Role of microRNAs in improved osteogenicity of topographically modified titanium implant surfaces

This project aimed at understanding the molecular mechanisms involved in the superior integration of micro-roughened titanium implant surfaces with the surrounding bone, when compared with their smooth surfaces. It involved studying the role of microRNAs and cell signaling pathways in the molecular regulation of bone cells on topographically modified titanium dental implants. The findings suggest a highly regulated microRNA-mediated control of molecular mechanisms during the process of bone formation that may be responsible for the superior osseointegration properties on micro-roughened titanium implant surfaces and indicate the possibility of using microRNA modulators to enhance osseointegration in clinically demanding circumstances.

miRPlant : an integrated tool for identification of plant miRNA from RNA sequencing data

Background Small RNA sequencing is commonly used to identify novel miRNAs and to determine their expression levels in plants. There are several miRNA identification tools for animals such as miRDeep, miRDeep2 and miRDeep*. miRDeep-P was developed to identify plant miRNA using miRDeep’s probabilistic model of miRNA biogenesis, but it depends on several third party tools and lacks a user-friendly interface. The objective of our miRPlant program is to predict novel plant miRNA, while providing a user-friendly interface with improved accuracy of prediction. Result We have developed a user-friendly plant miRNA prediction tool called miRPlant. We show using 16 plant miRNA datasets from four different plant species that miRPlant has at least a 10% improvement in accuracy compared to miRDeep-P, which is the most popular plant miRNA prediction tool. Furthermore, miRPlant uses a Graphical User Interface for data input and output, and identified miRNA are shown with all RNAseq reads in a hairpin diagram. Conclusions We have developed miRPlant which extends miRDeep* to various plant species by adopting suitable strategies to identify hairpin excision regions and hairpin structure filtering for plants. miRPlant does not require any third party tools such as mapping or RNA secondary structure prediction tools. miRPlant is also the first plant miRNA prediction tool that dynamically plots miRNA hairpin structure with small reads for identified novel miRNAs. This feature will enable biologists to visualize novel pre-miRNA structure and the location of small RNA reads relative to the hairpin. Moreover, miRPlant can be easily used by biologists with limited bioinformatics skills.

A novel saliva-based microRNA biomarker panel to detect head and neck cancers

Background MicroRNAs (miRNAs) are known to play an important role in cancer development by post-transcriptionally affecting the expression of critical genes. The aims of this study were two-fold: (i) to develop a robust method to isolate miRNAs from small volumes of saliva and (ii) to develop a panel of saliva-based diagnostic biomarkers for the detection of head and neck squamous cell carcinoma (HNSCC). Methods Five differentially expressed miRNAs were selected from miScript™ miRNA microarray data generated using saliva from five HNSCC patients and five healthy controls. Their differential expression was subsequently confirmed by RT-qPCR using saliva samples from healthy controls (n = 56) and HNSCC patients (n = 56). These samples were divided into two different cohorts, i.e., a first confirmatory cohort (n = 21) and a second independent validation cohort (n = 35), to narrow down the miRNA diagnostic panel to three miRNAs: miR-9, miR-134 and miR-191. This diagnostic panel was independently validated using HNSCC miRNA expression data from The Cancer Genome Atlas (TCGA), encompassing 334 tumours and 39 adjacent normal tissues. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic capacity of the panel. Results On average 60 ng/μL miRNA was isolated from 200 μL of saliva. Overall a good correlation was observed between the microarray data and the RT-qPCR data. We found that miR-9 (P <0.0001), miR-134 (P <0.0001) and miR-191 (P <0.001) were differentially expressed between saliva from HNSCC patients and healthy controls, and that these miRNAs provided a good discriminative capacity with area under the curve (AUC) values of 0.85 (P <0.0001), 0.74 (P < 0.001) and 0.98 (P < 0.0001), respectively. In addition, we found that the salivary miRNA data showed a good correlation with the TCGA miRNA data, thereby providing an independent validation. Conclusions We show that we have developed a reliable method to isolate miRNAs from small volumes of saliva, and that the saliva-derived miRNAs miR-9, miR-134 and miR-191 may serve as novel biomarkers to reliably detect HNSCC. © 2014 International Society for Cellular Oncology.

