Abstract

   Breast cancer is the most common cancer among women worldwide. We
   previously showed that early-life exposure to flaxseed (FS) or its
   components, FS oil (FSO) and secoisolariciresinol diglucoside (SDG),
   affects the mammary gland (MG) and is associated with the reduction of
   breast cancer risk during adulthood. However, the underlying mechanisms
   are not understood. This study aimed to investigate the effect of FS,
   FSO, and SDG on the MG miRNA signature at a late stage of development.
   Female C57BL/6 mice, 4–5 weeks of age, were randomized into four groups
   to receive: (i) basal AIN-93G, (ii) 10% FS, (iii) 3.67% FSO, or (iv)
   0.15% SDG. After 21 days, the mice were sacrificed and MG miRNAs were
   profiled. Diet-specific MG miRNA signatures were identified.
   Deregulated miRNAs were associated with breast cancer and targeted
   genes involved in MG development, growth, and cancer. The study allowed
   for the identification of potential biomarkers or novel therapeutic
   targets to prevent and/or reduce the risk of breast cancer.

   Keywords: breast cancer, flaxseed, mammary gland, microRNAs

1. Introduction

   Breast cancer is the second leading cause of cancer death in female
   patients after lung cancer [[32]1,[33]2]. Thus, much research is being
   conducted for its prevention and treatment, including the role of diet.
   Because of its unique composition, flaxseed (FS) has been studied for
   its effect on breast cancer (reviewed in [[34]3]).

   Flaxseed (FS; Linum usitatissimum) is an oilseed containing
   approximately 40% oil (FSO), more than half of which is the n-3
   polyunsaturated fatty acids (PUFA), α-linolenic acid (ALA), the highest
   level of the dietary phytoestrogen lignan, secoisolariciresinol
   diglucoside (SDG) (~1%), and approximately 30% dietary fiber [[35]4].
   Due to the elevated contents of ALA, FSO is considered to be an
   anti-inflammatory agent, while the lignan components have documented
   anti-oxidant and estrogenic/anti-estrogenic activities. Dietary fiber
   is generally recognized as having many beneficial effects, including
   modulation of the gut microbiota with the production of short-chain
   fatty acids (SCFA) [[36]5].

   We previously showed that FS consumption was associated with the
   reduction in breast cancer risk (reviewed in [[37]3]). In mice injected
   with breast tumor cells, 10% FS significantly decreased the tumor size
   and growth at a late stage of carcinogenesis [[38]6]. The 10%
   FS-induced inhibition of breast cancer growth and metastasis was
   related to the downregulation of insulin-like growth factor 1 and
   epidermal growth factor [[39]7]. The FS inhibitory effect on breast
   cancer was also attributed to both FSO and SDG components.

   In addition, studies from our group showed the link between early-life
   exposure to FS, mammary gland (MG) development, and breast cancer risk
   during adulthood. Notably, a 10% FS exposure in utero, during suckling,
   and throughout life could improve MG morphogenesis and differentiation,
   which in turn, helps to prevent MG carcinogenesis (reviewed in
   [[40]3]). In addition, SDG-based diet during pregnancy and lactation
   helped in reducing the susceptibility of mice to carcinogenesis and
   reduced tumorigenesis. This pattern was not observed with FSO-based
   dietary intervention [[41]8]. Taken together, these findings support
   the use of FS and SDG during MG development to reduce the future risk
   of breast cancer. However, the mechanisms involved in the protective
   effects of early-life exposure to FS and its components remain unclear.
   These may involve gene expression regulation via microRNAs (miRNAs).

   MiRNAs are a class of small non-coding RNAs that regulate gene
   expression at the post-transcriptional level [[42]9]. MiRNAs are
   involved in several physiological processes across multiple tissues,
   including playing a vital role during MG development through the
   regulation of cell differentiation, proliferation, and involution
   across different stages of life [[43]10,[44]11]. Their aberrant
   expression levels were associated with cancers, including breast cancer
   (reviewed in [[45]12]). In a recent systematic review, potential
   markers of breast cancer were identified for early detection and
   diagnosis, including miR-210 and miR-21 [[46]13].

