Systems biology based miRNA-mRNA expression pattern analysis of Emodin in breast cancer cell lines.

Breast cancer has been among the most prominent cancers with high mortality. Currently most of the offered therapeutics are toxic; hence, less toxic therapeutic intervention is required. Here, we studied the molecular mechanisms of the effect of a phytoestrogen Emodin on estrogen receptor positive MCF-7 and negative MDA-MB-231 cells by carrying out a comprehensive network assessment. Differentially expressed microRNAs along with their previously identified differentially expressed mRNAs were analyzed through microarrays by using integrative systems biology approach. For each cell line miRNA-target gene networks were built, gene ontology and pathway enrichment analyses were performed, enrichment maps were constructed and the potential key genes, miRNAs and miRNA-gene interactions were studied.

miR-770-5p regulates EMT and invasion in TNBC cells by targeting DNMT3A.

MicroRNAs (miRNAs) are shown to regulate various processes in cancer like motility and invasion that are key features of the metastatic triple negative breast cancer (TNBCs). Epithelial-mesenchymal transition (EMT) is one of the well-defined cellular transitioning processes characterized with reduced E-cadherin expression and increased mesenchymal molecules such as Vimentin or Snail thereby gives the cells mobility and invasive character. Aberrant DNA methylation by DNA methyltransferases (DNMTs) plays an important role in carcinogenesis. It is well known that DNMTs are required for transcriptional silencing of tumor-associated genes. DNMT3A-induced promoter hypermethylation of E-cadherin has also been known to improve cancer metastasis. Our results indicated that miR-770-5p could downregulate Vimentin and Snail expression levels, while increasing or restoring the expression of E-Cadherin hence, leading to inhibition of EMT phenotypes along with motility and invasion. Specifically, we showed that overexpression of miR-770-5p restored the expression of E-Cadherin in MDA-MB-231 cells via directly targeting DNMT3A. We also observed the change in the spindled shapes showing the loss of mesenchymal characteristics and gain of epithelial phenotype in miR-770-5p overexpressing cells. When considered together, our results show that miR-770-5p could effectively inhibit invasion potential driven by EMT.

MiR-25 and KLF4 relationship has early prognostic significance in the development of cervical cancer.

Cervical squamous cell carcinoma (SCC) is one of the common cancer types among women. MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in the formation and development of many cancer types by regulating expression of their targets. While many studies have investigated the relationship between miRNAs and cervical cancer, no robust miRNA biomarkers have been defined yet for diagnosis of cervical lesions. In this study, we performed a statistical meta-analysis to identify miRNAs and a class compassion analysis to evaluate mRNAs with the power to discriminate between normal, intraepithelial lesions and invasive cancer samples. Differentially expressed (DE) mRNAs were compared with the targets of meta-miRNAs. After bioinfomatics analysis and qRT-PCR validations with cytology samples and FFPE tissues, we defined miR-25 and its target KLF4 (Kruppel-like factor 4) as candidate biomarkers for in vitro studies. Our results showed that miR-25 expression was significantly higher in precancerous lesions and invasive carcinoma while presenting consistent expression patterns in both cytological and FFPE tissue samples. In line with this, its direct target KLF4 expression decreased in precancerous lesions in cytological samples and also in the invasive cancer group in FFPE tissues. Furthermore, in vitro studies showed that mir-25 inhibition decreased proliferation and motility of HeLa cells and promoted an increase in the protein level of KLF4. We conclude that inhibition of miR-25 may upregulate KLF4 expression and regulate cell proliferation and motility in cervical cancer.