Gene regulation by translational inhibition is determined by Dicer partnering proteins

MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment1,​2,​3,​4. In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition1. In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript5. However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown6. Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized7,​8,​9, but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic systems.

Study of miRNA polymorphisms and their potential association with breast cancer risk in Australian Caucasian populations

This project established a large and well characterised prospective breast cancer DNA biobank and used this biobank to conduct genetic studies in breast cancer. The thesis presented the results of these high-throughput genotyping studies in two separate Australian Caucasian case-control populations and identified association between three novel genetic variants in microRNA genes and breast cancer risk.

A large-scale analysis of genetic variants within putative miRNA binding sites in prostate cancer

Prostate cancer is the second most common malignancy among men worldwide. Genome-wide association studies have identified 100 risk variants for prostate cancer, which can explain approximately 33% of the familial risk of the disease. We hypothesized that a comprehensive analysis of genetic variations found within the 3' untranslated region of genes predicted to affect miRNA binding (miRSNP) can identify additional prostate cancer risk variants. We investigated the association between 2,169 miRSNPs and prostate cancer risk in a large-scale analysis of 22,301 cases and 22,320 controls of European ancestry from 23 participating studies. Twenty-two miRSNPs were associated (P<2.3×10(-5)) with risk of prostate cancer, 10 of which were within 7 genes previously not mapped by GWAS studies. Further, using miRNA mimics and reporter gene assays, we showed that miR-3162-5p has specific affinity for the KLK3 rs1058205 miRSNP T-allele, whereas miR-370 has greater affinity for the VAMP8 rs1010 miRSNP A-allele, validating their functional role. SIGNIFICANCE Findings from this large association study suggest that a focus on miRSNPs, including functional evaluation, can identify candidate risk loci below currently accepted statistical levels of genome-wide significance. Studies of miRNAs and their interactions with SNPs could provide further insights into the mechanisms of prostate cancer risk.

A genetic variant of MDM4 influences regulation by multiple microRNAs in prostate cancer

The oncogene MDM4, also known as MDMX or HDMX, contributes to cancer susceptibility and progression through its capacity to negatively regulate a range of genes with tumour-suppressive functions. As part of a recent genome-wide association study it was determined that the A-allele of the rs4245739 SNP (A>C), located in the 3'-UTR of MDM4, is associated with an increased risk of prostate cancer. Computational predictions revealed that the rs4245739 SNP is located within a predicted binding site for three microRNAs (miRNAs): miR-191-5p, miR-887 and miR-3669. Herein, we show using reporter gene assays and endogenous MDM4 expression analyses that miR-191-5p and miR-887 have a specific affinity for the rs4245739 SNP C-allele in prostate cancer. These miRNAs do not affect MDM4 mRNA levels, rather they inhibit its translation in C-allele-containing PC3 cells but not in LNCaP cells homozygous for the A-allele. By analysing gene expression datasets from patient cohorts, we found that MDM4 is associated with metastasis and prostate cancer progression and that targeting this gene with miR-191-5p or miR-887 decreases in PC3 cell viability. This study is the first, to our knowledge, to demonstrate regulation of the MDM4 rs4245739 SNP C-allele by two miRNAs in prostate cancer, and thereby to identify a mechanism by which the MDM4 rs4245739 SNP A-allele may be associated with an increased risk for prostate cancer.

miR-514a regulates the tumour suppressor NF1 and modulates BRAFi sensitivity in melanoma

To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.