   Interestingly, environmental factors such as diet have been shown to
   alter the expression of miRNAs. Recent studies have showed that dietary
   phytochemicals may inhibit breast cancer through the regulation of
   miRNAs expression (reviewed in [[47]14]). We recently found that n-3
   PUFA altered the expression of miR-21 in MCF-7 breast cancer cells
   [[48]15]. To date, no studies have investigated the effect of FS and
   its components, FSO and SDG, on the miRNA expression signature during
   MG development. Understanding the functions and contextual interactions
   between dietary components and MG miRNA signature (miRNome) during
   development could provide insights on the mechanism employed by
   bioactive agents to generate positive health outcomes and subsequently
   mitigate disease risk for lifelong health.

   The objective of this study was to investigate the effect of exposing
   female C57BL/6 mice to 10% FS, as well as FSO or SDG found at the level
   in 10% FS, during MG development on MG miRNA expression signature and
   to identify potential targets to prevent breast cancer development
   during adulthood.

2. Materials and Methods

2.1. Animal Study and Dietary Treatments

   Fifty-six C57BL/6 female mice were purchased from Charles River
   Laboratories (Senneville, QC, Canada) at 4–5 weeks of age in order to
   ensure the mammary glands (MGs) were entering the pubertal stage of
   development. Upon arrival, the mice were randomized into four groups (n
   = 14/group) and maintained on a basal modified AIN93G diet ([[49]16],
   BD) for 7 days acclimatization. The AIN93G diet was modified to
   increase the fat percentage from 7% to 20%, as in our previous studies
   [[50]17]. Due to its low n-3 PUFA and phytosterol content, corn oil was
   used instead of soybean oil to minimize the confounding effect that may
   otherwise derive from soybean oil. Mice were housed four per cage at 21
   °C and on a 14:10 light/dark cycle with HEPA (high efficiency
   particulate air) filtered air. On day 0, all mice were inspected for
   vaginal opening to confirm the onset of puberty and then fed either (a)
   BD, (b) 10% FS, (c) 3.67% FS oil (FSO), or (d) 0.15% SDG diets for 3
   weeks until sacrifice (Day 0 to 21). The FSO and SDG were at the same
   levels present in the FS diet.

   Omegalo^® Cold Milled FS and FSO, kindly provided by Omega Nutrition
   Inc. (Vancouver, BC, Canada), were used to supplement the 10% FS and
   3.67% FSO diets, respectively. SDG (99.5% pure) was prepared as
   previously described [[51]18]. The FSO fatty acid composition was
   analyzed using gas chromatography by Omega Nutrition. The proximate
   composition of FS was analyzed by Maxxam Analytics (Mississauga, ON,
   Canada), while SDG content was analyzed using HPLC by ChromaDex
   Analytics (Boulder, CO, USA) [52]Table S1). Diets were formulated based
   on the AIN-93G diet ([53]Table S2) using the FS compositional analysis
   and were prepared by Dyets Inc. (Bethlehem, PA, USA). The diets were
   isocaloric, with equal macronutrient levels. Diets were stored at 4 °C
   until needed. Diets in cages were replaced every 2 to 3 days.

   Food intake and body weight were measured twice a week. At day 21, mice
   were sacrificed by cervical dislocation and blood was collected via
   cardiac puncture in a serum vacutainer for lignan analysis. The fourth
   left MG was dissected and immediately stored at −80 °C for further
   analyses.

   All the animal procedures were performed in accordance with the
   Regulations of the Animals for Research Act in Ontario and the
   Guidelines of the Canadian Council on Animal Care, and were approved by
   the animal ethics committee of the University of Toronto (Animal Use
   Protocol #: 20011734).