Involvement of miR-770-5p in trastuzumab response in HER2 positive breast cancer cells.

miRNAs may play effective roles in breast cancer so modulating their expression levels could have therapeutic benefits. Recent studies have found the combination of miRNA-based therapeutics with conventional drugs as promising. This study aimed to find drug-responsive miRNAs, and explore their anticancer activities in HER2+ breast cancer cells and regulatory role in the trastuzumab response. qRT-PCR-array analysis was performed with effective concentrations of tamoxifen and trastuzumab treated BT-474, SK-BR-3 and MCF-7 cells. Motility and invasion analyses were performed with wound healing and xCELLigence impedance-based assays respectively. Viability of cells following mimic transfection and drug treatment was assessed by WST-1 assay. Western blot analysis was used to assess miR-770-5p regulation of proteins and their phosphorylated forms. The clinical relevance of miR-770-5p was examined by TCGA data analysis. The qRT-PCR-array results indicated that miR-770-5p was responsive in a drug and cell line independent manner. Overexpression of miR-770-5p inhibited the motility and cell invasion through regulation of AKT and ERK proteins. Additionally, miR-770-5p potentiated the effectiveness of trastuzumab. Thus, regulating the expression level of miR-770-5p in combination with trastuzumab treatment may simultaneously inhibit the downstream elements of PI3K and MAPK signalling, thereby blocking the proliferation, motility and invasion capacities of HER2+ breast cancer cells.

Determination of Usnic Acid Responsive miRNAs in Breast Cancer Cell Lines.

OBJECTIVE: Breast Cancer (BC) is the most common type of cancer diagnosed in women. A common treatment strategy for BC is still not available because of its molecular heterogeneity and resistance is developed in most of the patients through the course of treatment. Therefore, alternative medicine resources as being novel treatment options are needed to be used for the treatment of BC. Usnic Acid (UA) that is one of the secondary metabolites of lichens used for different purposes in the field of medicine and its anti-proliferative effect has been shown in certain cancer types, suggesting its potential use for the treatment. METHODS: Anti-proliferative effect of UA in BC cells (MDA-MB-231, MCF-7, BT-474) was identified through MTT analysis. Microarray analysis was performed in cells treated with the effective concentration of UA and UA-responsive miRNAs were detected. Their targets and the pathways that they involve were determined using a miRNA target prediction tool. RESULTS: Microarray experiments showed that 67 miRNAs were specifically responsive to UA in MDA-MB-231 cells while 15 and 8 were specific to BT-474 and MCF-7 cells, respectively. The miRNA targets were mostly found to play role in Hedgehog signaling pathway. TGF-Beta, MAPK and apoptosis pathways were also the prominent ones according to the miRNA enrichment analysis. CONCLUSION: The current study is important as being the first study in the literature which aimed to explore the UA related miRNAs, their targets and molecular pathways that may have roles in the BC. The results of pathway enrichment analysis and anti-proliferative effects of UA support the idea that UA might be used as a potential alternative therapeutic agent for BC treatment.

Meta-microRNA Biomarker Signatures to Classify Breast Cancer Subtypes.

Breast cancer is one of the leading causes of morbidity and mortality that is in need of novel diagnostics and therapeutics. Meta-analysis of microarray data offers promise to combine studies and provide more robust results. We report here a molecular classification of pathological subtypes (estrogen receptor [ER], progesterone receptor [PR], and Human Epidermal Growth Factor Receptor 2 [HER2]) of breast cancers with microRNA (miRNA)-dependent signatures. A ranking-based meta-analysis approach was applied to eight independent microarray data sets and meta-miRNA lists were obtained that are specific to each breast cancer subtype. The comparison of the lists with miRCancer and the PhenomiR 2.0 databases pointed out nine prominent miRNAs: let-7b-5p, let-7c-5p, let-7e-5p, miR-130a-3p, miR-30a-5p, miR-92a-1-5p, miR-211-5p, miR-500a-3p, and miR-516b-3p. Further analysis conducted with the TCGA data showed that these miRNAs can differentiate tumors from normal samples as well as discriminate the molecular subtypes of breast cancer. According to the PAM50 classification, three of these miRNAs (let-7b-5p, let-7c-5p, and miR-30a-5p) downregulated significantly, whereas miR-130a-3p, miR-92a-1-5p, miR-211-5p, and miR-500a-3p upregulated in tumors from the luminal A to the basal-like subtypes. When the prominent meta-miRNAs and their targets were analyzed, they appeared to be taking part in important signaling pathways in cancer such as the PI3K-Akt signaling and the p53 signaling pathways. Furthermore, the regulatory genes, which are key players for ER, PR, and ErBb signaling pathways, were found to be under control of several meta-miRNAs. These meta-miRNAs and the genes they are regulating offer new promise for future translational research and potential targets for precision medicine diagnostics.