Evaluation of polymorphisms in predicted target sites for micro RNAs differentially expressed in endometriosis

Previous microarray analyses identified 22 microRNAs (miRNAs) differentially expressed in paired ectopic and eutopic endometrium of women with and without endometriosis. To investigate further the role of these miRNAs in women with endometriosis, we conducted an association study aiming to explore the relationship between endometriosis risk and single-nucleotide polymorphisms (SNPs) in miRNA target sites for these differentially expressed miRNAs. A panel of 102 SNPs in the predicted miRNA binding sites were evaluated for an endometriosis association study and an ingenuity pathway analysis was performed. Fourteen rare variants were identified in this study. We found SNP rs14647 in the Wolf-Hirschhorn syndrome candidate gene1 (WHSC1) 3'UTR (untranslated region) was associated with endometriosis-related infertility presenting an odds ratio of 12.2 (95% confidence interval = 2.4-60.7, P = 9.03 x 10(-5)). SNP haplotype AGG in the solute carrier family 22, member 23 (SLC22A23) 3'UTR was associated with endometriosis-related infertility and more severe disease. With the individual genotyping data, ingenuity pathways analysis identified the tumour necrosis factor and cyclin-dependant kinase inhibitor as major factors in the molecular pathways. Significant associations between WHSC1 alleles and endometriosis-related infertility and SLC22A23 haplotypes and the disease severe stage were identified. These findings may help focus future research on subphenotypes of this disease. Replication studies in independent large sample sets to confirm and characterize the involvement of the gene variation in the pathogenesis of endometriosis are needed.

Integration of miRNA and mRNA expression with DNA copy number of Ewing sarcoma cell lines

Ewing sarcoma is an aggressive and poorly differentiated malignancy of bone and soft tissue. It primarily affects children, adolescents, and young adults, with a slight male predominance. It is characterized by a translocation between chromosomes 11 and 22 resulting in the EWSR1-FLI1fusion transcription factor. The aim of this study is to identify putative Ewing sarcoma target genes through an integrative analysis of three microarray data sets. Array comparative genomic hybridization is used to measure changes in DNA copy number, and analyzed to detect common chromosomal aberrations. mRNA and miRNA microarrays are used to measure expression of protein-coding and miRNA genes, and these results integrated with the copy number data. Chromosomal aberrations typically contain also bystanders in addition to the driving tumor suppressor and oncogenes, and integration with expression helps to identify the true targets. Correlation between expression of miRNAs and their predicted target mRNAs is also evaluated to assess the results of post-transcriptional miRNA regulation on mRNA levels. The highest frequencies of copy number gains were identified in chromosome 8, 1q, and X. Losses were most frequent in 9p21.3, which also showed an enrichment of copy number breakpoints relative to the rest of the genome. Copy number losses in 9p21.3 were found have a statistically significant effect on the expression of MTAP, but not on CDKN2A, which is a known tumor-suppressor in the same locus. MTAP was also down-regulated in the Ewing sarcoma cell lines compared to mesenchymal stem cells. Genes exhibiting elevated expression in association with copy number gains and up-regulation compared to the reference samples included DCAF7, ENO2, MTCP1, andSTK40. Differentially expressed miRNAs were detected by comparing Ewing sarcoma cell lines against mesenchymal stem cells. 21 up-regulated and 32 down-regulated miRNAs were identified, includingmiR-145, which has been previously linked to Ewing sarcoma. The EWSR1-FLI1 fusion gene represses miR-145, which in turn targets FLI1 forming a mutually repressive feedback loop. In addition higher expression linked to copy number gains and compared to mesenchymal stem cells, STK40 was also found to be a target of four different miRNAs that were all down-regulated in Ewing sarcoma cell lines compared to the reference samples. SLCO5A1 was identified as the only up-regulated gene within a frequently gained region in chromosome 8. This region was gained in over 90 % of the cell lines, and also with a higher frequency than the neighboring regions. In addition, SLCO5A1 was found to be a target of three miRNAs that were down-regulated compared to the mesenchymal stem cells.

