Expression of miRNAs Targeting <Emphasis Type="Italic">mTOR</Emphasis> and <Emphasis Type="Italic">S6K1</Emphasis> Genes of mTOR Signaling Pathway Including miR-96, miR-557, and miR-3182 in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3′-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.     

In-silico investigations of selective miRNA-gene targets and their validation studies in obstructive sleep apnea (OSA) patient cohorts

BackgroundObstructive sleep apnoea (OSA) is a prevalent form of sleep disordered breathing which results in sleep fragmentation and deprivation. Obesity and cardiovascular disorders are the major risk factors associated with OSA. Molecular analysis of the factors associated with OSA could demarcate the clinical analysis pattern in a population.ObjectiveThis study pertains to in-silico analyses of miRNA and their gene targets with validation for their potential role in OSA as putative biomarker candidates.MethodsmiRDB, TargetScan and miRanda databases were used to identify targets of miR-27 and let-7 that have documented role in OSA and co-related obesity and cardiovascular disorders. Quantitative PCR was used to analyze expression pattern of miR-27 and let-7 in obese and non-obese OSA patient cohorts with respective controls. In-silico analysis was done using PatchDoc to obtain atomic contact energy (ACE) scores that indicated the docked gene targets to the predicted miRNA structures. The docked structures were analysed using Maestro Suite 11 for the hydrogen and aromatic interactions.ResultsDownregulation of miR-27 and let-7 in OSA compared to controls was observed. In-silico data analysis was performed for gene targets (TGFBR1, TGFBR2, SMAD2, SMAD4, CRY2 and CNR1) of the selected miRNAs (miR-27 and let-7). Among all, CNR1 and CRY2 were found to be better targets for miR-27 and let-7 respectively as per ACE scores, ROC scores and expression fold change in OSA.ConclusionOur study gives insights to the expression profiling of miR-27 and let-7 and explore a set of potential target genes (CNR1 and CRY2) of these two miRNAs for a promising clinical relevance in OSA.     

MicroRNA-338-3p as a novel therapeutic target for intervertebral disc degeneration

Recent studies have demonstrated the pivotal role played by microRNAs (miRNAs) in the etiopathogenesis of intervertebral disc degeneration (IDD). The study of miRNA intervention in IDD models may promote the advancement of miRNA-based therapeutic strategies. The aim of the current study was to investigate whether intradiscal delivery of miRNA can attenuate IDD development. Our results showed that miR-338-3p expression was significantly increased in the nucleus pulposus (NP) of patients with IDD. Moreover, there was a statistically significant positive correlation between the expression level of miR-338-3p and the severity of IDD. Our functional studies showed that miR-338-3p significantly influenced the expression of extracellular matrix synthesis genes, as well as the proliferation and apoptosis of NP cells. Mechanistically, miR-338-3p aggravated IDD progression by directly targeting SIRT6, a negative regulator of the MAPK/ERK pathway. Intradiscal injection of antagomir-338-3p significantly decelerated IDD development in mouse models. Our study is the first to identify miR-338-3p as a mediator of IDD and thus may be a promising target for rescuing IDD.Subject terms: Genetic markers, Translational research, Gene therapy     

Suitable reference genes for relative quantification of miRNA expression in prostate cancer

Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa- miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa- miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.     

Polyphenol-Enriched Blueberry Preparation Controls Breast Cancer Stem Cells by Targeting FOXO1 and miR-145

Scientific evidence supports the early deregulation of epigenetic profiles during breast carcinogenesis. Research shows that cellular transformation, carcinogenesis, and stemness maintenance are regulated by epigenetic-specific changes that involve microRNAs (miRNAs). Dietary bioactive compounds such as blueberry polyphenols may modulate susceptibility to breast cancer by the modulation of CSC survival and self-renewal pathways through the epigenetic mechanism, including the regulation of miRNA expression. Therefore, the current study aimed to assay the effect of polyphenol enriched blueberry preparation (PEBP) or non-fermented blueberry juice (NBJ) on the modulation of miRNA signature and the target proteins associated with different clinical-pathological characteristics of breast cancer such as stemness, invasion, and chemoresistance using breast cancer cell lines. To this end, 4T1 and MB-MDM-231 cell lines were exposed to NBJ or PEBP for 24 h. miRNA profiling was performed in breast cancer cell cultures, and RT-qPCR was undertaken to assay the expression of target miRNA. The expression of target proteins was examined by Western blotting. Profiling of miRNA revealed that several miRNAs associated with different clinical-pathological characteristics were differentially expressed in cells treated with PEBP. The validation study showed significant downregulation of oncogenic miR-210 expression in both 4T1 and MDA-MB-231 cells exposed to PEBP. In addition, expression of tumor suppressor miR-145 was significantly increased in both cell lines treated with PEBP. Western blot analysis showed a significant increase in the relative expression of FOXO1 in 4T1 and MDA-MB-231 cells exposed to PEBP and in MDA-MB-231 cells exposed to NBJ. Furthermore, a significant decrease was observed in the relative expression of N-RAS in 4T1 and MDA-MB-231 cells exposed to PEBP and in MDA-MB-231 cells exposed to NBJ. Our data indicate a potential chemoprevention role of PEBP through the modulation of miRNA expression, particularly miR-210 and miR-145, and protection against breast cancer development and progression. Thus, PEBP may represent a source for novel chemopreventative agents against breast cancer.     

