isolating-sequencing-analyzing-extracellular-micrornas-from-human

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Aarhus University

Emerging evidence suggests functional regulation of the Hippo pathway by the actin cytoskeleton, although the detailed molecular mechanism remains incomplete. In a genetic screen, we identified a requirement for β-Spectrin in the posterior follicle cells for the oocyte repolarization process during Drosophila mid-oogenesis. β-spectrin mutations lead to loss of Hippo signaling activity in the follicle cells. A similar reduction of Hippo signaling activity was observed after β-Spectrin knockdown in mammalian cells. We further demonstrated that β-spectrin mutations disrupt the basal actin network in follicle cells. The abnormal stress fiber-like actin structure on the basal side of follicle cells provides a likely link between the β-spectrin mutations and the loss of the Hippo signaling activity phenotype.

β-Spectrin regulates the hippo signaling pathway and modulates the basal actin network.

Body size is one of the features that distinguish one species from another in the biological world. Animals have developed mechanisms to control their body size during normal development. However, how animals cope with genetic alterations and/or environmental stresses to develop into normal-sized adults remain poorly understood. The ability of the animals to develop into a normal-sized adult after the challenges of genetic alterations and/or environmental stresses reveals a robustness of body size control. Here we show that the mutation of dGPAT4, a de novo synthase of lysophosphatidic acid, is a genetic alteration that triggers such a robust response of the animals to body size challenges in Drosophila. Loss of dGPAT4 leads to a severe delay of development, slow growth and resultant small-sized animals during the larval stages, but results in normal-sized adult flies. The robust body size adjustment of the dGPAT4 mutant is likely achieved by corresponding changes in ecdysone and insulin signaling, which is also manifested by compromised food intake. Thus, we propose that a strategy has been evolved by the animals to reach final body size when challenged by genetic alterations, which requires the coordinated ecdysone and insulin signaling. 

Ecdysone and insulin signaling play essential roles in readjusting the altered body size caused by the dGPAT4 mutation in Drosophila.

The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed that decreased expression of miR-30a-3p and increased expression of miR-210-3p were significantly associated with the presence of distant metastases. miR-210-3p tumor cell specificity was evaluated by in situ hybridization and its biomarker potential was confirmed by ROC curve analysis (AUC = 0.839). Lastly, agreement between miRNA-seq and RT-qPCR for FFPE-derived RNA was evaluated and a high level of concordance was determined. In conclusion, this study has identified and validated metastasis-related miRNAs in LAC.

Small RNA sequencing reveals metastasis-related microRNAs in lung adenocarcinoma.

Cell delamination is a conserved morphogenetic process important for the generation of cell diversity and maintenance of tissue homeostasis. Here, we used  embryonic neuroblasts as a model to study the apical constriction process during cell delamination. We observe dynamic myosin signals both around the cell adherens junctions and underneath the cell apical surface in the neuroectoderm. On the cell apical cortex, the nonjunctional myosin forms flows and pulses, which are termed medial myosin pulses. Quantitative differences in medial myosin pulse intensity and frequency are crucial to distinguish delaminating neuroblasts from their neighbors. Inhibition of medial myosin pulses blocks delamination. The fate of a neuroblast is set apart from that of its neighbors by Notch signaling-mediated lateral inhibition. When we inhibit Notch signaling activity in the embryo, we observe that small clusters of cells undergo apical constriction and display an abnormal apical myosin pattern. Together, these results demonstrate that a contractile actomyosin network across the apical cell surface is organized to drive apical constriction in delaminating neuroblasts.

Apical constriction is driven by a pulsatile apical myosin network in delaminating  neuroblasts.

Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1).

Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis.

Age-dependent ectopic fat accumulation (EFA) in animals contributes to the progression of tissue aging and diseases such as obesity, diabetes, and cancer. However, the primary causes of age-dependent EFA remain largely elusive. Here, we characterize the occurrence of age-dependent EFA in Drosophila and identify HDAC6, a cytosolic histone deacetylase, as a suppressor of EFA. Loss of HDAC6 leads to significant age-dependent EFA, lipid composition imbalance, and reduced animal longevity on a high-fat diet. The EFA and longevity phenotypes are ameliorated by a reduction of the lipid-droplet-resident protein PLIN2. We show that HDAC6 is associated physically with the chaperone protein dHsc4/Hsc70 to maintain the proteostasis of PLIN2. These findings indicate that proteostasis collapse serves as an intrinsic cue to cause age-dependent EFA. Our study suggests that manipulation of proteostasis could be an alternative approach to the treatment of age-related metabolic diseases such as obesity and diabetes.

HDAC6 Suppresses Age-Dependent Ectopic Fat Accumulation by Maintaining the Proteostasis of PLIN2 in Drosophila.

