Table S1. MinimumInformation for Publication ofQuantitative Real-Time PCR Experiments (MIQE guidelines).

Table S2. Presence of risk factors for developing breast cancer in asymptomatic high-risk and patient groups.
Risk factors / BC (n=30) / RC (n=30) / P value
Early menarche <12y / 20 (66.7) / 15 (50) / 0.19
Late menopause >53 / 2 (6.7) / 0 (0) / 0.15
Nulliparous / 12 (40) / 13 (43.3) / 0.79
Late first pregnancy ≥30y / 2 (6.7) / 2 (6.7) / NA
No breast feeding / 18 (60) / 13 (43.3) / 0.19
Prior breast problems# / 5 (16.6) / 7 (23.3) / 0.518
Radiotherapy to the chest area / 0 (0) / 0 (0) / NA
Use of hormonal contraception / 4 (13.3) / 6 (20) / 0.48
Use of HRT / 0 (0) / 0 (0) / NA
Family history of BC or OC / 4 (13.3) / 9 (30) / 0.117
Obesity ≥ 30 Kg/m2 / 10 (33.3) / 7 (23.3) / 0.39
Smoking / 0 (0) / 0 (0) / NA
Night light exposure / 0 (0) / 2 (6.7) / 0.15
Alcohol consumption / 0 (0) / 0 (0) / 1.00
Physical activity / 0 (0) / 2 (6.7) / 0.15
Values are shown as number (percentage). BC; breast cancer, OC; ovarian cancer, RC; Risky control group, HRT; hormonal replacement therapy. # Breast problems existed in the study populations were fibrocystic disease, fat necrosis and fibroadenoma, no atypical hyperplasia. Two sided-Chi square test was used. All p values were >0.05.
Table S3. Breast cancer quantitative risk assessment.
Risk evaluator program / BC (n=30) / RC (n=30) / P value
Tyrer-Cuzick model
5-years risk / 0.9 ± 0.7 / 0.5 ± 0.6 / 0.02 *
10-years risk / 1.9 ± 1.4 / 1.2 ± 1.2 / 0.04 *
Lifetime risk / 12.3 ± 4.5 / 15.2 ± 5.9 / 0.03 *
Probability of BRCA1mutation / 0.1 ± 0.08 / 0.3 ±0.7 / 0.0001 *
Probability of BRCA2mutation / 0.14 ± 0.1 / 0.5 ± 0.9 / 0.0001 *
NCI model
5-years risk / 0.9 ± 0.3 / 1.1 ± 0.4 / 0.12
Lifetime risk / 9.2 ± 2.8 / 13.8 ± 8.6 / 0.0001 *
Values are shown as mean ± SD and as percentage. NCI; National Cancer Institute.Individuals below 35 years old could not be evaluated by NCI model. BC; breast cancer, RC; risky control, BRCA1 and BRCA2, breast cancer type 1 and 2 gene mutation
Table S4. MicroRNA-21 expression levels among the study groups.
Variables / BC (n=30) / RC (n=30) / HC (n=30) / P value
CT RNU6B / 34.84 (32.5-35.9) / 34.86 (33.6-35.4) / 35.12 (33.6-35.9) / 0.111
CT microRNA-21 / 26.04 (25.3-27.2) / 28.23 (27.7-28.6) / 29.0 (26.9-29.66) / <0.001*
-∆ CT / 8.84 (6.64-10.04) / 6.8 (5.64-6.96) / 6.10 (4.94-6.5) / <0.001*
-∆∆ CT / 3.62 (1.42-4.82) / 1.58 (0.42-1.74) / ‒‒ / <0.001*
Fold change / 12.72 (2.67-28.2) / 2.98 (1.33-3.33) / ‒‒ / <0.001*
Values are shown as median (1st-3rdquartiles). Mann-Whitney U and Kruskal-Wallis tests were used. BC; breast cancer group, RC; asymptomatic high-riskgroup, HC; healthy control group, CT; threshold cycle number. ∆∆CT and fold change calculated versus NC.
Table S5. MicroRNA-21 expression levels according to demographic and reproductive risk factors in BC and RC groups (n=60).
