MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response.

MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor-immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.

Analysis of Proliferation, Apoptosis, and Motility in Mouse Embryonic Melanocytic Precursor Cells.

In this chapter, we provide a method to purify and culture embryonic melanocytic stem cells that express green fluorescent protein in a cell-type specific manner. Isolation of melanocytic lineage cell populations that are >98% pure is accomplished through the use of GFP-based fluorescence activated cell sorting. We also provide a method to culture the purified melanoblasts and to analyze their proliferation, apoptosis, and motility properties.

miR-146a regulates mechanotransduction and pressure-induced inflammation in small airway epithelium.

Mechanical ventilation generates biophysical forces, including high transmural pressures, which exacerbate lung inflammation. This study sought to determine whether microRNAs (miRNAs) respond to this mechanical force and play a role in regulating mechanically induced inflammation. Primary human small airway epithelial cells (HSAEpCs) were exposed to 12 h of oscillatory pressure and/or the proinflammatory cytokine TNF-α. Experiments were also conducted after manipulating miRNA expression and silencing the transcription factor NF-κB or toll-like receptor proteins IRAK1 and TRAF6. NF-κB activation, IL-6/IL-8/IL-1ß cytokine secretion, miRNA expression, and IRAK1/TRAF6 protein levels were monitored. A total of 12 h of oscillatory pressure and TNF-α resulted in a 5- to 7-fold increase in IL-6/IL-8 cytokine secretion, and oscillatory pressure also resulted in a time-dependent increase in IL-6/IL-8/IL-1ß cytokine secretion. Pressure and TNF-α also resulted in distinct patterns of miRNA expression, with miR-146a being the most deregulated miRNA. Manipulating miR-146a expression altered pressure-induced cytokine secretion. Silencing of IRAK1 or TRAF6, confirmed targets of miR-146a, resulted in a 3-fold decrease in pressure-induced cytokine secretion. Cotransfection experiments demonstrate that miR-146a's regulation of pressure-induced cytokine secretion depends on its targeting of both IRAK1 and TRAF6. MiR-146a is a mechanosensitive miRNA that is rapidly up-regulated by oscillatory pressure and plays an important role in regulating mechanically induced inflammation in lung epithelia.

Isolation, Purification, and Culture of Embryonic Melanoblasts from Green Fluorescent Protein-expressing Reporter Mice.

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

Cadmium-mediated toxicity of lung epithelia is enhanced through NF-κB-mediated transcriptional activation of the human zinc transporter ZIP8.

Cadmium (Cd), a toxic heavy metal and carcinogen that is abundantly present in cigarette smoke, is a cause of smoking-induced lung disease. SLC39A8 (ZIP8), a zinc transporter, is a major portal for Cd uptake into cells. We have recently identified that ZIP8 expression is under the transcriptional control of the NF-κB pathway. On the basis of this, we hypothesized that cigarette-smoke induced inflammation would increase ZIP8 expression in lung epithelia, thereby enhancing Cd uptake and cell toxicity. Herein we report that ZIP8 is a central mediator of Cd-mediated toxicity. TNF-α treatment of primary human lung epithelia and A549 cells induced ZIP8 expression, resulting in significantly higher cell death attributable to both apoptosis and necrosis following Cd exposure. Inhibition of the NF-κB pathway and ZIP8 expression significantly reduced cell toxicity. Zinc (Zn), a known cytoprotectant, prevented Cd-mediated cell toxicity via ZIP8 uptake. Consistent with cell culture findings, a significant increase in ZIP8 mRNA and protein expression was observed in the lung of chronic smokers compared with nonsmokers. From these studies, we conclude that ZIP8 expression is induced in lung epithelia in an NF-κB-dependent manner, thereby resulting in increased cell death in the presence of Cd. From this we contend that ZIP8 plays a critical role at the interface between micronutrient (Zn) metabolism and toxic metal exposure (Cd) in the lung microenvironment following cigarette smoke exposure. Furthermore, dietary Zn intake, or a lack thereof, may be a contributing factor in smoking-induced lung disease.

Gene expression networks in COPD: microRNA and mRNA regulation.