2.2. Serum Lignan Analysis

   Serum concentrations of the lignans enterodiol (END), enterolactone
   (ENL), and secoisolariciresinol (SECO) were determined by liquid
   chromatography-mass spectrometry (LCMS/MS) as previously described
   [[54]19].

2.3. NanoString nCounter miRNA Profiling and Data Analysis

   Total RNA was extracted from the MG samples (n = 6/group) using the
   mirVana^TM miRNA isolation Kit (Ambion, Life Technologies, Waltham, MA,
   USA). RNA concentration and purity were measured using the NanoDrop
   ND-2000 (ThermoScientific, Wilmington, DE, USA).

   The expression profile of 578 miRNAs was assessed with the nCounter^®
   Mouse v1.5 miRNA Expression Assay Kit (NanoString Technologies,
   Seattle, WA, USA) (miRBase built v15). The experiments were performed
   at the Princess Margaret Genomics Centre Toronto, Canada
   ([55]www.pmgenomics.ca), using 200 ng of total RNA according to the
   manufacturer’s instructions.

   Data analysis was performed using nSolver^TM Analysis Software v4.0
   (NanoString Technologies). Background hybridization was corrected by
   subtracting the geometric mean calculated from eight negative controls.
   Data were then normalized to the geometric mean of all probes. To
   filter for expressed miRNAs, only probes that were above the background
   in 80% of the samples of any of the treatment groups were retained.
   Samples with less than 75% probes above the background were excluded.
   The final dataset was log2 transformed and used for statistical
   analysis.

   Unsupervised hierarchical clustering analysis was performed with the
   normalized and log2 transformed data using ClustVis
   ([56]https://biit.cs.ut.ee/clust vis/) [[57]20].

2.4. MiRNAs Target Prediction and Pathway Analysis

   Experimentally validated target genes of differentially expressed
   miRNAs were identified in silico using the miRWalk Release 2.0 and
   miRTarBase Release 7.0 databases in order to include all validated
   target genes that overlapped between and were exclusive to the two
   databases [[58]21,[59]22].

   Gene enrichment analysis was performed for miRNA gene targets using
   one-sided hypergeometric test conducted on Gene Ontology (GO)
   biological process (GObp), molecular function (GOmf), and cellular
   component (GOcc) gene sets using ClueGO, a Cytoscape plug-in
   application [[60]23]. The GO gene sets with a Benjamini–Hochberg
   adjusted q < 0.05 were considered significant and were clustered into
   ClueGO groups based on the similarity in the number of genes calculated
   by a Kappa score within ClueGO. Data were visualized using Cytoscape
   (v3.7.1) [[61]24].

2.5. Statistical Analysis

   Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad
   Software, Inc., La Jolla, CA, USA). Data are presented as means ±
   standard error of mean (SEM). To analyze body weight and food intake,
   one-way ANOVA with Bonferroni’s post-hoc for multiple comparisons test
   was used.

   Serum lignan concentration was analyzed by Kruskal–Wallis test with
   Dunn’s post-hoc for multiple comparisons and differences were
   considered significant with p ≤ 0.05. For miRNA, one-way ANOVA was used
   to calculate statistical significance of the normalized
   log2-transformed miRNA expression counts between the treatment groups.
   The p-values were corrected using the Benjamini–Hochberg method (FDR <
   0.2). Pairwise comparisons were used to compare between the treatment
   groups.

3. Results

3.1. Body Weights and Food Intake

   Body weights and food intake did not differ among the treatment groups
   ([62]Table S3).

3.2. Serum Lignan Concentration

   Serum concentrations of END, ENL, and SECO were detectable in the FS
   and SDG groups but not in the BD and FSO groups ([63]Table S4).
   Multiple comparison analysis showed significant differences in SECO,
   END, ENL, and total lignan concentrations between the 10% FS and the
   BD- or FSO-fed groups. Significant differences in SECO and ENL
   concentrations were found between the SDG-fed group and the BD- or
   FSO-fed group. No significant differences were found between the 10% FS
   group compared to the SDG group despite relatively much higher lignan
   levels in the 10%FS group due to large variability, particularly in the
   FS group.

