Role of Deregulated microRNAs in Breast Cancer Progression Using FFPE Tissue

MicroRNAs (miRNAs) contribute to cancer initiation and progression by silencing the expression of their target genes, causing either mRNA molecule degradation or translational inhibition. Intraductal epithelial proliferations of the breast are histologically and clinically classified into normal, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). To better understand the progression of ductal breast cancer development, we attempt to identify deregulated miRNAs in this process using Formalin-Fixed, Paraffin-Embedded (FFPE) tissues from breast cancer patients. Following tissue microdissection, we obtained 8 normal, 4 ADH, 6 DCIS and 7 IDC samples, which were subject to RNA isolation and miRNA expression profiling analysis. We found that miR-21, miR-200b/c, miR-141, and miR-183 were consistently up-regulated in ADH, DCIS and IDC compared to normal, while miR-557 was uniquely down-regulated in DCIS. Interestingly, the most significant miRNA deregulations occurred during the transition from normal to ADH. However, the data did not reveal a step-wise miRNA alteration among discrete steps along tumor progression, which is in accordance with previous reports of mRNA profiling of different stages of breast cancer. Furthermore, the expression of MSH2 and SMAD7, two important molecules involving TGF-β pathway, was restored following miR-21 knockdown in both MCF-7 and Hs578T breast cancer cells. In this study, we have not only identified a number of potential candidate miRNAs for breast cancer, but also found that deregulation of miRNA expression during breast tumorigenesis might be an early event since it occurred significantly during normal to ADH transition. Consequently, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, typically with rich clinical information, as a means of miRNA biomarker discovery.     

MicroRNA-155 is regulated by the transforming growth factor beta/Smad pathway and contributes to epithelial cell plasticity by targeting RhoA

Transforming growth factor β (TGF-β) signaling facilitates metastasis in advanced malignancy. While a number of protein-encoding genes are known to be involved in this process, information on the role of microRNAs (miRNAs) in TGF-β-induced cell migration and invasion is still limited. By hybridizing a 515-miRNA oligonucleotide-based microarray library, a total of 28 miRNAs were found to be significantly deregulated in TGF-β-treated normal murine mammary gland (NMuMG) epithelial cells but not Smad4 knockdown NMuMG cells. Among upregulated miRNAs, miR-155 was the most significantly elevated miRNA. TGF-β induces miR-155 expression and promoter activity through Smad4. The knockdown of miR-155 suppressed TGF-β-induced epithelial-mesenchymal transition (EMT) and tight junction dissolution, as well as cell migration and invasion. Further, the ectopic expression of miR-155 reduced RhoA protein and disrupted tight junction formation. Reintroducing RhoA cDNA without the 3′ untranslated region largely reversed the phenotype induced by miR-155 and TGF-β. In addition, elevated levels of miR-155 were frequently detected in invasive breast cancer tissues. These data suggest that miR-155 may play an important role in TGF-β-induced EMT and cell migration and invasion by targeting RhoA and indicate that it is a potential therapeutic target for breast cancer intervention.     

Real-Time Monitoring of miRNA Function in Pancreatic Cell Lines Using Recombinant AAV-Based miRNA Asensors

Background

MicroRNAs (miRNAs) are reportedly involved in pancreatic ductal adenocarcinoma (PDAC) development. Current methods do not allow us to reliably monitor miRNA function. Asensors are adeno-associated virus (AAV) vector miRNA sensors for real-time consecutive functional monitoring of miRNA profiling in living cells.

Methods

miR-200a, -200b, -21, -96, -146a, -10a, -155, and -221 in three PDAC cell lines (BxPC-3, CFPAC-1, SW1990), pancreatic epithelioid carcinoma cells (PANC-1), and human pancreatic nestin-expressing cells (hTERT-HPNE) were monitored by Asensors. Subsequently, the real-time consecutive functional profile of all miRNAs was evaluated.

Results

Selected miRNAs were detectable in all cell lines with high sensitivity and reproducibility. In the three PDAC cell lines, BxPC-3, CFPAC-1, and SW1990, the calibrated signal unit of the eight miRNAs Asensors was significantly lower than that of the Asensor control. However, in PANC-1 cells, miR-200a and -155 showed upregulation of target gene expression at 24 hours after infection with the sensors; at 48 hours, miR-200b and -155 displayed upregulation of reporter expression; and at 72 hours, reporter gene expression was upregulated by miR-200a and -200b. The result that miRNA could upregulate gene expression was further confirmed in miR-155 of hTERT-HPNE cells. Furthermore, miRNA activity varied among cell/tissue types and time.

