Novel miR-29b target regulation patterns are revealed in two different cell lines

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b, have been detected in various biological fluids and tissues from a large variety of disease models. However, how miRNAs “react” and function in different cellular environments is still largely unknown. In this study, the regulation patterns of miR-29b between human and mouse cell lines were compared for the first time. CRISPR/Cas9 gene editing was used to stably knockdown miR-29b in human cancer HeLa cells and mouse fibroblast NIH/3T3 cells with minimum off-targets. Genome editing revealed mir-29b-1, other than mir-29b-2, to be the main source of generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells

iSRAP - A one-touch research tool for rapid profiling of small RNA-seq data

Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of noncoding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes

Distribution of microRNA profiles in pre-clinical and clinical forms of murine and human prion disease

Prion diseases are distinguished by long pre-clinical incubation periods during which prions actively propagate in the brain and cause neurodegeneration. In the pre-clinical stage, we hypothesize that upon prion infection, transcriptional changes occur that can lead to early neurodegeneration. A longitudinal analysis of miRNAs in pre-clinical and clinical forms of murine prion disease demonstrated dynamic expression changes during disease progression in the affected thalamus region and serum. Serum samples at each timepoint were collected whereby extracellular vesicles (EVs) were isolated and used to identify blood-based biomarkers reflective of pathology in the brain. Differentially expressed EV miRNAs were validated in human clinical samples from patients with human sporadic Creutzfeldt-Jakob disease (sCJD), with the molecular subtype at codon 129 either methionine-methionine (MM, n = 14) or valine-valine (VV, n = 12) compared to controls (n = 20). EV miRNA biomarkers associated with prion infection predicted sCJD with an AUC of 0.800 (85% sensitivity and 66.7% specificity) in a second independent validation cohort (n = 26) of sCJD and control patients with MM or VV subtype. This study discovered clinically relevant miRNAs that benefit diagnostic development to detect prion-related diseases and therapeutic development to inhibit prion infectivity

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

Background: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multiomics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies

RAB27A promotes melanoma cell invasion and metastasis via regulation of pro-invasive exosomes

Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma

Defining the roles of long and small transcriptomes in neurodegenerative diseases

© 2016 Dr. Camelia QuekNeurodegenerative diseases belong to a group of disorders that are characterised by progressive degeneration of neurons, and often exhibit aberrant transcriptional regulatory programs prior to the manifestation of clinical symptoms. Owing to the evidence that RNAs are functionally rich class of molecules that dictates phenotypic changes, the identification of aberrant RNA signatures potentially provides mechanistic insights into disease pathogenesis. In this thesis, I present a comprehensive study of long and small transcriptomes in neurodegenerative diseases, with the objective of restructuring the approach of disease diagnosis and treatment. I determine the underlying disease mechanisms associated with misfolded protein aggregation and neuronal death using murine models. I then investigate whether these transcripts can be used as biomarkers and therapeutics to monitor disease progression. The first aspect of this thesis highlighted the challenges of large-scale transcriptomic data processing for biological analysis. I developed iSRAP, an integrated small RNA analysis pipeline, for rapid profiling of small RNA sequences generated from next-generation sequencing. Data sets from several small RNA studies were used to demonstrate iSRAP workflow, which covers from data quality assessment to differential expression analysis. The results revealed iSRAP features, including high-throughput capability, graphical result representations, convenience and reliability. iSRAP can serve as a platform for rapid analysis of transcriptomic data so that informed decision can be made on the downstream analyses of small RNA studies. The second aspect of my work emphasised the significant interest in utilising exosomes to identify small RNA biomarkers for diagnostics purposes. Exosomes are nano-sized extracellular vesicles of endocytic origin that involves in shuttling of RNA between cells within a biological system, facilitating disease spreading and pathogenesis. There are currently different methods available to isolate exosomes for studying small RNA profiles. In order to investigate the feasible exosome isolation method for biomarker discovery that required simple workflow, I implemented a combined approach of RNA sequencing and bioinformatics to profile a wide range of small RNA species in exosomes isolated by two different methods: differential ultracentrifugation and OptiPrep velocity ultracentrifugation. The analysis showed that these methods yielded exosomes with similar small RNA profiles to each other, suggesting that the higher purity of exosomes by OptiPrep method did not influence the small RNA profiles. Therefore, these findings revealed that the conventional ultracentrifugation-based method posed as a simple and sufficient protocol for biomarker discovery in exosomes. To better understand the disease mechanisms and therapeutic intervention in neurodegenerative diseases, a mouse model was used to recapitulate human prion diseases and Parkinson’s disease respectively. In the context of prion diseases, the study focused on the clinical relevance of microRNA (miRNA), which is a class of small RNAs that negatively regulates gene targets controlling fundamental pathways such as cellular signalling and neuronal development. The temporal distribution of miRNA expression profiles over the course of prion infection was determined. I established an analytical workflow to detect pre-clinical and clinical miRNA signatures in mice that were infected with prions over a time course. There were several pre-clinical and clinical miRNA signatures, such as let-7b, miR-223-3p and miR-362-5p, which were implicated in the early impairment of neurogenesis or terminal-stage disease progression. The detected miRNA signatures at different stages of prion disease can potentially serve as promising diagnostic and therapeutic measures for inhibiting and screening of prion infectivity. The final aspect of this thesis detailed the therapeutic relevance of the CuII(atsm) compound in Parkinson’s disease using whole transcriptomes analysis. I employed several computational and statistical methods to show a panel of protein-coding RNAs associated with neuronal development, dopamine synthesis and synaptic neurotransmission in the substantia nigra of the brain. Upon CuII(atsm) treatment, the expression of 40 genes involved in promoting dopamine synthesis, calcium signalling and synaptic plasticity were restored. To my knowledge, this is the first transcriptomic study that comprehensively reported the key therapeutic pathways targeted by CuII(atsm) compound that may provide therapeutic benefits for Parkinson’s disease and other neurodegenerative diseases. In summary, the collective findings from this thesis provide neurologists with mechanistic insights into the neuronal pathways, and thereby enabling the development of diagnostic and therapeutic measures that aim at inhibiting the progression of degenerating neurons in the brain. The present studies will therefore provide more informed treatment alternatives for neurodegenerative diseases

