MicroRNA-10a is reduced in breast cancer and regulated in part through retinoic acid

All experimental procedures involving tissue samples from human participants were approved by the Clinical Research Ethics Committee (University College Hospital, Galway). Written informed consent was obtained from each patient and all clinical investigation was performed according to the principles expressed in the Declaration of Helsinki.

Breast tissue specimens (n = 168) were obtained at University College Hospital, Galway. The clinical patient samples comprised of 103 malignant tissue biopsies, 30 normal mammary tissue biopsies obtained at reduction mammoplasty, and 35 fibroadenoma tissues which are benign breast disease tissues. Full patient demographics and clinicopathological details were collected and maintained prospectively (Table 1). Samples were immersed in RNAlater® (Qiagen) for 24 hours, then the RNAlater® was removed and the tissue stored at −80°C until required.

T47D and SK-BR-3 breast cancer cell lines were previously purchased from the American Type Culture Collection (Manassas, VA). T47D cells were cultured in RPMI-1640 media and SK-BR-3 cells were cultured in McCoy’s 5A. Both media types were supplemented with 10% fetal bovine serum (FBS) and 100U/ml penicillin/ 100 μg streptomycin (P/S). Cells were incubated at 37°C and 5% CO₂ with a media change performed twice weekly and passage every 7 days.

Breast tissue specimens or cell pellets were homogenised in 1 ml TRIzol® lysis reagent (Invitrogen) as previously described [27]. Total (large and micro) RNA was extracted from malignant (n = 103), normal (n = 30) and fibroadenoma (n = 35) mammary tissue using the RNeasy Mini Kit (QIAGEN) as per manufacturer’s instructions.

1 μg of large RNA was reverse transcribed using SuperScript III reverse transcriptase enzyme (200U/μl), 0.1 M DTT, RT-5x Buffer, RNaseOut Ribonulease Inhibitor (40U/μl), Random primers (3 μg/μl) and dNTP’s (100 mM)-Promega (Invitrogen, Carlsbad, CA, USA). TaqMan® Gene Expression Assays targeting RARβ and THRα (Table 2) were used in TaqMan® Universal Mastermix (Applied Biosystems). 100 ng of mature miR-10a (Table 2) was reverse transcribed using the MultiScribe™-based High-Capacity cDNA Archive Kit (dNTP 100 mM, RT Buffer 10x, RNase Inhibitor 20U/μl, Stem loop primer 50nM, MultiScribe RT 50U/μl) (Appied Biosystems). The resulting cDNA for both mRNA and microRNA was analysed using the ABI 79000 Fast real-time PCR system (Applied Biosystems). These reactions were carried out in a final volume of 10 μl comprising of 0.7 μl cDNA, 5 μl TaqMan® Universal PCR fast Master Mix (2×), 0.5 μl TaqMan® primer-probe mix (0.2 μM), Forward primer (1.5 μM), and Reverse Primer (0.7 μM) (Applied Biosystems). The RQ-PCR cycle comprised of 10-minute incubation at 95°C followed by a 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds. The use of an Inter-assay control derived from a breast cancer cell line (T47D) on each reaction allowed comparison of data across plates, and all reactions were carried out in triplicate with a standard deviation of < 0.3 between replicates considered acceptable. The relative quantity of mRNA and miRNA expression was calculated using the comparative cycle threshold (ΔΔCt) [37]. The endogenous controls used for gene expression were Mitochondrial Ribosomal Protein L19 (MRPL19) and Peptidyl-Prolyl Isomerase A (PPIA) [38]. For the miRNA analysis, let-7a was employed as an endogenous control [39]. The geometric mean of the Ct value was used to normalise the data and the sample with the lowest expression level was applied as a calibrator.

T47D and SK-BR-3 cell lines were seeded at 2.4×10⁴ cells/cm² in a 6-well plate. The following day, the cells were exposed to all-trans Retinoic Acid (ATRA, 0.1 μM, 1 μM, 5 μM) or L-Thyroxine (T₄, 0.1 μM, 0.5 μM, 5 μM) for 24 hours. This was carried out to establish optimal concentrations for the assay [27]. Once optimal concentrations were established (ATRA, 1 μM, 5 μM and T₄ 0.5 μM), the assay was then performed in triplicate in both cell lines. The cells were also exposed to a combination of ATRA (1 μM or 5 μM) and T₄ (0.5 μM) for 24 hours. Controls included cells cultured in the appropriate diluents used for each stimulant. Dimethyl sulphoxide (DMSO, 0.5%) for was used to dilute ATRA. Ammonium hydroxide (NH₄OH, 0.1%) was the appropriate diluent for T₄. Cells were harvested at the appropriate time point by trypsination, centrifuged at 120 × g for 4 mins and the cell pellet stored at −80°C. Total large and microRNA was extracted, and the corresponding cDNA analysed using RQ-PCR targeting miR-10a, RARβ and THRα. The endogenous controls used were let-7a for miRNA analysis, and MPRL19 and PPIA for gene expression analysis as previously described. The data was expressed relative to cells cultured in appropriate diluent controls.