3.3. Mammary Gland miRNome

   Among the 578 miRNAs profiled in MG, 238 were detected ([64]Table S5),
   with 45 of the top 50 expressed miRNAs shared between the groups
   ([65]Figure 1A). After applying the cut-off, one sample from the BD
   group and another from the FSO group were excluded. Ten miRNAs were
   found to be significantly differentially expressed between the
   treatment groups (p < 0.05, FDR < 0.2). Unsupervised hierarchical
   clustering based on the expression profiles of these miRNAs produced a
   separation between samples from different treatment groups, even if not
   perfect ([66]Figure 1B). This separation was clear when using the three
   groups (FS, FSO, and SDG) to construct the heatmap ([67]Figure S1).

Figure 1.

   [68]Figure 1
   [69]Open in a new tab

   Mammary gland (MG) miRNAs changes in response to the three experimental
   diets. (A) Top miRNAs detected in MG using NanoString Technologies
   (mean values ± SEM); (B). Heatmap showing unsupervised hierarchical
   clustering of the 10 significantly deregulated miRNAs between the
   treatment groups (p value < 0.05, FDR < 0.2), n = 5–6/group. For each
   miRNA, the expression values were transformed to Z-scores, where red
   indicates higher expression and blue indicates lower expression.

   Pairwise comparisons showed a diet-specific effect on miRNAs in the MG
   ([70]Figure S2). In the FS group, both miR-297c and miR-500 were
   downregulated compared to the BD and FSO groups, while miR-1 was
   upregulated compared to the FSO, with no significant change compared to
   the BD. In addition, the 10% FS diet significantly increased the
   expression of miR-210 compared to the BD and the SDG diets.

   FSO significantly deregulated the expression of four miRNAs: miR-30b,
   miR-324-5p, miR-382, and miR-423-3p. MiR-30b and miR-324-5p were
   significantly increased by FSO compared to BD and SDG. While miR-382
   and miR-423 were significantly down- and upregulated, respectively,
   compared to the FS group, their expression levels were not
   significantly changed compared to the BD group ([71]Figure S2).

   The SDG diet, in turn, significantly increased miR-142-5p and decreased
   miR-1966 expression levels compared to the BD and FSO groups.

3.4. Identification of Gene Targets of Deregulated miRNAs and Pathway
Enrichment Analysis

   In silico analysis using miRWalk Release 2.0 and miRTarBase Release 7.0
   databases identified a total of 162 experimentally validated genes
   ([72]Table S6).

   To identify the pathways enriched in response to each treatment, we
   completed the analysis using the list of validated targets of miRNA
   significantly changed in FS, FSO, and SDG groups separately. Using the
   Cytoscape plug-in application ClueGO, we identified significantly
   enriched GO gene sets, which were grouped together based on common
   function.

   The FS group had the highest number of sets significantly enriched for
   the genes targeted by miR297c, miR-500, miR-1, and miR-210 ([73]Figure
   2A). These 47 GO gene sets were clustered into 17 CluGo groups, with
   the top five most significant GO terms being as follows: regulation of
   growth, regulation of cell development, tube development, tube
   morphogenesis (GObp), and protein kinase binding (GOmf).

Figure 2.

   [74]Figure 2
   [75]Open in a new tab

   Functional enrichment analysis of miRNA target genes. Network analysis
   for target genes of miRNAs deregulated in FS (A), FSO (B), SDG (C), and
   for all validated targets identified in the study (D). The enrichment
   analysis was performed across gene ontology (GO) molecular function
   (GOmf), biological process (GObp), and cellular component (GOcc)
   pathways. Significantly enriched genes (Benjamini–Hochberg (HB)
   adjusted q < 0.01) were used to construct the network. Network node
   layout is based on the similarity between the genes within the GO gene
   sets. Terms with a similarity score >0.5 were linked by an edge. Nodes
   of the same color belong to the same cluster.