Conclusion

It is possible that miRNA participates in the pathophysiology of pancreatic cancer, but the current popular methods do not accurately reveal the real-time miRNA function. Thus, this report provided a convenient, accurate, and sensitive approach to miRNA research.     

Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization

MicroRNAs (miRNAs) have emerged as key regulators in the pathogenesis of cancers where they can act as either oncogenes or tumor suppressors. Most miRNA measurement methods require total RNA extracts which lack critical spatial information and present challenges for standardization. We have developed and validated a method for the quantitative analysis of miRNA expression by in situ hybridization (ISH) allowing for the direct assessment of tumor epithelial expression of miRNAs. This co-localization based approach (called qISH) utilizes DAPI and cytokeratin immunofluorescence to establish subcellular compartments in the tumor epithelia, then multiplexed with the miRNA ISH, allows for quantitative measurement of miRNA expression within these compartments. We use this approach to assess miR-21, miR-92a, miR-34a, and miR-221 expression in 473 breast cancer specimens on tissue microarrays. We found that miR-221 levels are prognostic in breast cancer illustrating the high-throughput method and confirming that miRNAs can be valuable biomarkers in cancer. Furthermore, in applying this method we found that the inverse relationship between miRNAs and proposed target proteins is difficult to discern in large population cohorts. Our method demonstrates an approach for large cohort, tissue microarray-based assessment of miRNA expression.     

Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer

Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b, and miR-155, and down-regulation of miR-216a, miR-216b, miR-217, and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP, and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.     

Identification and Validation of Oncologic miRNA Biomarkers for Luminal A-like Breast Cancer

Introduction

Breast cancer is a common disease with distinct tumor subtypes phenotypically characterized by ER and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression, and altered miRNA expression has been demonstrated in a variety of cancer states presenting the potential for exploitation as cancer biomarkers. Blood provides an excellent medium for biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A-like (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting.

Methods

Blood samples were prospectively collected from patients with Luminal A-like breast cancer (n = 54) and controls (n = 56). RNA was extracted, reverse transcribed and subjected to microarray analysis (n = 10 Luminal A-like; n = 10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Expression of specific miRNAs was validated by RQ-PCR (n = 44 Luminal A; n = 46 Control) and potential relationships between circulating miRNA levels and clinicopathological features of breast cancer were investigated.

Results

Microarray analysis identified 76 differentially expressed miRNAs. ANN revealed 10 miRNAs for further analysis (miR-19b, miR-29a, miR-93, miR-181a, miR-182, miR-223, miR-301a, miR-423-5p, miR-486-5 and miR-652). The biomarker potential of 4 miRNAs (miR-29a, miR-181a, miR-223 and miR-652) was confirmed by RQ-PCR, with significantly reduced expression in blood of women with Luminal A-like breast tumors compared to healthy controls (p = 0.001, 0.004, 0.009 and 0.004 respectively). Binary logistic regression confirmed that combination of 3 of these miRNAs (miR-29a, miR-181a and miR-652) could reliably differentiate between cancers and controls with an AUC of 0.80.

Conclusion

This study provides insight into the underlying molecular portrait of Luminal A-like breast cancer subtype. From an initial 76 miRNAs, 4 were validated with altered expression in the blood of women with Luminal A-like breast cancer. The expression profiles of these 3 miRNAs, in combination with mammography, has potential to facilitate accurate subtype-specific breast tumor detection.     

Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth

MicroRNAs (miRNAs) play important roles in cancer development. By cloning and sequencing of a HPV16(+) CaSki cell small RNA library, we isolated 174 miRNAs (including the novel miR-193c) which could be grouped into 46 different miRNA species, with miR-21, miR-24, miR-27a, and miR-205 being most abundant. We chose for further study 10 miRNAs according to their cloning frequency and associated their levels in 10 cervical cancer- or cervical intraepithelial neoplasia-derived cell lines. No correlation was observed between their expression with the presence or absence of an integrated or episomal HPV genome. All cell lines examined contained no detectable miR-143 and miR-145. HPV-infected cell lines expressed a different set of miRNAs when grown in organotypic raft cultured as compared to monolayer cell culture, including expression of miR-143 and miR-145. This suggests a correlation between miRNA expression and tissue differentiation. Using miRNA array analyses for age-matched normal cervix and cervical cancer tissues, in combination with northern blot verification, we identified significantly deregulated miRNAs in cervical cancer tissues, with miR-126, miR-143, and miR-145 downregulation and miR-15b, miR-16, miR-146a, and miR-155 upregulation. Functional studies showed that both miR-143 and miR-145 are suppressive to cell growth. When introduced into cell lines, miR-146a was found to promote cell proliferation. Collectively, our data indicate that downregulation of miR-143 and miR-145 and upregulation of miR-146a play a role in cervical carcinogenesis.     