Genetics and Genomics of Melanoma: Current Progress and Future Directions

Melanoma is a form of skin cancer that develops in the skin’s pigment cells, known as melanocytes, and can spread via blood and the lymphatic system to nearby tissues or distant organs in the body [...

Distinct molecular profiles and immunotherapy treatment outcomes of V600E and V600K BRAF-mutant melanoma

Purpose: BRAF V600E and V600K melanomas have distinct clinicopathologic features, and V600K appear to be less responsive to BRAFi±MEKi. We investigated mechanisms for this and explored whether genotype affects response to immunotherapy. Experimental Design: Pretreatment formalin-fixed paraffin-embedded tumors from patients treated with BRAFi±MEKi underwent gene expression profiling and DNA sequencing. Molecular results were validated using The Cancer Genome Atlas (TCGA) data. An independent cohort of V600E/K patients treated with anti–PD-1 immunotherapy was examined. Results: Baseline tissue and clinical outcome with BRAFi±MEKi were studied in 93 patients (78 V600E, 15 V600K). V600K patients had numerically less tumor regression (median, −31% vs. −52%, P = 0.154) and shorter progression-free survival (PFS; median, 5.7 vs. 7.1 months, P = 0.15) compared with V600E. V600K melanomas had lower expression of the ERK pathway feedback regulator dual-specificity phosphatase 6, confirmed with TCGA data (116 V600E, 17 V600K). Pathway analysis showed V600K had lower expression of ERK and higher expression of PI3K-AKT genes than V600E. Higher mutational load was observed in V600K, with a higher proportion of mutations in PIK3R1 and tumor-suppressor genes. In patients treated with anti–PD-1, V600K (n = 19) had superior outcomes than V600E (n = 84), including response rate (53% vs. 29%, P = 0.059), PFS (median, 19 vs. 2.7 months, P = 0.049), and overall survival (20.4 vs. 11.7 months, P = 0.081). Conclusions: BRAF V600K melanomas appear to benefit less from BRAFi±MEKi than V600E, potentially due to less reliance on ERK pathway activation and greater use of alternative pathways. In contrast, these melanomas have higher mutational load and respond better to immunotherapy.J.S. Wilmott is supported by an NHMRC Research Fellowship. J.L. McQuade is supported by an ASCO/CCF Career Development Award, a Melanoma SPORE Developmental Research Program Award, and an NIH T32 Training Grant CA009666. R.A. Scolyer is supported by an NHMRC Practitioner Fellowship. J.Y.H. Yang is supported by NHMRC CDF and ARC Discovery Project grant (DP170100654). G.V. Long is supported by an NHMRC Practitioner Fellowship and the University of Sydney Medical Foundation. A.M. Menzies is supported by a Cancer Institute NSW Fellowship. This work was supported by a Pfizer Australia grant (WS2345795 to A.M. Menzies), and a Cancer Council NSW grant (RG17-04 to J. Holst, J.S. Wilmott, and A.M. Menzies). This research was also supported by an Australian National Health and Medical Research Council program grant

Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery

<p>Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30–55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.</p