WNT1 Gene from WNT Signaling Pathway Is a Direct Target of miR-122 in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an invasive form of hepatic cancer arising from the accumulation of multiple genetic alterations. In this study, the causal role of disturbed canonical Wnt/β-catenin pathway was approved, and some of HCC-driven important gene candidates were determined. MicroRNAs (miRNAs), small non-coding RNAs, are the key regulators of important cancer genes, and their participation in tumorigenesis has been shown. By reviewing literature, WNT1 gene with functional significance was selected to approve miRNAs as new subjects for targeted therapy.For proper and fast miRNA detection and also confirmation of the role of bioinformatics in obtaining practical data, we benefited from different bioinformatics tools such as TargetScan, miRanda, and DIANA. In order to use an HCC model, we used HepG2 cell line. Luciferase assay was applied to assess the ability of the selected miRNAs in targeting WNT1 3′-UTR. To overexpress the selected miRNA in HepG2 cell line, viral construct was prepared. Quantitative real-time PCR was performed to evaluate selected miRNA and target gene expression levels. miR-122 was selected according to data concerning various bioinformatics tools.miR-122 was downregulated and WNT1 gene expression was upregulated in HepG2 cell line. After viral construct transduction, miR-122 expression was elevated and WNT1 expression was notably declined. Finally, we introduced WNT1 gene as one of the important genes in HCC, and also, we showed that miR-122 can regulate WNT1 gene expression.Moreover, our study determines the potential of bioinformatics analyses in providing accurate and reliable data for miRNA: messenger RNA (mRNA) prediction.     

Isoform specific gene auto-regulation via miRNAs: a case study on miR-128b and ARPP-21

In this study, we investigate whether miRNAs located within “host” protein-coding genes may regulate the expression of their host genes. We find that 43 of 174 miRNAs encoded within RefSeq genes are predicted to target their host genes. Statistical analysis of this phenomenon suggests that gene auto-regulation via miRNAs may be under positive selective pressure. Our analysis also indicates that several of the 43 miRNAs have a much lower expectation of targeting their host genes by chance than others. Among these examples, we identify miR-128b:ARPP-21 (cyclic AMP-regulated phosphoprotein, 21 kD) as a case in which both the miRNA and the target site are also evolutionarily conserved. We provide experimental support for this miRNA:target interaction via reporter silencing assays, and present evidence that this isoform-specific gene auto-regulation has been preserved in vertebrate species in order to prevent detrimental consequences of ARPP-21 over-expression in brain.     

DNA methylation-regulated microRNA pathways in ovarian serous cystadenocarcinoma: A meta-analysis

Epigenetic regulation has been linked to the initiation and progression of cancer. Aberrant expression of microRNAs (miRNAs) is one such mechanism that can activate or silence oncogenes (OCGs) and tumor suppressor genes (TSGs) in cells. A growing number of studies suggest that miRNA expression can be regulated by methylation modification, thus triggering cancer development. However, there is no comprehensive in silico study concerning miRNA regulation by direct DNA methylation in cancer. Ovarian serous cystadenocarcinoma (OSC) was therefore chosen as a tumor model for the present work.Twelve batches of OSC data, with at least 35 patient samples in each batch, were obtained from The Cancer Genome Atlas (TCGA) database. The Spearman rank correlation coefficient (SRCC) was used to quantify the correlation between the CpG DNA methylation level and miRNA expression level. Meta-analysis was performed to reduce the effects of biological heterogeneity among different batches. MiRNA-target interactions were also inferred by computing SRCC and meta-analysis to assess the correlation between miRNA expression and cancer-associated gene expression and the interactions were further validated by a query against the miRTarBase database.A total of 26 potential epigenetic-regulated miRNA genes that can target OCGs or TSGs in OSC were found to show biological relevance between DNA methylation and miRNA gene expression. Furthermore, some of the identified DNA-methylated miRNA genes; for instance, the miR-200 family, were previously identified as epigenetic-regulated miRNAs and correlated with poor survival of ovarian cancer. We also found that several miRNA target genes, BTG3, NDN, HTRA3, CDC25A, and HMGA2 were also related to the poor outcomes in ovarian cancer.The present study proposed a systematic strategy to construct highly confident epigenetic-regulated miRNA pathways for OSC. The findings are validated and are in line with the literature. The inclusion of direct DNA methylated miRNA events may offer another layer of explanation that along with genetics can give a better understanding of the carcinogenesis process.     