Motile cilia impairment is a common condition in patients with chronically inflamed airways, such as is seen in nasal polyps (NPs). The mechanism underlying this pathogenic condition is complex and not fully understood.

Absence or mislocalization of DNAH5 is a characteristic marker for motile ciliary abnormality in nasal polyps.

Tau protein participates in microtubule stabilization, axonal transport, and protein trafficking. Loss of normal tau function will exert a negative effect. However, current knowledge on the impact of tau deficiency on the motor behavior and related neurobiological changes is controversial. In this study, we examined motor functions and analyzed several proteins implicated in the maintenance of midbrain dopaminergic (DA) neurons (mDANs) function of adult and aged tau, tau, tau mice. We found tau deficiency could not induce significant motor disorders. However, we discovered lower expression levels of transcription factors Orthodenticle homeobox 2 (OTX2) of mDANs in older aged mice. Compared with age-matched tau mice, there were 54.1% lower (p = 0.0192) OTX2 protein (OTX2-fluorescence intensity) in VTA DA neurons of tau mice and 43.6% lower (p = 0.0249) OTX2 protein in VTA DA neurons of tau mice at 18 months old. Combined with the relevant reports, our results suggested that tau deficiency alone might not be enough to mimic the pathology of Parkinson's disease. However, OTX2 down-regulation indicates that mDANs of tau-deficient mice will be more sensitive to toxic damage from MPTP.

Tau Deficiency Down-Regulated Transcription Factor Orthodenticle Homeobox 2 Expression in the Dopaminergic Neurons in Ventral Tegmental Area and Caused No Obvious Motor Deficits in Mice.

Tight junctions (TJs) alteration is commonly seen in airway inflammatory diseases. Oncostatin M (OSM) is an inflammatory mediator associated with chronic rhinosinusitis with nasal polyps (CRSwNP). We have previously shown that human nasal epithelial cells (hNECs) are highly permissive cells for influenza A virus (IAV). However, its role in TJs alteration and the effects of IAV on inducing OSM expression in nasal epithelium remains to be further investigated. In this study, OSM and TJs expression was measured and compared between inferior turbinate from healthy controls and nasal polyps from CRSwNP. Additionally, hNECs cultured at air-liquid interface (ALI) were infected with H3N2 influenza virus to study the role of influenza virus in inducing epithelial OSM expression as a possible means of exacerbation. The expression of ZO-1, claudin-1, and occludin was markedly decreased and correlated negatively with that of OSM in CRSwNP. By using the in vitro hNEC model, H3N2 infection resulted in significantly increased OSM expression (2.2-, 4.7- and 3.9-fold higher at 8, 24, and 48 h post-infection vs. mock infection). Furthermore, OSM is found to co-localize with ciliated and goblet cells in hNECs infected with H3N2 influenza virus. Our findings demonstrated that increased OSM expression is implicated in CRSwNP as a possible mechanism of TJs' impairment, which can be further augmented following influenza infection via epithelial OSM expression, possibly contributing to exacerbations.

H3N2 influenza virus infection enhances oncostatin M expression in human nasal epithelium.

The prevalent porcine helminth, , compromises pig health and reduces farm productivity worldwide. The closely related human parasite, , infects more than 800 million people representing a disease burden of 1.31 million disability-adjusted life years. The infections are often chronic in nature, and the parasites have a profound ability to modulate their hosts' immune responses. This study provides the first in-depth characterisation of extracellular vesicles (EVs) from different developmental stages and body parts of  and proposes the role of these vesicles in the host-parasite interplay. The release of EVs from the third- (L3) and fourth-stage (L4) larvae and adults was demonstrated by transmission electron microscopy (TEM), and sequencing of EV-derived RNA identified a number of microRNAs (miRNAs) and transcripts of potential host immune targets, such as IL-13, IL-25 and IL-33, were identified. Furthermore, proteomics of EVs identified several proteins with immunomodulatory properties and other proteins previously shown to be associated with parasite EVs. Taken together, these results suggest that  EVs and their cargo may play a role in host-parasite interactions. This knowledge may pave the way to novel strategies for helminth infection control and knowledge of their immune modulatory potential.

Exploration of extracellular vesicles from  provides evidence of parasite-host cross talk.