All data are presented as Mean (SEM), and graphically represented using box plots and linear scatter plots. A general ANOVA model was used to compare mean responses. Scatter plots were displayed using Linear Regression and Lowess smoother to determine the relationships between different populations. The level of relationship was determined using Pearson correlation coefficients.

MicroRNA extracted from malignant (n = 103), normal (n = 30) and fibroadenoma (n = 35) breast tissue biopsies (Table 1) was analysed by RQ-PCR. Levels of miR-10a were significantly decreased in breast cancer tissues (n = 103, 2.3(0.08) Log₁₀ RQ) compared to both normal (n = 30, 3.1(0.17) Log₁₀ RQ, p < 0.001) and fibroadenoma tissues (n = 35, 2.9(0.15) Log₁₀ RQ, p < 0.001, Figure 1). miR-10a expression was further stratified based on patient clinicopathological details. Expression of miR-10a was not dysregulated across epithelial subtype (p = 0.168), tumour grade (p = 0.299), tumour stage (p = 0.340) or menopausal status (p = 0.126, results not shown).

Expression levels of RARβ and THRα were previously reported by this group, on a total of n = 100 breast tissues, consisting of n = 75 breast cancers, n = 10 fibroadenoma tissues and n = 15 normal breast tissues [27]. Supplementary data shows results from increasing patient sample number to include a total of n = 168 breast tissues for the analysis. This RNA was extracted from an additional n = 27 breast tumours, n = 20 fibroadenoma and n = 15 normal breast tissues was quantified by RQ-PCR targeting RARβ and THRα. RARβ gene expression was found to be significantly down-regulated in breast cancer (n = 101, 0.83 (0.04) Log₁₀ RQ) compared to both normal (n = 27, 1.35 (0.09) Log₁₀ RQ, p < 0.001) and fibroadenoma tissue (n = 32, 1.49 (0.07) Log₁₀ RQ, p < 0.001, Additional file 1: Figure S1.). No significant association was observed with epithelial subtype (p = 0.122), tumour grade (p = 0.363), tumour stage (p = 0.614) or menopausal status (p = 0.635, results not shown).

Levels of THRα were found to be significantly decreased in breast cancer (n = 101, 0.90 (0.03) Log₁₀ RQ) compared to both normal (n = 27, 1.50 (0.06) Log₁₀ RQ, p < 0.001) and fibroadenoma tissues (n = 32, 1.28(0.07), p < 0.001, Additional file 2: Figure S2.). Further analysis revealed THRα expression was not significantly deregulated across epithelial subtype (p = 0.116), tumour stage (p = 0.859) or menopausal status (p = 0.679, results not shown).

Expression of miR-10a across 168 breast tissues was correlated with the gene expression results for RARβ and THRα. A significant positive correlation with RARβ gene expression was observed (r = 0.31, p < 0.001, Figure 2A). miR-10a expression also revealed a robust positive correlation with THRα gene expression (r = 0.32, p < 0.001, Figure 2B).

This study was performed to determine the impact of all-trans retinoic acid (RA) and L-Thyroxine (T₄) on miR-10a expression in vitro. The following concentrations were selected for the analysis, ATRA (1 μM and 5 μM) and T₄ (0.5 μM). This was determined based on preliminary studies, and reflected the most effective doses. Cells were harvested, and changes in miR-10a expression were quantified by RQ-PCR.

In the case of the T47D cells, miR-10a expression was shown to be stimulated at 1 μM ATRA (2.2 fold, SEM(0.6), p = 0.11) and 5 μM ATRA (2.3 fold, SEM(0.7), p = 0.2). In the presence of 0.5 μM T₄, no stimulation of miR-10a expression was observed (0.64 fold, (0.08), p < 0.05). When combining both reagents, a significant synergistic increase was shown at 1 μM ATRA+ 0.5 μM T₄ (3.1 fold, (0.3), p < 0.005) and at 5 μM ATRA+ 0.5 μM T₄ (3.4 fold, (0.8), p < 0.05).

In the SK-BR-3 cells, ATRA alone had a significant impact on miR-10a expression (3.5-4.1 fold (1.2) p < 0.005, Figure 3B). Similar to the T47D cells, SK-BR-3 cells did not show a change in miR-10a expression following stimulation with T₄ alone (0.7 fold (0.5)). Combining ATRA and T₄ resulted in a synergistic impact on miR-10a expression (2.5-2.6 fold (0.2), p < 0.005).

The impact of ATRA or T₄ on receptors was also determined. In the T47D cells, RARβ gene expression was increased by 99–183 fold following stimulation with ATRA alone (Figure 4A). The addition of T₄ abrogated this effect (76–92 fold increase). Stimulation with T₄ alone or in combination had no impact on expression of THRα (0.8-1.5 fold, Figure 4B). In the SK-BR-3 cells, combination of ATRA and T₄ stimulated increased RARβ expression (37–48 fold increase, Figure 4C), with no change observed in THRα (Figure 4D) or RARβ in any other conditions.