Brain-Derived Extracellular Vesicle microRNA Signatures Associated with In Utero and Postnatal Oxycodone Exposure

Oxycodone (oxy) is a semi-synthetic opioid commonly used as a pain medication that is also a widely abused prescription drug. While very limited studies have examined the effect of in utero oxy (IUO) exposure on neurodevelopment, a significant gap in knowledge is the effect of IUO compared with postnatal oxy (PNO) exposure on synaptogenesis—a key process in the formation of synapses during brain development—in the exposed offspring. One relatively unexplored form of cell–cell communication associated with brain development in response to IUO and PNO exposure are extracellular vesicles (EVs). EVs are membrane-bound vesicles that serve as carriers of cargo, such as microRNAs (miRNAs). Using RNA-Seq analysis, we identified distinct brain-derived extracellular vesicle (BDEs) miRNA signatures associated with IUO and PNO exposure, including their gene targets, regulating key functional pathways associated with brain development to be more impacted in the IUO offspring. Further treatment of primary 14-day in vitro (DIV) neurons with IUO BDEs caused a significant reduction in spine density compared to treatment with BDEs from PNO and saline groups. In summary, our studies identified for the first time, key BDE miRNA signatures in IUO- and PNO-exposed offspring, which could impact their brain development as well as synaptic function.

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INTEGRINS MEDIATE PLACENTAL EXTRACELLULAR VESICLE TRAFFICKING TO LUNG AND LIVER IN VIVO

10.1101/2020.09.22.309047 ◽

2020 ◽

Author(s):

Sean L. Nguyen ◽

Soo Hyun Ahn ◽

Jacob W. Greenberg ◽

Benjamin W. Collaer ◽

Dalen W. Agnew ◽

...

Keyword(s):

Immune Cells ◽

Extracellular Vesicle ◽

Dependent Manner ◽

Cellular Phenotype ◽

Reporter Mouse ◽

Membrane Bound ◽

First Time

ABSTRACTMembrane-bound extracellular vesicles (EVs) mediate intercellular communication in all organisms, and those produced by placental mammals have become increasingly recognized as significant mediators of fetal-maternal communication. Here, we aimed to identify maternal cells targeted by placental EVs and elucidate the mechanisms by which they traffic to these cells. Exogenously administered pregnancy-associated EVs traffic specifically to the lung; further, placental EVs associate with lung interstitial macrophages and liver Kupffer cells in an integrin-dependent manner. Localization of EV to maternal lungs was confirmed in unmanipulated pregnancy using a transgenic reporter mouse model, which also provided in situ and in vitro evidence that fetally-derived EVs, rarely, may cause genetic alteration of maternal cells. These results provide for the first time direct in vivo evidence for targeting of placental EVs to maternal immune cells, and further, evidence that EVs can alter cellular phenotype.

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Integrins mediate placental extracellular vesicle trafficking to lung and liver in vivo

Scientific Reports ◽

10.1038/s41598-021-82752-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sean L. Nguyen ◽

Soo Hyun Ahn ◽

Jacob W. Greenberg ◽

Benjamin W. Collaer ◽

Dalen W. Agnew ◽

...

Keyword(s):

Immune Cells ◽

Extracellular Vesicle ◽

Dependent Manner ◽

Cellular Phenotype ◽

Reporter Mouse ◽

Membrane Bound ◽

First Time

AbstractMembrane-bound extracellular vesicles (EVs) mediate intercellular communication in all organisms, and those produced by placental mammals have become increasingly recognized as significant mediators of fetal-maternal communication. Here, we aimed to identify maternal cells targeted by placental EVs and elucidate the mechanisms by which they traffic to these cells. Exogenously administered pregnancy-associated EVs traffic specifically to the lung; further, placental EVs associate with lung interstitial macrophages and liver Kupffer cells in an integrin-dependent manner. Localization of EV to maternal lungs was confirmed in unmanipulated pregnancy using a transgenic reporter mouse model, which also provided in situ and in vitro evidence that fetally-derived EVs, rarely, may cause genetic alteration of maternal cells. These results provide for the first time direct in vivo evidence that placental EVs target maternal immune cells, and further, that EVs can alter cellular phenotype.