   In both, FSO and SDG groups, 22 and 10 gene sets were identified,
   respectively; all of them were identified in the FS and are shared
   between the groups, including regulation of growth, tube morphogenesis,
   and tube development ([76]Figure 2B,C).

   The miRNA-mRNA network shows that significantly deregulated miRNAs in
   the FS and FSO groups target genes involved in the MG development (red
   circles) ([77]Figure 3) and/or associated with breast cancer (blue
   circles), including Egfr, Igf-1, Igf-1R, Hif1a, Ets1, and pgr.

Figure 3.

   [78]Figure 3
   [79]Open in a new tab

   Regulatory network of miRNAs and their experimentally validated targets
   in enriched pathways (miR-423 is excluded due to no significantly
   enriched genes having been identified). Gene Ontology GOmf, GObp, and
   GOcc were used to color the nodes. The network was generated using
   Cytoscape 3.7.1 and consisted of the miRNAs deregulated in FS (green),
   FSO (blue), and SDG (purple), with the miRNA node size representing the
   expression level in the specific group. Nodes with red circles
   correspond to genes involved in MG development. Nodes with blue circles
   correspond to genes associated with breast cancer.

4. Discussion

   To investigate mechanisms underlying beneficial effects of FS in the
   context of breast health, we analyzed miRNA responses in the MG of
   pubertal mice to two main FS components, FSO and SDG, versus FS. We
   found overlap in miRNA expression across the three diets, but three
   diet-specific miRNA signatures could be identified. Specifically, a
   four-miRNA signature (higher miR-1 and miR-210 and lower miR-297c and
   miR-500 expression) significantly associated with the FS diet, a
   different four-miRNA signature (higher miR-30b, miR-324-5p and
   miR-423-3p and lower miR-382 expression) significantly associated with
   the FSO diet, and a two-miRNA signature (higher miR-142-5p and
   miR-1966) significantly associated with the SDG diet. This shows that
   the effects of FS components on MG miRNA expression are not additive.
   Upon ingestion, FS and its components are processed in the intestine
   mainly through the action of the gut microbiota. The microbiota
   ferments FS fiber, producing short-chain fatty acids and gases, and
   converts SDG to the enterolignans ED and EL, which may travel to distal
   organs. Here, despite being fed the same amount of SDG, the 10% FS
   group had relatively higher serum enterolignans concentrations compared
   to SDG-fed group, in line with our previously published studies,
   showing significantly higher urinary enterolignan levels in female mice
   receiving 10% FS compared to SDG-fed mice [[80]25,[81]26]. The fiber in
   FS may have enhanced the conversion of SDG to enterolignans due to
   enhanced microbial fermentation.

   In this study, we examined the MG miRNome at the end of the MG
   developmental period. MiRNAs are known as one of the regulators of MG
   development. MG miRNA profiling in C57BL/6 female mice during
   development showed a distinctive miRNA signature based on developmental
   stages [[82]27]. This study identified seven temporally co-expressed
   clusters, which were enriched in miRNAs associated with breast cancer
   including, miR-210, miR-30b, and miR-21. The breast cancer-associated
   miRNAs may play a role in cell proliferation and invasion during normal
   MG development, but their expression may become deregulated in breast
   cancer.

   Taken together, this shows that miRNA expression regulation is critical
   throughout various stages of life in the MG development. Alterations in
   MG during the early stages of development have been associated with
   increased susceptibility to breast cancer [[83]28]. Exposures to
   environmental factors, including hormones, chemicals, and diets, could
   play an important role in increasing this susceptibility
   [[84]28,[85]29].