Do Perineuronal Net Elements Contribute to Pathophysiology of Spinal Muscular Atrophy? In Vitro and Transcriptomics Insights.

Spinal muscular atrophy (SMA) is one of the most common childhood onset neurodegenerative disorders in global health whereby novel biomarkers and therapeutic targets are sorely needed. SMA is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons in the brain stem and spinal cord that leads to mortality in infants worldwide. In majority of the patients, SMA is caused by homozygous deletion of the SMN1 gene. The clinical spectrum of the SMA displays, however, large person-to-person variations where the underlying mechanisms are poorly understood. We report in this study transcriptomics insights gleaned from patients with the severe type I (GM03813 and GM09677) and the mild type III. Pathway enrichment and functional analysis showed that especially extracellular matrix (ECM), synapse organization, and ECM receptor interaction pathways were affected. Among the neural ECM components, hyaluronan and proteoglycan link protein (HAPLN1), which is a key triggering molecule of the perineuronal net (PNN), was significantly downregulated in type I fibroblasts compared to type III. PNN is a specialized form of neural ECM around the neuronal cell bodies and dendrites in the central nervous system. In addition, we evaluated the PNN expression in vitro in a model established by SMN silencing in the PC12 rat pheochromocytoma cell line which can be differentiated into neurons with nerve growth factor treatment. In this neuronal in vitro model, we found that HAPLN1 showed a significant 50% decrease. Our results describe the association between PNN elements, especially HAPLN1, and SMA pathophysiology for the first time. These observations collectively inform future translational research on SMA for discovery of novel molecular targets for diagnostics and precision medicine innovation.

hsa-miR-301a- and SOX10-dependent miRNA-TF-mRNA regulatory circuits in breast cancer.

Breast cancer is the most common cancer among women and the molecular pathways that play main roles in breast cancer regulation are still not completely understood. MicroRNAs (miRNAs) and transcription factors (TFs) are important regulators of gene expression. It is important to unravel the relation of TFs, miRNAs, and their targets within regulatory networks to clarify the processes that cause breast cancer and the progression of it. In this study, mRNA and miRNA expression studies including breast tumors and normal samples were extracted from the GEO microarray database. Two independent mRNA studies and a miRNA study were selected and reanalyzed. Differentially expressed (DE) mRNAs and miRNAs between breast tumor and normal samples were listed by using BRBArray Tools. CircuitsDB2 analysis conducted with DE miRNAs and mRNAs resulted in 3 significant circuits that are SOX10- and hsamiR-301a-dependent. The following significant circuits were characterized and validated bioinformatically by using web-based tools: SOX10→hsa-miR-301a→HOXA3, SOX10→hsa-miR-301a→KIT, and SOX10→hsa-miR-301a→NFIB. It can be concluded that regulatory motifs involving miRNAs and TFs may be useful for understanding breast cancer regulation and for predicting new biomarkers.

DICER1 gene and miRNA dysregulation in mesenchymal stem cells of patients with myelodysplastic syndrome and acute myeloblastic leukemia.

