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

Obesity Is Associated with Altered Tumor Metabolism in Metastatic Melanoma

PURPOSE: Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICI) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient body mass index (BMI). EXPERIMENTAL DESIGN: Associations between BMI [normal (NL \u3c 25) or OW/OB (BMI ≥ 25)] and tumor or microbiome characteristics were examined in specimens from 782 patients with metastatic melanoma across 7 cohorts. DNA associations were evaluated in The Cancer Genome Atlas cohort. RNA sequencing from 4 cohorts (n = 357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x = 36) by LC/MS, and in flow-sorted melanoma tumor cells (x = 37) and patient-derived melanoma cell lines (x = 17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n = 371). RESULTS: DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI. CONCLUSIONS: These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies

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

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 [...

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

Understanding the Tumor Microenvironment in Melanoma Patients with In-Transit Metastases and Its Impacts on Immune Checkpoint Immunotherapy Responses

Melanoma is the leading cause of global skin cancer-related death and currently ranks as the third most commonly diagnosed cancer in Australia. Melanoma patients with in-transit metastases (ITM), a type of locoregional metastasis located close to the primary tumor site, exhibit a high likelihood of further disease progression and poor survival outcomes. Immunotherapies, particularly immune checkpoint inhibitors (ICI), have demonstrated remarkable efficacy in ITM patients with reduced occurrence of further metastases and prolonged survival. The major challenge of immunotherapeutic efficacy lies in the limited understanding of melanoma and ITM biology, hindering our ability to identify patients who likely respond to ICIs effectively. In this review, we provided an overview of melanoma and ITM disease. We outlined the key ICI therapies and the critical immune features associated with therapy response or resistance. Lastly, we dissected the underlying biological components, including the cellular compositions and their communication networks within the tumor compartment, to enhance our understanding of the interactions between immunotherapy and melanoma, providing insights for future investigation and the development of drug targets and predictive biomarkers