WIPF1, WNT10A

ATM, GNAI2, GNB2, MAP2K1,MAPK12, NRAS, PLCB2, PXN

A) Axonal guidance signalingB) P2Y Purinergic Receptor signaling pathway

ARHGEF12, ARPC3, ARHGEF15, ATM, FARP2, GNAI1, GNB2, MAP2K1, NRAS, PLCB2, PLCD3, PXN, TUBA4A, UNC5B, WAS, WIPF1, WNT10A

ATM, GNAI2, GNB2, MAP2K1, NRAS, PLCB2, PLCD3

Table S3

Figure S1 miRNA-429 is upregulated, while miR-190 and miR-584 are downregulated in HER2+ BC cell lines. The expression levels of miR-429 (A), miR-190-5p (B) and miR-584 (C) were evaluated in SKBR3 HER2+ cell line compared to MCF10A ‘normal-like’ cell line, by RT-PCR analysis. The results are the average of three independent experiments in triplicate(t test, p value <0.05, *; <0.01, **, <0.001, ***).

*

C

0

0,001

0,002

0,003

0,004

0,005

MCF10A SKBR-3

miR

-584

rela

tive

expr

essi

on**A

0

0,0002

0,0004

0,0006

0,0008

0,001

0,0012

MCF10A SKBR3

miiR

-429

rela

tive

expr

essi

on

4.02x10-

5±1.68x10-5

1.76x10-

5±4.91x10-5

B

***

0

0,00002

0,00004

0,00006

0,00008

0,0001

0,00012

MCF10A SKBR-3

mIR

-190

rela

tive

expr

essi

on

2.18x10-12±6.13x10-12

9.95x10-5±5.04x10-6

0.001±0.001

0.025±0.001

Figure S2 RT-PCR analysis revealed that miR-429 is specific for HER2+ BC. RT-PCR analysis of miR-429 expression level in Luminal A, Luminal B and HER2+ BC compared to normal-like cell lines. The results are the average of three independentexperiments in triplicate. T test, p value<0.05,*; <0.01, **; <0.001, ***.

0

0,5

1

1,5

2

MCF10A T47D MCF7 BT474 SKBR3 MDA-MB-453

miR

-429

fold

cha

nge

expr

essi

on

***** ***

***

*

HER2+Luminal BLuminal ANormal-like

0

100

200

300

400

500

600

Normal Stage1 Stage2 Stage3

miR

-429

aver

age

expr

essi

on ***

******

Luminal ALuminal BHER2+Basal

Figure S3: miR-429 is significantly overexpressed in each of the 3 considered stages compared to normal samples (t test p value<0.001, ***) (3A). Each stage composition is variable, containing either Luminal A Luminal B, Her2+ and basal. We observed that miR-429 expression is not associated with different stages of BC.

66 13 3 13135 65 28 6146 37 10 9

LumA: 69.6%, LumB: 13.6%; Her2+:3.1%, Basal: 13.7% LumA: 46.7%, LumB: 22.5%; Her2+: 9.7%; Basal: 21.1%LumA: 45.1%; LumB: 36.2%; Her2+: 9.8%; Basal: 8.9%

Normal (n=87)Stage 1 (n=95)Stage 2 (n=289)Stage 3 (n=102)

A

B

LumA: 247 totalLumB: 115 totalHer2+: 41 totalBasal: 83 total

**

0

0,0002

0,0004

0,0006

0,0008

0,001

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0,0014

SKBR3Scramble 72h

SKBR3 ASmiR-429 72h

miR

-429

fold

cha

nge

expr

essi

on

Figure S4 miRNA-429 controls HER2+ BC cell proliferation blocking G1/S transition. The SKBR-3 cells were treated with 200nM of As miR-429. The expression level of miR-429 was evaluated by RT-PCR analysis (t test compared to scramble treated cells, p value <0.01, **; <0.001, ***) (A). Wound healing test was performed on SKBR-3 treated with 200nM scramble oligonucleotide or As miR-429 in culture for 9 days. In each sample, the wound area was quantified by Image J analysis and normalized on the same area at time 0 (t test compared to scramble-treated cells; n=3 experiments in triplicate, p value <0.01, **) (B).Cell count was performed on HER2+ MDA-MB-453 cell line at 0, 24, 48, 72, 96 hours of 200nM scramble or As miR-420 oligonucleotide treatment. The results are the average of three independent experiments in triplicate(t test performed on scramble treated cells, p value<0.05, *)(C).

A B

AS m

iR-4

29U

ntre

ated

24h 96h 9days

Scra

mbl

eC

**

0

10000

20000

30000

40000

50000

60000

0 24 48 72

Cell

coun

t

MDA-MB-453 untreated

MDA-MB-453 scramble

MDA-MB-453 As miR-429

HER2+ cells xenografted in mice

Palpable tumor

Scramble S miR/As miR-429 local injection in atelogene vehicle

Sacrifice

Tumor volume measurements

Figure S5(A) Schematic representation of tumor injection and As miR-429 treatment workflow (B) The pictures represent the tumors, treated with As miR-429 (on the left) or with scramble oligonucleotides (on the

right) after explanting from the mouse (n=5 tumors from SKBR3 cells, T test, p value<0.05, *; p value <0.01, **).

A

B

SKBR3 tumors + AS miR-429 SKBR3 tumors + scramble miR

Figure S6 VHL is the direct target of miR-429.Sequence analysis of the 3’ UTR of human VHL revealed a seed for miR-429 (TargetScan prediction)(A).

VHL 3’UTR 822-828 …UUUGAUUAUAGUAUUAA…

Hsa-miR-429 (MIMAT0001536) AAUGGUCUGUCAUAAU

00,10,20,30,40,50,60,70,80,9

1

SKBR3tumor+scramble

SKBR3 tumor+ AsmiR429

miR

-429

expr

essi

on

A

Figure S7.RT-PCR analysis on tumors formed by scramble- or As miR-429 treated SKBR3 cells, explanted from mouse. The tumors, in which we succeeded in miR-429 reduction (A), have a higher VHL expression (B), reduction in HIF1a-target SLUG (C), VEGF (D) and SNAIL (E) expression in respect to Scramble-treated tumors. T test, p value <0.05, *; < 0.01, **.

C

0123456789

SKBR3 tumors+Scramble

SKBR3 tumors+ AsmiR-429

SLUG

exp

ress

ion

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0

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0,2

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