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lncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22031407 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1407

Author(s):

Hongxia Liu ◽

Wang Zheng ◽

Qianping Chen ◽

Yuchuan Zhou ◽

Yan Pan ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Cell Line ◽

Malignant Tumors ◽

Underlying Mechanism ◽

Mtor Pathway ◽

Rna Seq ◽

Cellular Autophagy ◽

First Time

Nasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors and is majorly treated by radiotherapy. However, radiation resistance remains a serious obstacle to the successful treatment of NPC. The aim of this study was to discover the underlying mechanism of radioresistance and to elucidate novel genes that may play important roles in the regulation of NPC radiosensitivity. By using RNA-seq analysis of NPC cell line CNE2 and its radioresistant cell line CNE2R, lncRNA CASC19 was screened out as a candidate radioresistance marker. Both in vitro and in vivo data demonstrated that a high expression level of CASC19 was positively correlated with the radioresistance of NPC, and the radiosensitivity of NPC cells was considerably enhanced by knockdown of CASC19. The incidence of autophagy was enhanced in CNE2R in comparison with CNE2 and another NPC cell line HONE1, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, CASC19 siRNA (siCASC19) suppressed cellular autophagy by inhibiting the AMPK/mTOR pathway and promoted apoptosis through the PARP1 pathway. Our results revealed for the first time that lncRNA CASC19 contributed to the radioresistance of NPC by regulating autophagy. In significance, CASC19 might be a potential molecular biomarker and a new therapeutic target in NPC.

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Quercetin inhibits intrahepatic cholangiocarcinoma by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation via the NF-κB pathway

10.21203/rs.3.rs-947377/v1 ◽

2021 ◽

Author(s):

Yinghui Song ◽

Zhihua Zhang ◽

Qin Chai ◽

GuoYi Xia ◽

Zhangtao Yu ◽

...

Keyword(s):

Intrahepatic Cholangiocarcinoma ◽

Clonogenic Assay ◽

Direct Interaction ◽

Rna Seq ◽

Wound Healing Assay ◽

Tumor Bearing ◽

Mesenchymal Transformation ◽

First Time

Abstract Intrahepatic cholangiocarcinoma (ICC) is a rare high-fatal hepatobiliary malignancy, the treatment option of ICC is very limited, and the prognosis is also poor. Recently, emerging evidence has shown the potential of quercetin (QE) for cancer therapy. We explored the effect and mechanism of QE on ICC in vitro and in vivo. CCK-8 assay and Clonogenic assay showed that QE could inhibit ICC cells proliferation and survival. PI staining suggested QE could induce ICC cells arrest in G1 phase. AV/PI staining suggested QE could promote ICC cells apoptosis. Wound Healing Assay and Transwell chamber experiment suggested QE could inhibit ICC cells EMT. RNA-seq, the changes in the structure of mitochondria by electron microscopy and the key markers of ferroptosis (free iron ions, MDA, SOD, GPX4) were supported QE could promote ferroptosis in ICC cells. Molecular docking showed that QE had direct interaction with NF-κB and GPX4. In vivo, treatment with QE inhibited tumor growth and prolonged survival time of tumor-bearing nude mice. Our data for the first time suggest that QE is a new ferroptosis inducer and combinative treatment of inhibiting NF-κB in ICC cells by inducing ferroptosis and inhibiting EMT, which will hopefully provide a prospective strategy for ICC patients.

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Advances in microglia cellular models: focus on extracellular vesicle production

Biochemical Society Transactions ◽

10.1042/bst20210203 ◽

2021 ◽

Author(s):

Lorenzo Ceccarelli ◽

Laura Marchetti ◽

Chiara Giacomelli ◽

Claudia Martini

Keyword(s):

Cell Communication ◽

Extracellular Vesicle ◽

Cellular Models ◽

Advantages And Disadvantages ◽

Inflammatory Processes ◽

The Central Nervous System ◽

Harmful Effects ◽

Brain Homeostasis

Microglia are the major component of the innate immune system in the central nervous system. They promote the maintenance of brain homeostasis as well as support inflammatory processes that are often related to pathological conditions such as neurodegenerative diseases. Depending on the stimulus received, microglia cells dynamically change their phenotype releasing specific soluble factors and largely modify the cargo of their secreted extracellular vesicles (EVs). Despite the mechanisms at the basis of microglia actions have not been completely clarified, the recognized functions exerted by their EVs in patho-physiological conditions represent the proof of the crucial role of these organelles in tuning cell-to-cell communication, promoting either protective or harmful effects. Consistently, in vitro cell models to better elucidate microglia EV production and mechanisms of their release have been increased in the last years. In this review, the main microglial cellular models that have been developed and validated will be described and discussed, with particular focus on those used to produce and derive EVs. The advantages and disadvantages of their use will be evidenced too. Finally, given the wide interest in applying EVs in diagnosis and therapy too, the heterogeneity of available models for producing microglia EVs is here underlined, to prompt a cross-check or comparison among them.

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Tiny Actors in the Big Cellular World: Extracellular Vesicles Playing Critical Roles in Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21207688 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7688 ◽

Cited By ~ 2

Author(s):

Ancuta Jurj ◽

Cecilia Pop-Bica ◽

Ondrej Slaby ◽

Cristina D. Ştefan ◽

William C. Cho ◽

...

