Jan Stenvang

Silencing of microRNA families by seed-targeting tiny LNAs

The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.

Inhibition of microRNA function by antimiR oligonucleotides

MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in many developmental and cellular processes. Moreover, there is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human diseases. Indeed, disease-associated miRNAs represent a new class of targets for the development of miRNA-based therapeutic modalities, which may yield patient benefits unobtainable by other therapeutic approaches. The recent explosion in miRNA research has accelerated the development of several computational and experimental approaches for probing miRNA functions in cell culture and in vivo. In this review, we focus on the use of antisense oligonucleotides (antimiRs) in miRNA inhibition for loss-of-function studies. We provide an overview of the currently employed antisense chemistries and their utility in designing antimiR oligonucleotides. Furthermore, we describe the most commonly used in vivo delivery strategies and discuss different approaches for assessment of miRNA inhibition and potential off-target effects. Finally, we summarize recent progress in antimiR mediated pharmacological inhibition of disease-associated miRNAs, which shows great promise in the development of novel miRNA-based therapeutics.

MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo.

The utility of LNA in microRNA-based cancer diagnostics and therapeutics.

MicroRNAs (miRNAs) are a novel class of small endogenous non-coding RNAs that regulate gene expression post-transcriptionally by binding to their cognate target mRNAs. Emerging evidence implies that miRNAs play important roles in cancer and thus, miRNAs have rapidly emerged as valuable markers for cancer diagnostics and promising targets for therapeutics. Locked nucleic acid (LNA) is a conformational RNA analoque that binds complementary RNA with unprecedented affinity and specificity. These properties make LNA well suited for miRNA detection and analysis for cancer diagnostics. Furthermore, recent studies on LNA-mediated silencing of miRNA function in vitro and in vivo support the potential of LNA in therapeutic intervention of cancer-associated miRNAs.

Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

MiR-155 is overexpressed in patients with atopic dermatitis and modulates T-cell proliferative responses by targeting cytotoxic T lymphocyte–associated antigen 4

BACKGROUND MicroRNAs (miRNAs) are short noncoding RNAs that suppress gene expression at the posttranscriptional level. Atopic dermatitis is a common chronic inflammatory skin disease characterized by the presence of activated T cells within the skin. OBJECTIVE We sought to explore the role of miRNAs in the pathogenesis of atopic dermatitis. METHODS Global miRNA expression in healthy and lesional skin of patients with atopic dermatitis was compared by using TaqMan MicroRNA Low Density Arrays. miR-155 expression in tissues and cells was quantified by means of quantitative real-time PCR. The cellular localization of miR-155 was analyzed by means of in situ hybridization. The regulation of cytotoxic T lymphocyte-associated antigen (CTLA-4) by miR-155 was investigated by using luciferase reporter assays and flow cytometry. CTLA-4 expression and functional assays were performed on T(H) cells overexpressing miR-155. RESULTS miR-155 was one of the highest-ranked upregulated miRNAs in patients with atopic dermatitis. In the skin miR-155 was predominantly expressed in infiltrating immune cells. miR-155 was upregulated during T-cell differentiation/activation and was markedly induced by T-cell activators in PBMCs in vitro and by superantigens and allergens in the skin in vivo. CTLA-4, an important negative regulator of T-cell activation, was identified as a direct target of miR-155. Overexpression of miR-155 in T(H) cells resulted in decreased CTLA-4 levels accompanied by an increased proliferative response. CONCLUSION miR-155 is significantly overexpressed in patients with atopic dermatitis and might contribute to chronic skin inflammation by increasing the proliferative response of T(H) cells through the downregulation of CTLA-4.

Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant

Seven fulvestrant resistant cell lines derived from the estrogen receptor α positive MCF-7 human breast cancer cell line were used to investigate the importance of epidermal growth factor receptor (ErbB1-4) signaling. We found an increase in mRNA expression of EGFR and the ErbB3/ErbB4 ligand heregulin2 (hrg2) and a decrease of ErbB4 in all resistant cell lines. Western analyses confirmed the upregulation of EGFR and hrg2 and the downregulation of ErbB4. Elevated activation of EGFR and ErbB3 was seen in all resistant cell lines and the ErbB3 activation occurred by an autocrine mechanism. ErbB4 activation was observed only in the parental MCF-7 cells. The downstream kinases pAkt and pErk were increased in five of seven and in all seven resistant cell lines, respectively. Treatment with the EGFR inhibitor gefitinib preferentially inhibited growth and reduced the S phase fraction in the resistant cell lines concomitant with inhibition of Erk and unaltered Akt activation. In concert, inhibition of Erk with U0126 preferentially reduced growth of resistant cell lines. Treatment with ErbB3 neutralizing antibodies inhibited ErbB3 activation and resulted in a modest but statistically significant growth inhibition of two resistant cell lines. These data indicate that ligand activated ErbB3 and EGFR, and Erk signaling play important roles in fulvestrant resistant cell growth. Furthermore, the decreased level of ErbB4 in resistant cells may facilitate heterodimerization of ErbB3 with EGFR and ErbB2. Our data support that a concerted action against EGFR, ErbB2 and ErbB3 may be required to obtain complete growth suppression of fulvestrant resistant cells.

MicroRNAs as targets for antisense-based therapeutics

MicroRNAs (miRNAs) are a novel class of endogenous non-coding single-stranded RNAs, which regulate gene expression post-transcriptionally by base pairing with their target mRNAs. So far > 5000 miRNA entries have been registered and miRNAs have been implicated in most, if not all, central cellular processes and several diseases. As the mechanism of action for miRNA regulation of target mRNAs is mediated by Watson-Crick base pairing, antisense oligonucleotides targeting the miRNAs appear as an obvious choice to specifically inhibit miRNA function. Indeed, miRNAs can be antagonized in vivo by oligonucleotides composed of high-affinity nucleotide mimics. Lessons learned from traditional antisense strategies and small-interfering RNA approaches, that is from potent nucleotide mimics, design rules, pharmacokinetics, administration and safety issues, are likely to pave the way for future clinical trials of miRNA-antagonizing oligonucleotides.

Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer

Objective To evaluate the potential for diagnosing colorectal cancer (CRC) from faecal metagenomes. Design We performed metagenome-wide association studies on faecal samples from 74 patients with CRC and 54 controls from China, and validated the results in 16 patients and 24 controls from Denmark. We further validated the biomarkers in two published cohorts from France and Austria. Finally, we employed targeted quantitative PCR (qPCR) assays to evaluate diagnostic potential of selected biomarkers in an independent Chinese cohort of 47 patients and 109 controls. Results Besides confirming known associations of Fusobacterium nucleatum and Peptostreptococcus stomatis with CRC, we found significant associations with several species, including Parvimonas micra and Solobacterium moorei. We identified 20 microbial gene markers that differentiated CRC and control microbiomes, and validated 4 markers in the Danish cohort. In the French and Austrian cohorts, these four genes distinguished CRC metagenomes from controls with areas under the receiver-operating curve (AUC) of 0.72 and 0.77, respectively. qPCR measurements of two of these genes accurately classified patients with CRC in the independent Chinese cohort with AUC=0.84 and OR of 23. These genes were enriched in early-stage (I–II) patient microbiomes, highlighting the potential for using faecal metagenomic biomarkers for early diagnosis of CRC. Conclusions We present the first metagenomic profiling study of CRC faecal microbiomes to discover and validate microbial biomarkers in ethnically different cohorts, and to independently validate selected biomarkers using an affordable clinically relevant technology. Our study thus takes a step further towards affordable non-invasive early diagnostic biomarkers for CRC from faecal samples.

The therapeutic potential of microRNAs in cancer.

AbstractMicroRNAs (miRNAs) have been uncovered as important posttranscriptional regulators of nearly every biological process in the cell. Furthermore, mounting evidence implies that miRNAs play key roles in the pathogenesis of cancer and that many miRNAs can function either as oncogenes or tumor suppressors. Thus, miRNAs have rapidly emerged as promising targets for the development of novel anticancer therapeutics. The development of miRNA-based cancer therapeutics relies on restoring the activity of tumor suppressor miRNAs using double-stranded miRNA mimics or inhibition of oncogenic miRNAs using single-stranded antisense oligonucleotides, termed antimiRs. In the present review, we focus on recent advancements in the discovery and development of miRNA-based cancer therapeutics using these 2 approaches. In addition, we summarize selected studies, in which modulation of miRNA activity in preclinical cancer models in vivo has demonstrated promising therapeutic potential.