Variables / Categories / Number / Fold change / P value
Age / <35 years old / 22 / 3.4 (1.35-20.7) / 0.111
≥35 years old / 38 / 3.16 (1.64-8.6)
Residence / Rural / 10 / 13.1 (2.9-28.1) / 0.026*
Urban / 50 / 3.16 (1.5-5.57)
Occupation / Employed / 23 / 3.1 (1.51-3.53) / 0.001*
Housewife / 37 / 3.46 (1.6-23.1)
Age at menarche / <12 years old / 35 / 3.34 (2.2-20.7) / 0.001*
≥12
years old / 25 / 3.16 (1.32-3.5)
Marital status / Single / 15 / 3.53 (1.2-20.7) / 0.127
Married / 45 / 3.16 (1.64-8.6)
Parity / Nullipara / 25 / 3.53 (1.7-23.1) / 0.002*
Parous / 35 / 3.1 (1.56-7.52)
Age at 1st pregnancy / <30 years old / 31 / 2.22 (1.48-3.2) / 0.683
≥30
years old / 4 / 2.95 (1.6-3.38)
Lactation / Yes / 29 / 3.01 (1.5-3.46) / 0.001*
No / 31 / 3.53 (1.6-28.3)
Contraceptive use / Yes / 10 / 1.76 (1.2-2.31) / 0.142
No / 50 / 3.83 (2.4-18.7)
Menopausal status / Premenopausal / 48 / 3.29 (1.5-12.7) / 0.654
Postmenopausal / 12 / 3.16 (1.6-17.7)
BBD $ / Yes / 12 / 2.27 (1.17-4.8) / 0.863
No / 48 / 3.32 (1.9-17.3)
FH of cancer / Yes / 19 / 3.16 (2.38-3.5) / 0.583
No / 41 / 3.32 (1.6-23.1)
Values are shown as median (1st -3rd quartiles). Mann-Whitney U test was used. BBD; benign breast disease, FH; family history.$ Benign breast disease included fibrocystic disease, fibroadenoma, and fat necrosis.
Table S6. Correlation between microRNA-21 expression level and clinicopathological features in BC patients (n=30).
Variables / Categories / Number (%) / Fold change / P value
Age at diagnosis / <35 years old / 8 (26.7) / 28.9 (15.1-42.7) / 0.016*
≥35 years old / 22 (73.3) / 7.39 (1.67-25.1)
Tumor side / Right / 18 (60) / 12.73 (2.7-28.1) / 0.755
Left / 12 (40) / 13.97 (2.3-30.6)
Tumor location / UOQ / 14 (46.6) / 9.04 (3.16-28.3) / 0.455
UIQ / 8 (26.7) / 17.38 (4.8-43.2)
LOQ / 2 (6.7) / 2.07 (1.56-2.57)
Areolar / 6 (20) / 22.9 (1.24-28.1)
Multi-centricity / No / 24 (80) / 9.04 (1.92-26.6) / 0.296
Yes / 6 (20) / 23.45 (7.2-32.9)
Histological type / IDC / 20 (66.7) / 8.01 (2.12-26.6) / 0.561
ILC / 6 (20) / 25.7 (2.16-32.9)
Mixed / 2 (6.7) / 39.6 (18.6-60.6))
Inflammatory / 2 (6.7) / 8.49 (7.52-9.45)
Skin invasion / No / 25 (83.3) / 7.57 (2.16-25.1) / 0.085
Yes / 5 (16.7) / 28.3 (18.6-32.9)
LV invasion / No / 14 (46.6) / 2.87 (1.24-8.63) / <0.001*
Yes / 16 (53.4) / 28.2 (19.7-39.4)
Pathological grade / Grade 2 / 23 (76.7) / 7.52 (1.67-28.1) / 0.002*
Grade 3 / 7 (23.3) / 23.1 (18.8-38.8)
Tumor size / T2 / 16 (53.3) / 2.78 (1.4-26.58) / 0.232
T3 / 6 (20) / 21.9 (7.26-34.8)
T4 / 8 (26.7) / 18.7 (12.7-30.6)
LN status / N0 / 5 (16.7) / 1.56 (1.24-2.57) / 0.029*
N1 / 10 (33.3) / 7.39 (2.16-9.45)
N2 / 15 (50) / 28.3 (20.7-39.9)
Metastasis / M0 / 16 (53.3) / 2.78 (1.40-8.08) / <0.001*
M1 / 14 (46.7) / 26.6 (18.7-39.9)
Site of metastasis / Bone / 6 (42.9) / 31.4 (25.1-39.9) / 0.576
Lung / 6 (42.9) / 18.7 (16.0-60.6)