Keyword(s):

Extracellular Vesicles ◽

Molecular Mechanisms ◽

Recipient Cell ◽

Cell Communication ◽

Therapeutic Agents ◽

Bioactive Molecules ◽

Cellular Functions ◽

Membrane Bound

Communications among cells can be achieved either via direct interactions or via secretion of soluble factors. The emergence of extracellular vesicles (EVs) as entities that play key roles in cell-to-cell communication offer opportunities in exploring their features for use in therapeutics; i.e., management and treatment of various pathologies, such as those used for cancer. The potential use of EVs as therapeutic agents is attributed not only for their cell membrane-bound components, but also for their cargos, mostly bioactive molecules, wherein the former regulate interactions with a recipient cell while the latter trigger cellular functions/molecular mechanisms of a recipient cell. In this article, we highlight the involvement of EVs in hallmarks of a cancer cell, particularly focusing on those molecular processes that are influenced by EV cargos. Moreover, we explored the roles of RNA species and proteins carried by EVs in eliciting drug resistance phenotypes. Interestingly, engineered EVs have been investigated and proposed as therapeutic agents in various in vivo and in vitro studies, as well as in several clinical trials.

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Transcriptomic Analysis of mRNA-lncRNA-miRNA Interactions in Hepatocellular Carcinoma

Scientific Reports ◽

10.1038/s41598-019-52559-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 14

Author(s):

Xia Tang ◽

Delong Feng ◽

Min Li ◽

Jinxue Zhou ◽

Xiaoyuan Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Rna Seq ◽

Pairwise Interactions ◽

Micro Rnas ◽

Non Coding Rnas ◽

First Time

Abstract Fully elucidating the molecular mechanisms of non-coding RNAs (ncRNAs), including micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs), underlying hepatocarcinogenesis is challenging. We characterized the expression profiles of ncRNAs and constructed a regulatory mRNA-lncRNA-miRNA (MLMI) network based on transcriptome sequencing (RNA-seq) of hepatocellular carcinoma (HCC, n = 9) patients. Of the identified miRNAs (n = 203) and lncRNAs (n = 1,090), we found 16 significantly differentially expressed (DE) miRNAs and three DE lncRNAs. The DE RNAs were highly enriched in 21 functional pathways implicated in HCC (p < 0.05), including p53, MAPK, and NAFLD signaling. Potential pairwise interactions between DE ncRNAs and mRNAs were fully characterized using in silico prediction and experimentally-validated evidence. We for the first time constructed a MLMI network of reciprocal interactions for 16 miRNAs, three lncRNAs, and 253 mRNAs in HCC. The predominant role of MEG3 in the MLMI network was validated by its overexpression in vitro that the expression levels of a proportion of MEG3-targeted miRNAs and mRNAs was changed significantly. Our results suggested that the comprehensive MLMI network synergistically modulated carcinogenesis, and the crosstalk of the network provides a new avenue to accurately describe the molecular mechanisms of hepatocarcinogenesis.

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Astrocytes donate mitochondria to increase glioblastoma tumorigenicity

10.21203/rs.3.rs-923533/v1 ◽

2021 ◽

Author(s):

Justin Lathia ◽

Dionysios Watson ◽

Defne Bayik ◽

Sarah Williford ◽

Adam Lauko ◽

...

Keyword(s):

Tumor Microenvironment ◽

Cell Communication ◽

Treatment Resistance ◽

Extracellular Vesicle ◽

Host Cells ◽

Molecular Events ◽

Mitochondrial Transfer ◽

New Research

Abstract Glioblastoma (GBM), the most common primary brain cancer in adults1, is characterized by numerous cell-intrinsic/extrinsic interactions that drive tumorigenesis. In addition to cell-surface and secreted protein/extracellular vesicle interactions2,3, treatment resistance of GBM is augmented by the formation of cytoplasmic interconnections and junctions among tumor cells4. These cytoplasmic bridges among tumor cells enable exchange of cellular metabolites as well as mitochondria4, which can play a key role in metabolic function and cellular programming in GBM5,6. However, the contribution of the tumor microenvironment to mitochondrial transfer, as well as the downstream impact of mitochondrial transfer on GBM remains poorly characterized. Here we identified horizontal mitochondrial transfer from the tumor microenvironment as a mechanism that enhances tumorigenesis in glioblastoma. We found that this transfer occurs primarily from brain-resident cells, including astrocytes, and can be appreciated in vitro and in vivo through intercellular connections between GBM cells and non-malignant host cells. The acquisition of astrocyte mitochondria drives an overall enhancement of mitochondrial membrane potential and metabolic capacity, while increasing glioblastoma cell self-renewal and tumor-initiating capacity. Collectively, our findings demonstrate that astrocyte mitochondrial transfer augments the tumorigenic capacity of glioblastoma cells and reveals a previously unknown cell-cell communication mechanism that drives tumor growth. We anticipate our findings will open new research directions to decipher the molecular events linking mitochondria acquisition from non-malignant cells to increased tumorigenicity of recipient GBM cells. This line of research will lead to new therapeutic opportunities targeting this understudied phenomenon and its sequelae in GBM.