Multipotent mesenchymal stem cells (MSC) are key components of the bone marrow (BM) microenvironment. The contribution of this microenvironment to the pathophysiology of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is not well defined. A recent study in mice demonstrated that DICER1 gene deletion in osteoprogenitor cells from the BM microenvironment suppressed osteogenic differentiation and induced MDS and AML-like haematological findings. The present study evaluated the expression profiles of microRNAs (miRNAs) and DICER1 gene in BM-derived MSC of patients with AML (n=12), MDS (n=10) and healthy controls (HC) (n=8).miRNA expression profiles were analyzed by microarray and confirmations were performed using quantitative real-time PCR (qRT-PCR). Patient MSC displayed impaired proliferative and differentiation potential compared to HC. DICER1 gene expression was lower in MSC from MDS and AML patients than HC and some differentially expressed miRNAs indicated the potential involvement of DICER1 in the pathogenesis of MDS and AML. qRT-PCR confirmation revealed down-regulated miRNAs (hsa-miR-30d-5p, hsa-miR-222-3p and hsa-miR-30a-3p in MDS; hsa-miR-1275, hsa-miR-4725-5p and hsa-miR-143-3p in AML) and over-expressed miRNAs (hsa-miR-4462 in MDS; hsa-miR-134-5p and hsa-miR-874-3p in AML) in MDS and AML. Thus, our findings validate the results of the aforementioned animal study and demonstrate downregulation of DICER1 gene and abnormal miRNA profile in MDS and AML, which may have implications for understanding MDS and AML pathogenesis and contribute to developing targeted treatment strategies.

Construction of miRNA-miRNA networks revealing the complexity of miRNA-mediated mechanisms in trastuzumab treated breast cancer cell lines.

Trastuzumab is a monoclonal antibody frequently used to prevent the progression of HER2+ breast cancers, which constitute approximately 20% of invasive breast cancers. microRNAs (miRNAs) are small, non-coding RNA molecules that are known to be involved in gene regulation. With their emerging roles in cancer, they are recently promoted as potential candidates to mediate therapeutic actions by targeting genes associated with drug response. In this study we explored miRNA-mediated regulation of trastuzumab mechanisms by identifying the important miRNAs responsible for the drug response via homogenous network analysis. Our network model enabled us to simplify the complexity of miRNA interactions by connecting them through their common pathways. We outlined the functionally relevant miRNAs by constructing pathway-based miRNA-miRNA networks in SKBR3 and BT474 cells, respectively. Identification of the most targeted genes revealed that trastuzumab responsive miRNAs favourably regulate the repression of targets with longer 3'UTR than average considered to be key elements, while the miRNA-miRNA networks highlighted central miRNAs such as hsa-miR-3976 and hsa-miR-3671 that showed strong interactions with the remaining members of the network. Furthermore, the clusters of the miRNA-miRNA networks showed that trastuzumab response was mostly established through cancer related and metabolic pathways. hsa-miR-216b was found to be the part of the most powerful interactions of metabolic pathways, which was defined in the largest clusters in both cell lines. The network based representation of miRNA-miRNA interactions through their shared pathways provided a better understanding of miRNA-mediated drug response and could be suggested for further characterization of miRNA functions.

Identification of Polycystic Ovary Syndrome (PCOS) Specific Genes in Cumulus and Mural Granulosa Cells.

Polycystic ovary syndrome (PCOS) is a metabolic and endocrine disorder which affects women of reproductive age with prevalence of 8-18%. The oocyte within the follicle is surrounded by cumulus cells (CCs), which connect with mural granulosa cells (MGCs) that are responsible for secreting steroid hormones. The main aim of this study is comparing gene expression profiles of MGCs and CCs in PCOS and control samples to identify PCOS-specific differentially expressed genes (DEGs). In this study, two microarray databases were searched for mRNA expression microarray studies performed with CCs and MGCs obtained from PCOS patients and control samples. Three independent studies were selected to be integrated with naive meta-analysis since raw meta-data from these studies were found to be highly correlated. DEGs in these somatic cells were identified for PCOS and control groups. This study enabled us to reveal dysregulation in MAPK (mitogen activated protein kinase), insulin and Wnt signaling pathways between CCs and MGCs in PCOS. The meta-analysis results together with qRT-PCR validations provide evidence that molecular signaling is dysregulated through MGCs and CCs in PCOS, which is important for follicle and oocyte maturation and may contribute to the pathogenesis of the syndrome.

The ability to generate senescent progeny as a mechanism underlying breast cancer cell heterogeneity.

BACKGROUND: Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. METHODOLOGY/PRINCIPAL FINDINGS: A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21(Cip1) correlated with senescence in these cell lines. p21(Cip1) knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. CONCLUSIONS/SIGNIFICANCE: Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.

Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues.

Quantitative gene expression measurements from tumor tissue are frequently compared with matched normal and/or adjacent tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in breast cancer for making comparisons among tumor subtypes, cell lines, and benign/malignant tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB, TBP, GAPDH, SDHA, HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals, on average. The expression of the studied RGs in breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRT-PCR data in the analysis of normal matched tumor breast tissue pairs by both programs. In addition, the expression of the gelsolin (GSN) gene, a well-known downregulated target in breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of tumor-normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in tumor-normal paired breast cancer qRT-PCR studies.

Functional genomics in translational cancer research: focus on breast cancer.

Conventional molecular and genetic methods for studying cancer are limited to the analysis of one locus at a time. A cluster of genes that are regulated together can be identified by DNA microarray, and the functional relationships can uncover new aspects of cancer biology. Breast cancer can be used to provide a model to demonstrate the current approaches to the molecular analysis of cancer. Meta-analysis is an important tool for the identification and validation of differentially expressed genes to increase power in clinical and biological studies across different sets of data. Recently, meta-analysis approaches have been applied to large collections of microarray datasets to investigate molecular commonalities of multiple cancer types not only to find the common molecular pathways in tumour development but also to compare the individual datasets to other cancer datasets to identify new sets of genes. Several investigators agree that microarray results should be validated. One commonly used method is quantitative reverse transcription PCR (qRT-PCR) to validate the expression profiles of the target genes obtained through microarray experiments. qRT-PCR is attractive for clinical use, since it can be automated and performed on fresh or archived formalin-fixed, paraffin-embedded tissue samples. The outcome of these analyses might accelerate the application of basic research findings into daily clinical practice through translational research and may have an impact on foreseeing the clinical outcome, predicting tumour response to specific therapy, identification of new prognostic biomarkers, discovering targets for the development of novel therapies and providing further insights into tumour biology.

A resampling-based meta-analysis for detection of differential gene expression in breast cancer.

BACKGROUND: Accuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures. METHODS: A resampling-based meta-analysis strategy, which involves the use of resampling and application of distribution statistics in combination to assess the degree of significance in differential expression between sample classes, was developed. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. Expression of the genes, selected from the gene list for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes were tested on 10 independent primary IDC samples and matched non-tumor controls by real-time qRT-PCR. Other existing breast cancer microarray datasets were used in support of the resampling-based meta-analysis. RESULTS: The two independent microarray studies were found to be comparable, although differing in their experimental methodologies (Pearson correlation coefficient, R = 0.9389 and R = 0.8465 for ductal and lobular samples, respectively). The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. The expression results of the selected genes obtained through real-time qRT-PCR supported the meta-analysis results. CONCLUSION: The proposed meta-analysis approach has the ability to detect a set of differentially expressed genes with the least amount of within-group variability, thus providing highly stable gene lists for class prediction. Increased statistical power and stringent filtering criteria used in the present study also make identification of novel candidate genes possible and may provide further insight to improve our understanding of breast cancer development.

Lack of association between the MDM2-SNP309 polymorphism and breast cancer risk.

BACKGROUND: A T-to-G polymorphism (SNP309) at the promoter region of MDM2 has been recently reported to extend the Sp1 binding site that positively regulates the MDM2 transcription level and consequently, its expression level. MDM2 is the negative regulator of p53 tumor suppressor protein and elevated levels of MDM2 hamper the stress response driven by the p53 pathway. Whether MDM2-SNP309 was associated with breast cancer as a predisposing factor was investigated. PATIENTS AND METHODS: A case-control study of 223 females diagnosed with breast cancer and 149 female controls sampled from the Turkish population was carried out and the T/G MDM2-SNP309 genotype of participants was determined. RESULTS: There was no significant association of the G/G or G/T genotypes with breast cancer risk (odds ratio (OR) 1.14, 95% confidence interval (CI) 0.59-2.22, and OR 1.20, 95% CI 0.67-2.12, respectively). Stratification of the data for onset age or for menopausal status at the time of diagnosis also revealed no association for either group.