Plasma MicroRNA Panel for Minimally Invasive Detection of Breast Cancer

Over the last few years, circulating microRNAs (miRNAs) have emerged as promising novel and minimally invasive markers for various diseases, including cancer. We already showed that certain miRNAs are deregulated in the plasma of breast cancer patients when compared to healthy women. Herein we have further explored their potential to serve as breast cancer early detection markers in blood plasma. Circulating miR-127-3p, miR-376a and miR-652, selected as candidates from a miRNA array-based screening, were found to be associated with breast cancer for the first time (n = 417). Further we validated our previously reported circulating miRNAs (miR-148b, miR-376c, miR-409-3p and miR-801) in an independent cohort (n = 210) as elevated in the plasma of breast cancer patients compared to healthy women. We described, for the first time in breast cancer, an over-representation of deregulated miRNAs (miR-127-3p, miR-376a, miR-376c and miR-409-3p) originating from the chromosome 14q32 region. The inclusion of patients with benign breast tumors enabled the observation that miR-148b, miR-652 and miR-801 levels are even elevated in the plasma of women with benign tumors when compared to healthy controls. Furthermore, an analysis of samples stratified by cancer stage demonstrated that miR-127-3p, miR-148b, miR-409-3p, miR-652 and miR-801 can detect also stage I or stage II breast cancer thus making them attractive candidates for early detection. Finally, ROC curve analysis showed that a panel of these seven circulating miRNAs has substantial diagnostic potential with an AUC of 0.81 for the detection of benign and malignant breast tumors, which further increased to 0.86 in younger women (up to 50 years of age).     

Identification of postoperative prognostic microRNA predictors in hepatocellular carcinoma

Comparison of microRNA (miRNA) expression profiles in the noncancerous liver tissues adjacent to hepatocelluar carcinomas (HCCs) was a strategy to identify postoperative prognostic predictors in this study. Expression profiles of 270 miRNAs were determined in the paraneoplastic liver tissues of 12 HCC patients with known postoperative prognosis. A panel of candidate miRNA predictors was identified. The prognostic predictive value of these candidate miRNAs was then verified in 216 postoperative HCC patients. Univariate analysis identified 8 and 3 miRNA predictors for recurrence-free (RFS) and overall (OS) survivals, respectively. Multivariate analysis revealed high expression levels of miR-155 (HR, 2.002 [1.324-3.027]; P?=?.001), miR-15a (HR, 0.478 [0.248-0.920]; P?=?.027), miR-432 (HR, 1.816 [1.203-2.740]; P?=?.015), miR-486-3p (HR, 0.543 [0.330-0.893]; P?=?.016), miR-15b (HR, 1.074 [1.002-1.152]; P?=?.043) and miR-30b (HR, 1.102 [1.025-1.185]; P?=?.009) were significantly associated with RFS. When clinicopathological predictors were included, multivariate analysis revealed that tumor number and miR-432, miR-486-3p, and miR-30b expression levels remained significant as independent predictors for RFS. Additionally, expression knockdown of miR-155 in J7 and Mahlavu hepatoma cells resulted in decreased cell growth and enhanced cell death in xenograft tumors, suggesting an oncogenic effect of miR-155. In conclusion, significant prognostic miRNA predictors were identified through examination of miRNA expression levels in paraneoplastic liver tissues. Functional analysis of a miRNA predictor, miR-155, suggested that the prognostic miRNA predictors identified under this strategy could serve as potential molecular targets for anticancer therapy.     