Circulating miRNA profile in HCV infected serum: novel insight into pathogenesis

Changes in circulating miRNA profiles have been associated with different diseases. Here we demonstrate the circulating miRNA profile in serum of HCV infected individuals using a microRNA array that profiles the expression of 940 miRNAs. Serum samples from two HCV genotype -1 and two HCV genotype -3 infected individuals were compared with healthy controls. Expression levels of miR-134, miR-198, miR-320c and miR-483-5p that were commonly upregulated in case of both genotypes were validated in 36 individual patient serum samples. Serum miR-134, miR-320c and miR-483-5p were significantly upregulated during HCV infection. miR-320c and miR-483-5p were also upregulated in HCV-JFH1 infected cells and cell culture supernatant. Pathway analysis of putative target genes of these miRNAs indicated involvement of PI3K-Akt, NFKB and MAPK signaling pathways. Results revealed novel insights on the role of circulating miRNAs in mediating pathogenesis in HCV-infected cells.

Cyclic Cationic Peptides Containing Sugar Amino Acids Selectively Distinguishes and Inhibits Maturation of Pre-miRNAs of the Same Family

The discovery of microRNAs (miRNAs) has added a new dimension to the gene regulatory networks, making aberrantly expressed miRNAs as therapeutically important targets. Small molecules that can selectively target and modulate miRNA levels can thus serve as lead structures. Cationic cyclic peptides containing sugar amino acids represent a new class of small molecules that can target miRNA selectively. Upon treatment of these small molecules in breast cancer cell line, we profiled 96 therapeutically important miRNAs associated with cancer and observed that these peptides can selectively target paralogous miRNAs of the same seed family. This selective inhibition is of prime significance in cases when miRNAs of the same family have tissue-specific expression and perform different functions. During these conditions, targeting an entire miRNA family could lead to undesired adverse effects. The selective targeting is attributable to the difference in the three-dimensional structures of precursor miRNAs. Hence, the core structure of these peptides can be used as a scaffold for designing more potent inhibitors of miRNA maturation and hence function.

Epigenetics and miRNA during bacteria-induced host immune responses

Various cellular processes including the pathogen-specific immune responses, host-pathogen interactions and the related evasion mechanisms rely on the ability of the immune cells to be reprogrammed accurately and in many cases instantaneously. In this context, the exact functions of epigenetic and miRNA-mediated regulation of genes, coupled with recent advent in techniques that aid such studies, make it an attractive field for research. Here, we review examples that involve the epigenetic and miRNA control of the host immune system during infection with bacteria. Interestingly, many pathogens utilize the epigenetic and miRNA machinery to modify and evade the host immune responses. Thus, we believe that global epigenetic and miRNA mapping of such host-pathogen interactions would provide key insights into their cellular functions and help to identify various determinants for therapeutic value.

SNPs in microRNAs and susceptibility to Late-Onset Alzheimer´s disease

Case-control association study of SNPs in microRNAs and susceptibility to Late-Onset Alzheimer´s disease.

La participación de los microRNAs en la regulación de la plasticidad en la corteza visual en un modelo de ambliopía experimental

La aplicaci�n de campos geomagn�ticos con la polaridad cambiante promueve la plasticidad de la corteza visual de animales privados visualmente mediante la cr�a en oscuridad, o bien a trav�s de la sutura palpebral de un ojo.Esto es debido a que la estimulaci�n magn�tica produce la down-regulaci�n de la expresi�n de los microRNAs let-7b*, miR-330, miR-338* y miR-376c en la corteza visual de animales sometidos a la cr�a en oscuridad, as� como de animales sujetos a sutura palpebral de un ojo. Como consecuencia de la down-regulaci�n de la expresi�n de los microRNAs citados anteriormente, se ven incrementados los niveles de expresi�n de sus correspondientes mol�culas diana, Conexina 26 para el caso de let-7b*, Tenascina R en el caso de miR-330, Contactina 4 para el caso de miR-338* y Matriz metalopeptidasa 9 y �-sinucle�na, ambas mol�culas diana de miR-376c. El aumento de expresi�n de estas mol�culas diana a nivel de RNA mensajero, as� como a nivel de prote�na en la corteza visual promueve la capacidad pl�stica de la corteza visual, ya que estas mol�culas diana se encuentran implicadas en procesos de crecimiento/elongaci�n de las neuritas y en la regulaci�n de la morfolog�a de las espinas dendr�ticas.