Liver / 2 (14.2) / 30.9 (23.1-38.9)
Clinical stage a / Stage II / 8 (26.7) / 1.40 (0.75-2.37) / <0.001*
Stage III / 8 (26.7) / 7.39 (3.1-18.85)
Stage IV / 14 (46.6) / 26.6 (18.7-39.9)
Values are shown as median (1st -3rd quartiles) or as number (percentage). UOQ, upper outer quadrant; UIQ, upper inner quadrant; LOQ, lower outer quadrant; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; LV, lymphovascular; T, tumor size; LN, lymph node; N, nodal stage; M, metastasis. P value was generated by using Mann-Whitney U and Kruskal-Wallis tests.
aClinical staging according to the American Joint Committee on Cancer.
Table S7. Association between microRNA-21 expression level and prognosis in BC patients (n=30).
Variables / Categories / Number / Fold change / P value
NPI / Moderate prognosis / 12 (40) / 8.08 (1.4-23.45) / 0.158
Poor prognosis / 18 (60) / 19.73 (3.2-34.8)
IHPI / Good prognosis / 18 (60) / 8.10 (2.57-20.7) / 0.296
Moderate prognosis / 10 (33.3) / 20.87 (2.2-32.9)
Poor prognosis / 2 (6.7) / 43.2 (39.9-46.5)
ESMO / Intermediate risk / 10 (33.3) / 5.60 (1.24-18.6) / 0.10
High risk / 20 (66.7) / 21.9 (5.2-33.84)
Values are shown as median (1st -3rd quartiles). NPI, Nottingham Prognostic Index;IHPI, Immunohistochemical Prognostic Index; ESMO; European Society of Medical Oncology recurrence risk. Mann-Whitney U and Kruskal-Wallis tests were used.
Table S8. Association between microRNA-21 expression level and hormonal status in BC patients (n=30).
Variables / Categories / Number / Fold change / P value
ER / Positive / 12 (40) / 25.68 (2.6-39.4) / 0.950
Negative / 18 (60) / 8.10 (2.57-20.7)
PR / Positive / 12 (40) / 25.68 (2.6-39.4) / 0.950
Negative / 18 (60) / 8.10 (2.57-20.7)
HER2+ / 0 / 24 (80) / 8.08 (1.92-24.1) / 0.015*
1+ / 6 (20) / 39.40 (9.5-46.5)
Molecular subtype / Luminal A / 14 (46.7) / 7.52 (1.56-20.7) / 0.122
Luminal B / 4 (13.3) / 9.45 (7.57-63.6)
HER2+ subset / 2 (6.7) / 39.9 (38.8-46.5)
Basal-like / 10 (33.3) / 18.64 (2.2-28.3)
Values are shown as median (1st -3rd quartiles). ER, estrogen receptor; PR, progesterone receptor; HER2+, Human Epidermal growth factor Receptor 2; Luminal A, ER+, PR+, HER2-; Luminal B (triple positive), ER+, PR+, HER2+; HER2+ subset, ER-, PR-, HER2+; Basal-like (triple negative), ER-, PR-, HER2-. Mann-Whitney U and Kruskal-Wallis tests were used.

Supplementary figures:

Fig. S1. Family pedigrees of some individuals enrolled in the study. These pictures are part of family pedigrees of study subjects having family history of breast cancer showing family members affected with BC (shaded circles) and their relation to the proband (signed by the arrow). Their age at diagnosis of BC is written below the affected relative. Deceased relative have slashed line. The individual ID is written at the top of each pedigree.