Human tumors exhibit plasticity and evolving capacity over time. It is difficult to study the mechanisms of how tumors change over time in human patients, in particular during the early stages when a few oncogenic cells are barely detectable. Here, we used a  tumor model caused by loss of  (), a highly conserved apicobasal cell polarity gene, to investigate the spatial-temporal dynamics of early tumorigenesis events. The fly  mutant tumors have been successfully used to model many aspects of tumorigenesis processes. However, it is still unknown whether  mutant tumors exhibit plasticity and evolvability along the temporal axis. We found that  mutant tumors displayed different growth rates and cell cycle profiles over time, indicative of a growth arrest-to-proliferation transition as the  mutant tumors progress. Longitudinal bulk and single-cell transcriptomic analysis of  mutant tumors revealed that the MAPK pathway, including JNK and ERK signaling activities, showed quantitative changes over time. We found that high JNK signaling activity caused G2/M cell cycle arrest in early  mutant tumors. In addition, JNK signaling activity displayed a radial polarity with the JNK cells located at the periphery of  mutant tumors, providing an inherent mechanism that leads to an overall decrease in JNK signaling activity over time. We also found that ERK signaling activity, in contrast to JNK activity, increased over time and promoted growth in late-stage  mutant tumors. Furthermore, high JNK signaling activity repressed ERK signaling activity in early  mutant tumors. Together, these data demonstrate that dynamic MAPK signaling activity, fueled by intratumor heterogeneity derived from tissue topological differences, drives a growth arrest-to-proliferation transition in  mutant tumors.This article has an associated First Person interview with the joint first authors of the paper.

Dynamic MAPK signaling activity underlies a transition from growth arrest to proliferation in  mutant tumors.

Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at the individual cell level remains unclear. In the past decades, studies using  wing imaginal discs as a model system have provided valuable insights into pattern formation, growth control and regeneration. Here, we provide single cell transcriptomic landscapes of pattern formation, proliferation and growth of wing imaginal discs. We found that patterning information is robustly maintained in the single cell transcriptomic data and can provide reference matrices for computationally mapping single cells into discrete spatial domains. Assignment of wing disc single cells to spatial subregions facilitates examination of patterning refinement processes. We also clustered single cells into different proliferation and growth states and evaluated the correlation between cell proliferation/growth states and spatial patterning. Furthermore, single cell transcriptomic analyses allowed us to quantitatively examine disturbances of differentiation, proliferation and growth in a well-established tumor model. We provide a database to explore these datasets at http://drosophilayanlab-virtual-wingdisc.ust.hk:3838/v2/This article has an associated 'The people behind the papers' interview.

Single cell transcriptomic landscapes of pattern formation, proliferation and growth in  wing imaginal discs.

Recent advances in single-cell RNA sequencing (scRNA-seq) techniques lead to an explosion of single-cell atlases from diverse biological contexts. The information of cell-cell signaling events, which underlie multicellular organism function, is embedded in these atlases. Here, we review current strategies of mining cell-cell signaling events from single-cell transcriptomics datasets and highlight examples where functions of predicted cell-cell signaling events from single-cell atlases are further pursued to yield new insights into biological processes.

Article	Year
β-Spectrin regulates the hippo signaling pathway and modulates the basal actin network. The Journal of biological chemistry\| PubMed ID: 25589787	2015
Ecdysone and insulin signaling play essential roles in readjusting the altered body size caused by the dGPAT4 mutation in Drosophila. Journal of genetics and genomics = Yi chuan xue bao\| PubMed ID: 26408093	2015
Small RNA sequencing reveals metastasis-related microRNAs in lung adenocarcinoma. Oncotarget\| PubMed ID: 28460486	2017
Apical constriction is driven by a pulsatile apical myosin network in delaminating neuroblasts. Development (Cambridge, England)\| PubMed ID: 28506995	2017
Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis. Frontiers in immunology\| PubMed ID: 28791012	2017
HDAC6 Suppresses Age-Dependent Ectopic Fat Accumulation by Maintaining the Proteostasis of PLIN2 in Drosophila. Developmental cell\| PubMed ID: 28966044	2017
Absence or mislocalization of DNAH5 is a characteristic marker for motile ciliary abnormality in nasal polyps. The Laryngoscope\| PubMed ID: 29148098	2018
Tau Deficiency Down-Regulated Transcription Factor Orthodenticle Homeobox 2 Expression in the Dopaminergic Neurons in Ventral Tegmental Area and Caused No Obvious Motor Deficits in Mice. Neuroscience\| PubMed ID: 29337233	2018
H3N2 influenza virus infection enhances oncostatin M expression in human nasal epithelium. Experimental cell research\| PubMed ID: 30142324	2018
Exploration of extracellular vesicles from provides evidence of parasite-host cross talk. Journal of extracellular vesicles\| PubMed ID: 30815237	2019
Dynamic MAPK signaling activity underlies a transition from growth arrest to proliferation in mutant tumors. Disease models & mechanisms\| PubMed ID: 31371383	2019
Single cell transcriptomic landscapes of pattern formation, proliferation and growth in wing imaginal discs. Development (Cambridge, England)\| PubMed ID: 31455604	2019
Mining cell-cell signaling in single-cell transcriptomics atlases. Current opinion in cell biology\| PubMed ID: 35609365	2022

Yan Yan

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