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.     

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.     

Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.     

miR-9 and let-7g enhance the sensitivity to ionizing radiation by suppression of NFκB1

The activation of nuclear factor-kappa B1 (NFkB1) in cancer cells may confer resistance to ionizing radiation (IR). To enhance the therapeutic efficiency of IR in lung cancer, we screened for microRNAs (miRNAs) that suppress NFkB1 and observed their effects on radiosensitivity in a human lung cancer cell line. From time series data of miRNA expression in γ-irradiated H1299 human lung cancer cells, we found that the expression of miR-9 was inversely correlated with that of NFκB1. Overexpression of miR-9 down-regulated the level of NFκB1 in H1299 cells, and the surviving fraction of γ-irradiated cells was decreased. Interestingly, let-7g also suppressed the expression of NFκB1, although there was no canonical target site for let-7g in the NFκB1 3' untranslated region. From these results, we conclude that the expression of miR-9 and let-7g could enhance the efficiency of radiotherapy for lung cancer treatment through the inhibition of NFκB1.     

Identifying the target mRNAs of microRNAs in colorectal cancer

MicroRNAs (miRNAs) play an important role in gene regulatory networks by inhibiting the expression of target mRNAs. There is a growing interest in identifying the relationship between miRNAs and their target mRNAs. Various experimental studies have been carried out to discover miRNAs involved in cancer and to identify their target genes. At the same time, a large volume of miRNA and mRNA expression profiles have become available owing to the development of high-throughput measurement technologies. So, there is now a pressing need to develop a computational method by which we can identify the target mRNAs of given miRNAs from such massive expression data sets. In this respect, we propose an effective linear model based identification method to unravel the relationship between miRNAs and their target mRNAs in colorectal cancer by using microarray expression profiles and sequence data.     

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.     

Comprehensive analysis of microRNA-mRNA co-expression in circadian rhythm

To investigate the potential role of microRNA (miRNA) in the regulation of circadian rhythm, we performed microarray-based expression profiling study of both miRNA and mRNA in mouse liver for 48 h at 4-hour intervals. Circadian miRNA-mRNA target pair is defined as the pair both elements of which show circadian expression patterns and the sequence-based target relationship of which can be predicted. Circadian initiators, Clock and Bmal1, showed inversely correlated circadian expression patterns against their corresponding miRNAs, miR-181d and miR-191, targeting them. In contrast, circadian suppressors, Per, Cry, CKIe and Rev-erba, exhibited positively correlated circadian expression patterns to their corresponding miRNAs. Genomic location analysis revealed that intronic region showed higher abundance of cyclic than non-cyclic miRNAs targeting circadian genes while other (i.e., 3''-UTR, exon and intergenic) regions showed no difference. It is suggested that miRNAs are involved in the regulation of peripheral circadian rhythm in mouse liver by modulating Clock:Bmal1 complex. Identifying specific miRNAs and their targets that are critically involved in circadian rhythm will provide a better understanding of the regulation of circadian-clock system.     

<Emphasis Type="Italic">Insilco</Emphasis> Prediction and Characterization of microRNAs from <Emphasis Type="Italic">Oncopeltus fasciatus</Emphasis> (Hemiptera: Lygaeidae) Genome

For studies on functional genomics, small RNAs, especially microRNAs (miRNAs), have emerged as a hot topic due to their importance in cellular and developmental processes. Identification of insect miRNAs largely depends on the availability of genomic sequences in the public domain. The large milkweed bug, Oncopeltus fasciatus (Dallas) is a hemimetabolous insect which has become a model hemipteran system for various molecular studies. In this study, we identified 96 candidate mature miRNAs from O. fasciatus genome using a blast search with the previously reported animal miRNAs. The secondary structure of predicted miRNA sequences was determined online using “mfold” web server and verified by calculating the minimal free energy index (MFEI). Six miRNAs let-7e, miR-133c, miR-219b, mir-466d, mir-669f, and mir-669l are reported for the first time in Insecta. Comparison of O. fasciatus mir-2 and mir-71 family clusters to those of diverse insect species showed that they are highly conserved. The phylogenetic analysis of miRNAs revealed the evolutionary relationship of conserved miRNAs of O. fasciatus with other insect species. Using a classical rule-based algorithm method, we predicted the possible targets of the new miRNAs. Our study not only identified the list of miRNAs in O. fasciatus but also provides a basic platform for developing novel pest management strategies based on artificial miRNAs.