Investigation of the efficacy of paclitaxel on some miRNAs profiles in breast cancer stem cells

Understanding of the functions of microRNAs in breast cancer and breast cancer stem cells have been a hope for the development of new molecular targeted therapies. Here, it is aimed to investigate the differences in the expression levels of let-7a, miR-10b, miR-21, miR-125b, miR-145, miR-155, miR-200c, miR-221, miR-222 and miR-335, which associated with gene and proteins in MCF-7 (parental) and MCF-7s (Mammosphere/stem cell-enriched population/CD44+/CD24-cells) cells treated with paclitaxel. MCF-7s were obtained from parental MCF-7 cells. Cytotoxic activity of paclitaxel was determined by ATP assay. Total RNA isolation and cDNA conversion were performed from the samples. Changes in expression levels of miRNAs were examined by RT-qPCR. Identified target genes and proteins of miRNAs were analyzed with RT-qPCR and western blot analysis, respectively. miR-125b was significantly expressed (2.0946-fold; p = 0.021) in MCF-7s cells compared to control after treatment with paclitaxel. Downregulation of SMO, STAT3, NANOG, OCT4, SOX2, ERBB2 and ERBB3 and upregulation of TP53 genes were significant after 48 h treatment in MCF-7s cells. Protein expressions of SOX2, OCT4, SMAD4, SOX2 and OCT4 also decreased. Paclitaxel induces miR-125b expression in MCF-7s cells. Upregulation of miR-125b may be used as a biomarker for the prediction of response to paclitaxel treatment in breast cancer.     

MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer

Cancer-associated fibroblasts (CAFs) promote tumorigenesis, growth, invasion and metastasis of cancer, whereas normal fibroblasts (NFs) are thought to suppress tumor progression. Little is known about miRNAs expression differences between CAFs and NFs or the patient-to-patient variability in miRNAs expression in breast cancer. We established primary cultures of CAFs and paired NFs from six resected breast tumor tissues that had not previously received radiotherapy or chemotherapy treatment and analyzed with miRNAs microarrays. The array data were analyzed using paired SAM t-test and filtered according to α and q values. Pathway analysis was conducted using DAVID v6.7. We identified 11 dysregulated miRNAs in CAFs: three were up-regulated (miR-221-5p, miR-31-3p, miR-221-3p), while eight were down-regulated (miR-205, miR-200b, miR-200c, miR-141, miR-101, miR-342-3p, let-7g, miR-26b). Their target genes are known to affect cell differentiation, adhesion, migration, proliferation, secretion and cell-cell interaction. By our knowledge it is firstly identify the expression profiles of miRNAs between CAFs and NFs and revealed their regulation on the associated signaling pathways.     

Expression patterns of microRNAs 155 and 451 during normal human erythropoiesis

To clarify the roles of microRNAs (miRNAs) in erythropoiesis, the expression of miR-155, miR-221, miR-223, and miR-451 were analyzed during the differentiation of purified normal human erythroid progenitors in a liquid culture system. Cells increased almost 500-fold in a number, and differentiated to benzidine-positive mature erythroblasts. Analyses of miRNA expression using the quantitative real-time polymerase chain reaction showed that the expression level of miR-155 decreased about 200-fold, and that the expression of miR-451 increased about 270-fold during 12 days of cultures. A moderate down-regulation of miR-221 and miR-223 was observed. MiR-451 was expressed in red blood cells about 10⁴-fold more than in granulocytes, obtained from normal human peripheral blood. These observations suggest that miR-155 and miR-451 are key molecules for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be a relevant method for analyzing pathological erythropoiesis.     

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

Alterations in Circulating miRNA Levels following Early-Stage Estrogen Receptor-Positive Breast Cancer Resection in Post-Menopausal Women

Introduction

Circulating microRNAs (miRNAs) exhibit remarkable stability and may serve as biomarkers in several clinical cancer settings. The aim of this study was to investigate changes in the levels of specific circulating miRNA following breast cancer surgery and evaluate whether these alterations were also observed in an independent data set.

Methods

Global miRNA analysis was performed on prospectively collected serum samples from 24 post-menopausal women with estrogen receptor-positive early-stage breast cancer before surgery and 3 weeks after tumor resection using global LNA-based quantitative real-time PCR (qPCR).

Results

Numbers of specific miRNAs detected in the samples ranged from 142 to 161, with 107 miRNAs detectable in all samples. After correction for multiple comparisons, 3 circulating miRNAs (miR-338-3p, miR-223 and miR-148a) exhibited significantly lower, and 1 miRNA (miR-107) higher levels in post-operative vs. pre-operative samples (p<0.05). No miRNAs were consistently undetectable in the post-operative samples compared to the pre-operative samples. Subsequently, our findings were compared to a dataset from a comparable patient population analyzed using similar study design and the same qPCR profiling platform, resulting in limited agreement.

Conclusions

A panel of 4 circulating miRNAs exhibited significantly altered levels following radical resection of primary ER+ breast cancers in post-menopausal women. These specific miRNAs may be involved in tumorigenesis and could potentially be used to monitor whether all cancer cells have been removed at surgery and/or, subsequently, whether the patients develop recurrence.     