   This is the first comprehensive study of the effects of FS as a whole
   food compared to its isolated components, FSO and SDG, on MG miRNA
   signature in healthy female mice during MG development. Our key
   findings include the identification of (1) diet-specific MG miRNA
   signatures, (2) diet-dependent deregulation of selected breast
   cancer-associated miRNAs, and (3) enriched gene sets targeted by these
   miRNAs in each dietary intervention group.

   Interestingly, most of the miRNAs identified in this study were shown
   to be deregulated in several types of cancers, including in breast
   cancer.

   In the FS group, miR-1 was upregulated. This miRNA is known as a tumor
   suppressor in breast cancer, but was significantly downregulated in
   breast cancer compared to non-tumor tissues [[86]30]. Breast cancer
   patients with low miR-1 expression levels were found to have a poor
   survival time [[87]31]. Moreover, previous studies suggest that
   upregulation of miR-1 beneficially counters several types of cancers by
   repressing the expression of Egfr (coding for an epidermal growth
   factor receptor) to inhibit cell proliferation, migration, and invasion
   [[88]32,[89]33,[90]34,[91]35]. Diet-induced restoration of miR-1 may
   have a potential therapeutic significance.

   Increased miR-500 serum levels have been linked to hepatocellular
   carcinoma [[92]36], while its downregulation was found to suppress lung
   cancer proliferation [[93]37]. However, it is not known if this miRNA
   is deregulated in breast cancer. FSO-associated miR-500 downregulation
   could potentially protect against mammary gland carcinogenesis and
   breast cancer development.

   MiR-297c inhibition in human or animal subjects was found to be
   correlated with the efficacy of NSAIDs (non-steroidal anti-inflammatory
   drugs) in cancer chemoprevention treatment. This miRNA has, however,
   been suggested for use as a marker to assess the effectiveness of
   treatment [[94]38]. Interestingly, in our study, miR-297c expression
   was significantly decreased in FS-fed mice.

   In the FSO group, with mice receiving the equivalent amount of 3.67%
   FSO as in the FS group, both miR-30b and miR-324-5p were upregulated
   compared to the BD and SDG groups, with no significant changes in the
   expression levels when compared to the FS group.

   MiR-30b is a member of the miR-30 family, identified as tumor
   suppressors, which have frequently been found to be downregulated in
   different types of tumors, including breast cancers (reviewed in
   [[95]30]). This miRNA was shown to be able to alter drug resistance in
   breast cancer. A previous study found that a 6-day treatment of breast
   cancer cell lines with trastuzumab (Herceptin) resulted in a
   significant upregulation of miR-30b. Trastuzumab-induced miR-30b
   expression could, in consequence, induce cell growth inhibition by
   targeting cyclin E2 (CCNE2) [[96]39]. MiR-30b was also found to be
   involved in MG development; its upregulation in the tissue led to an
   impaired MG function and structure during lactation and involution
   [[97]40].

   In a recent study, miRNA profiling of triple-negative breast cancer
   (TNBC) tissues showed high miR-324-5p expression levels. This
   expression was significantly correlated with decreased overall survival
   in breast cancer patients [[98]41].

   MiR-382 was reported to be upregulated in the serum and plasma of
   patients with breast cancer and suggested to be used as a non-invasive
   marker for the diagnosis of breast cancer [[99]42]. This miRNA was also
   shown to promote cell viability, invasion, and survival in breast
   cancer patients [[100]43].

   MiR-142-5p is one of the oncogenic miRNAs targeting cancer-related
   pathways. Its expression levels are significantly higher in breast
   cancer cell lines compared to controls [[101]44]. This miRNA was also
   identified as a negative regulator of TGF-β signaling pathway and
   suggested to be used as a therapeutic target in breast cancer
   [[102]44].

   For the SDG-induced miR-1966, very limited data are available to
   describe the role of this miRNA in MG development and/or in cancers. A
   very recent study showed that miR-1966 is significantly downregulated
   in toosendanin-induced liver injury in mice [[103]45].