Genome-wide computational analyses of microRNAs and their targets from Canis familiaris

By computational analyses, we identified 357 miRNA candidates from Canis familiaris genome, among which 300 are homology of characterized human miRNAs, the remains are not reported in any other animal. Of the 357 miRNA genes, 142 are organized into 53 clusters, and two clusters locate in the paternally imprinted region. These dog miRNAs may regulate more than 800 possible targets, which are involved in a wide range of cellular processes. Remarkably, miR-186 resides in the eighth intron of its target gene in the same orientation, suggesting a feedback regulation of miRNA on its host gene.     

High-throughput quantitative detection of triple-negative breast cancer-associated expressed miRNAs by rolling circle amplification on fluorescence-encoded microspheres

Compared with other types of breast cancer, triple-negative breast cancer(TNBC) has the characteristics of a high degree of malignancy and poor prognosis. Early diagnosis of TNBC through biological markers and timely development of effective treatment methods can reduce its mortality. Many Research experiments have confirmed that some specific mi RNA expression profiles in TNBC can used as markers for early diagnosis. However, detecting the expression profiles of multiple groups of miRNAs accord...     

Genome-wide computational analyses of microRNAs and their targets from Canis familiaris

By computational analyses, we identified 357 miRNA candidates from Canis familiaris genome, among which 300 are homology of characterized human miRNAs, the remains are not reported in any other animal. Of the 357 miRNA genes, 142 are organized into 53 clusters, and two clusters locate in the paternally imprinted region. These dog miRNAs may regulate more than 800 possible targets, which are involved in a wide range of cellular processes. Remarkably, miR-186 resides in the eighth intron of its target gene in the same orientation, suggesting a feedback regulation of miRNA on its host gene.     

Sequence-based analysis of 5′UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5′UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5′UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p’s binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5′UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5′UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.     

Prebiotic Oligosaccharides: Special Focus on Fructooligosaccharides,Its Biosynthesis and Bioactivity

MicroRNAs (miRNAs) are important nonprotein-coding genes involved in almost all biological processes during biotic and abiotic stresses in plants. To investigate the miRNA-mediated plant response to drought stress, two drought-tolerant (C-306 and NI-5439) and two drought-sensitive (HUW-468 and WL-711) wheat genotypes were exposed to 25 % PEG 6000 for 1, 12 and 24 h. Temporal expression patterns of 12 drought-responsive miRNAs and their corresponding nine targets were monitored by quantitative real-time PCR (qRT-PCR). The results showed differential expression of miRNAs and their targets with varying degree of upregulation and downregulation in drought-sensitive genotypes. Likewise, in drought-tolerant wheat genotypes, maximum accumulation of miR393a and miR397a was observed at 1 h of stress. In addition, nearly perfect negative correlation was observed in four miRNA and target pairs (miR164-NAC, miR168a-AGO, miR398-SOD and miR159a-MYB) across all the temporal period studied which could be a major player during drought response in wheat. We, for the first time, validated the presence of miR529a and miR1029 in wheat. These findings gives a clue for temporal and variety-specific differential regulation of miRNAs and their targets in wheat in response to osmotic shock and could help in defining the potential roles of miRNAs in plant adaptation to osmotic stress in future.     

Analysis of the NCI-60 dataset for cancer-related microRNA and mRNA using expression profiles

BackgroundRecent studies have indicated that microRNA (miRNA) may play an oncogenic or tumor suppressor role in human cancer. To study the regulatory role of miRNAs in tumorigenesis, an integrated platform has been set up to provide a user friendly interface for query. The main advantage of the present platform is that all the miRNA target genes’ information and disease records are drawn from experimentally verified or high confidence records.ResultsMiRNA target gene results are annotated with reference to the disease gene as well as the pathway database. The correlation strength between miRNA and target gene expression profile is quantified by computing the correlation coefficient using the NCI-60 expression profiling data. Comprehensive analysis of the NCI-60 data found that the cumulative percentage of negative correlation coefficients for cleavage regulation is slightly higher than its positive counterpart; which indicated that the mRNA degradation mechanism is slightly dominant. In addition, the RNAHybrid and TargetScans scores are computed which potentially served as quantitative estimators for miRNA–mRNA binding events.Three scores are defined for each miRNA–mRNA pair, which are based on the disease gene and pathway information. These three scores allow user to sort out high confidence cancer-related miRNA–mRNA pairs.Statistical tests were applied to investigate the relations of three chromosomal features, i.e., CpG island, fragile site, and miRNA cluster, with cancer-related miRNAs. A web-based interface has been set up for query, which can be accessed at: http://ppi.bioinfo.asia.edu.tw/mirna_target/ConclusionsThe main advantage of the present platform on miRNA–mRNA targeting information is that all the target genes’ information and disease records are experimentally verified. Although this may limit the number of miRNA–mRNA relationships, the results provided here are more solid and have fewer false positive events. Certain novel cancer-related miRNA–mRNA pairs are identified and confirmed in the literature. Fisher's exact test suggests that CpG island and fragile site associated miRNAs tend to associate with cancer formation. In summary, the present platform provides an easy means of investigating cancer-related miRNAs.