Validation of Expression Patterns for Nine miRNAs in 204 Lymph-Node Negative Breast Cancers

Introduction

Although lymph node negative (LN-) breast cancer patients have a good 10-years survival (∼85%), most of them still receive adjuvant therapy, while only some benefit from this. More accurate prognostication of LN- breast cancer patient may reduce over- and under-treatment. Until now proliferation is the strongest prognostic factor for LN- breast cancer patients. The small molecule microRNA (miRNA) has opened a new window for prognostic markers, therapeutic targets and/or therapeutic components. Previously it has been shown that miR-18a/b, miR-25, miR-29c and miR-106b correlate to high proliferation.

Methods

The current study validates nine miRNAs (miR-18a/b miR-25, miR-29c, miR-106b, miR375, miR-424, miR-505 and let-7b) significantly correlated with established prognostic breast cancer biomarkers. Total RNA was isolated from 204 formaldehyde-fixed paraffin embedded (FFPE) LN- breast cancers and analyzed with quantitative real-time Polymerase Chain Reaction (qPCR). Independent T-test was used to detect significant correlation between miRNA expression level and the different clinicopathological features for breast cancer.

Results

Strong and significant associations were observed for high expression of miR-18a/b, miR-106b, miR-25 and miR-505 to high proliferation, oestrogen receptor negativity and cytokeratin 5/6 positivity. High expression of let-7b, miR-29c and miR-375 was detected in more differentiated tumours. Kaplan-Meier survival analysis showed that patients with high miR-106b expression had an 81% survival rate vs. 95% (P = 0.004) for patients with low expression.

Conclusion

High expression of miR-18a/b are strongly associated with basal-like breast cancer features, while miR-106b can identify a group with higher risk for developing distant metastases in the subgroup of Her2 negatives. Furthermore miR-106b can identify a group of patients with 100% survival within the otherwise considered high risk group of patients with high proliferation. Using miR-106b as a biomarker in conjunction to mitotic activity index could thereby possibly save 18% of the patients with high proliferation from overtreatment.     

Circulating MicroRNAs as Easy-to-Measure Aging Biomarkers in Older Breast Cancer Patients: Correlation with Chronological Age but Not with Fitness/Frailty Status

Circulating microRNAs (miRNAs) hold great promise as easily accessible biomarkers for diverse (patho)physiological processes, including aging. We have compared miRNA expression profiles in cell-free blood from older versus young breast cancer patients, in order to identify “aging miRNAs” that can be used in the future to monitor the impact of chemotherapy on the patient’s biological age. First, we assessed 175 miRNAs that may possibly be present in serum/plasma in an exploratory screening in 10 young and 10 older patients. The top-15 ranking miRNAs showing differential expression between young and older subjects were further investigated in an independent cohort consisting of another 10 young and 20 older subjects. Plasma levels of miR-20a-3p, miR-30b-5p, miR106b, miR191 and miR-301a were confirmed to show significant age-related decreases (all p≤0.004). The remaining miRNAs included in the validation study (miR-21, miR-210, miR-320b, miR-378, miR-423-5p, let-7d, miR-140-5p, miR-200c, miR-374a, miR376a) all showed similar trends as observed in the exploratory screening but these differences did not reach statistical significance. Interestingly, the age-associated miRNAs did not show differential expression between fit/healthy and non-fit/frail subjects within the older breast cancer cohort of the validation study and thus merit further investigation as true aging markers that not merely reflect frailty.     

Differentially Expressed MicroRNAs in Postpartum Breast Cancer in Hispanic Women

The risk of breast cancer transiently increases immediately following pregnancy; peaking between 3-7 years. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to be dysregulated in breast cancer. We conducted miRNA profiling of 56 tumors from a case series of multiparous Hispanic women and assessed the pattern of expression by time since last full-term pregnancy. A data-driven splitting analysis on the pattern of 355 miRNAs separated the case series into two groups: a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n = 12), and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n = 44). We identified 15 miRNAs with significant differential expression between the early and late postpartum groups; 60% of these miRNAs are encoded on the X chromosome. Ten miRNAs had a two-fold or higher difference in expression with miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were underexpressed in the early versus the late postpartum group. The DNA methylation of three out of five tested miRNAs (miR-31, miR-135b, and miR-138) was lower in the early versus late postpartum group, and negatively correlated with miRNA expression. Here we show that miRNAs are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic dysfunction may be operative in postpartum breast cancers.