   Interestingly, six of the ten miRNAs identified in this study (with the
   exception of miR-210 in the FS group, miR-423-3p in the FSO group, as
   well as the SDG-induced miR142-5p and miR-1966), were expressed in the
   opposite direction compared to their expression in the breast cancer
   tissues and/or cell lines. Therefore, it is speculated that FS and FSO
   dietary interventions could be used either to antagonize breast
   cancer-associated alterations of selected miRNAs or to prevent their
   deregulation in the MG during development. Note that mice used in this
   study were 4–5 weeks old, which corresponds to late stages of pubertal
   development. This supports the notion of early-life dietary
   intervention, using FS, to mitigate the risks of mammary gland
   malignancies and breast cancer.

   This study brings a new concept of diet-dependent MG response that is
   mediated by miRNAs. Our data discern the differences between the
   consumption of purified FS components, SDG and FSO, and whole FS on the
   MG miRNA signature. However, the mechanisms behind these specific
   responses are not clear. This is likely due to the gut-microbiota
   derived metabolites resulting from the ingestion of FS as whole food
   and its isolated FSO (3.67%) or SDG (0.148%) components.

   Overall, the gut microbiota outcomes may have a significant consequence
   on the amount and types of metabolites entering the systemic
   circulation, which could in consequence influence the miRNA-mediated
   mammary gland response (MG). Though, given the complexity and because
   the effect of FS and its components, FSO and SDG, on the gut-MG axis
   has not been completely elucidated yet, gut microbial profiling in
   response to different dietary interventions is needed for a better
   understanding of the mechanisms driving our diet-associated significant
   miRNA signatures.

   To further explore the role of miRNAs in mediating MG response to
   different diets, we identified a regulatory network of the deregulated
   miRNAs with their experimentally validated targets. Although
   distinctive miRNA signatures were associated with different diets, the
   in silico analysis revealed the presence of shared gene sets between
   the three groups. Growth regulation and tube morphogenesis were
   identified among the most significantly enriched pathways. Thus, it
   could be postulated that deregulated miRNAs may be involved in
   promoting growth properties such as cell proliferation and
   differentiation in the MG.

   It is not surprising to see that the FS-fed group had the greatest
   number of enriched gene sets followed by the FSO and SDG groups.
   Compared to isolated FSO and SDG, the FS contains soluble and insoluble
   dietary fiber, digestible proteins, and lignans, which may also have
   either a direct or an indirect effect on the MG response.

   Interestingly, enrichment analysis shows that FS- and FSO- dependent
   miRNAs are associated with gene sets involved in MG development,
   including Bcl2l11, Csf1, Gja1, Hif1a, Igf1, Igf1r, Pax3, Pgr, Slc12a2,
   and Vdr.

   Finally, constructing the miRNA-mRNA interaction network served not
   only to visualize the interaction between miRNAs and their targets, but
   also to identify potential biomarkers or novel therapeutic targets to
   prevent and/or reduce the risk of breast cancers. Of these, FS-induced
   miR-1 may be a potential candidate. In addition to its association with
   MG development, this miRNA also targets genes involved in breast cancer
   development, including Igf-1 and Igf-1R coding for insulin-like growth
   factor 1 and its receptor (reviewed in [[104]46] and [[105]47]), as
   well as Cdk9 coding for cyclin-dependent kinase 9 (CDK9), involved in
   cell survival and the regulation of apoptosis. Cdk9 inhibitors are
   considered to be anticancer therapeutics [[106]48].

5. Conclusions

   This research is significant, as it provides evidence that FS and its
   FSO and SDG components have different physiological consequences, which
   should be considered when used as a preventive or therapeutic agent.
   With the current rise of dietary supplements, this study reinforces the
   importance of discerning the difference in health outcomes between
   whole foods compared to isolated components. Furthermore, by taking a
   global approach, it suggests a potential mechanism via miRNA for FS to
   impact developing MG, a mechanism that has only been marginally
   considered so far.

Acknowledgments