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In Silico and In Vitro Analysis of lncRNA XIST Reveals a Panel of Possible Lung Cancer Regulators and a Five-Gene Diagnostic Signature

Cancers ◽

10.3390/cancers12123499 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3499

Author(s):

Periklis Katopodis ◽

Qiduo Dong ◽

Heerni Halai ◽

Cristian I. Fratila ◽

Andreas Polychronis ◽

...

Keyword(s):

Lung Cancer ◽

Cell Lines ◽

In Silico ◽

Cell Biology ◽

Tumour Progression ◽

Cell Communication ◽

Nucleic Acid Metabolism ◽

Rna Seq ◽

Diagnostic Potential

Long non-coding RNAs (lncRNAs) perform a wide functional repertoire of roles in cell biology, ranging from RNA editing to gene regulation, as well as tumour genesis and tumour progression. The lncRNA X-inactive specific transcript (XIST) is involved in the aetiopathogenesis of non-small cell lung cancer (NSCLC). However, its role at the molecular level is not fully elucidated. The expression of XIST and co-regulated genes TSIX, hnRNPu, Bcl-2, and BRCA1 analyses in lung cancer (LC) and controls were performed in silico. Differentially expressed genes (DEGs) were determined using RNA-seq in H1975 and A549 NSCLC cell lines following siRNA for XIST. XIST exhibited sexual dimorphism, being up-regulated in females compared to males in both control and LC patient cohorts. RNA-seq revealed 944 and 751 DEGs for A549 and H1975 cell lines, respectively. These DEGs are involved in signal transduction, cell communication, energy pathways, and nucleic acid metabolism. XIST expression associated with TSIX, hnRNPu, Bcl-2, and BRCA1 provided a strong collective feature to discriminate between controls and LC, implying a diagnostic potential. There is a much more complex role for XIST in lung cancer. Further studies should concentrate on sex-specific changes and investigate the signalling pathways of the DEGs following silencing of this lncRNA.

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High-Resolution Insights Into the in vitro Developing Blood-Brain Barrier: Novel Morphological Features of Endothelial Nanotube Function

Frontiers in Neuroanatomy ◽

10.3389/fnana.2021.661065 ◽

2021 ◽

Vol 15 ◽

Author(s):

Shireen Mentor ◽

David Fisher

Keyword(s):

High Resolution ◽

Blood Brain Barrier ◽

Cell Communication ◽

High Resolution Electron Microscopy ◽

Brain Barrier ◽

Transport Systems ◽

Functional Roles ◽

Blood Brain ◽

First Time

High-resolution electron microscopy (HREM) imaging of the in vitro blood-brain barrier (BBB), is a promising modality for investigating the dynamic morphological interplay underpinning BBB development. The successful establishment of BBB integrity is grounded in the brain endothelial cells (BEC’s) ability to occlude its paracellular spaces of brain capillaries through the expression of the intercellular tight junction (TJ) proteins. The impermeability of these paracellular spaces are crucial in the regulation of transcellular transport systems to achieve homeostasis of the central nervous system. To-date research describing morphologically, the dynamics by which TJ interaction is orchestrated to successfully construct a specialized barrier remains undescribed. In this study, the application of HREM illuminates the novel, dynamic and highly restrictive BEC paracellular pathway which is founded based on lateral membrane alignment which is the functional imperative for the mechanical juxtapositioning of TJ zones that underpin molecular bonding and sealing of the paracellular space. For the first time, we report on the secretion of a basement membrane in vitro, which allow BECs to orientate themselves into distinct basolateral and apicolateral domains and establish a 3-dimensional BEC construct. We report for the first time, on the expression of nanovesicles bound to the plasma membrane surfaces of the BECs. These membrane-bound vesicles are reported to possess an array of DNA/RNA constituents and chemotaxic properties affecting the formation of nanotubes that span the paracellular space between BECs, facilitating BBB construction, alluding to a functional role in mediating cell-to-cell communication. This study suggests that novel, ultrathin nanotubular (NT) structures are involved in functional roles in bringing into alignment the paracellular space of BECs. Immortalized mouse BECs (b.End3, b.End5) and primary rat cardiac microvascular ECs were used to further validate the in vitro BBB model by profiling variances in peripheral EC monolayer development. These cardiac capillary ECs presented with an opposite topographical profile: large fenestra and intercellular spaces, devoid of morphological ultrastructures. This comparative study alludes to the role of NT facilitation in TJ-induced hemifusion of apicolateral BEC membranes, as a structural event forming the basis for establishing a polarized BBB.

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