BACKGROUND: The mechanisms underlying chronic obstructive pulmonary disease (COPD) remain unclear. MicroRNAs (miRNAs or miRs) are small non-coding RNA molecules that modulate the levels of specific genes and proteins. Identifying expression patterns of miRNAs in COPD may enhance our understanding of the mechanisms of disease. A study was undertaken to determine if miRNAs are differentially expressed in the lungs of smokers with and without COPD. miRNA and mRNA expression were compared to enrich for biological networks relevant to the pathogenesis of COPD. METHODS: Lung tissue from smokers with no evidence of obstructive lung disease (n=9) and smokers with COPD (n=26) was examined for miRNA and mRNA expression followed by validation. We then examined both miRNA and mRNA expression to enrich for relevant biological pathways. RESULTS: 70 miRNAs and 2667 mRNAs were differentially expressed between lung tissue from subjects with COPD and smokers without COPD. miRNA and mRNA expression profiles enriched for biological pathways that may be relevant to the pathogenesis of COPD including the transforming growth factor ß, Wnt and focal adhesion pathways. miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD compared with smokers without obstruction. miR-15b was increased in COPD samples compared with smokers without obstruction and localised to both areas of emphysema and fibrosis. miR-15b was differentially expressed within GOLD classes of COPD. Expression of SMAD7, which was validated as a target for miR-15b, was decreased in bronchial epithelial cells in COPD. CONCLUSIONS: miRNA and mRNA are differentially expressed in individuals with COPD compared with smokers without obstruction. Investigating these relationships may further our understanding of the mechanisms of disease.

Differential expression of microRNA and predicted targets in pulmonary sarcoidosis.

BACKGROUND: Recent studies show that various inflammatory diseases are regulated at the level of RNA translation by small non-coding RNAs, termed microRNAs (miRNAs). We sought to determine whether sarcoidosis tissues harbor a distinct pattern of miRNA expression and then considered their potential molecular targets. METHODS AND RESULTS: Genome-wide microarray analysis of miRNA expression in lung tissue and peripheral blood mononuclear cells (PBMCs) was performed and differentially expressed (DE)-miRNAs were then validated by real-time PCR. A distinct pattern of DE-miRNA expression was identified in both lung tissue and PBMCs of sarcoidosis patients. A subgroup of DE-miRNAs common to lung and lymph node tissues were predicted to target transforming growth factor (TGFß)-regulated pathways. Likewise, the DE-miRNAs identified in PBMCs of sarcoidosis patients were predicted to target the TGFß-regulated "wingless and integrase-1" (WNT) pathway. CONCLUSIONS: This study is the first to profile miRNAs in sarcoidosis tissues and to consider their possible roles in disease pathogenesis. Our results suggest that miRNA regulate TGFß and related WNT pathways in sarcoidosis tissues, pathways previously incriminated in the pathogenesis of sarcoidosis.

Correction: MiR-34a/c-Dependent PDGFR-α/ß Downregulation Inhibits Tumorigenesis and Enhances TRAIL-Induced Apoptosis in Lung Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0067581.].

MicroRNA delivery by cationic lipoplexes for lung cancer therapy.

Lung cancer is the leading cause of cancer deaths in western countries and carries a poor overall five year survival rate. Several studies demonstrate that microRNAs (miRNAs or miRs) are actively involved in tumor development by serving as tumor suppressors, oncogenes or both. In lung cancer, miRNAs may serve as both diagnostic and prognostic biomarkers as well as regulate in vitro and in vivo tumor progression. However, miRNA-based therapy is faced with several challenges including lack of tissue specificity, lack of optimal delivery systems, poor cellular uptake and risk of systemic toxicity. Here, we report a cationic lipid based miRNA delivery system to address some of these challenges. Among many lung cancer related miRNAs, miR-133b, a tumor suppressor, was selected as a therapeutic target because it directly targets the prosurvival gene MCL-1 thus regulating cell survival and sensitivity of lung cancer cells to chemotherapeutic agents. The efficacy of pre-miR-133b containing lipoplexes was evaluated in A549 non-small cell lung cancer (NSCLC) cells. Compared with siPORT NeoFX transfection agent, lipoplexes delivered pre-miR-133b in a more efficient manner with ~2.3-fold increase in mature miR-133b expression and ~1.8-fold difference in MCL-1 protein downregulation in vitro. In the in vivo biodistribution study, lipoplexes achieved ~30% accumulation in lung tissue, which was ~50-fold higher than siPORT NeoFX transfection agent. Mice treated with pre-miR-133b containing lipoplexes had mature miR-133b expression in lung ~52-fold higher than untreated mice. Our results demonstrated that cationic lipoplexes are a promising carrier system for the development of miRNA-based therapeutics in lung cancer treatment.

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods.

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.

Changes in Body Mass Index Associated with Antiretroviral Regimen Switch Among Treatment-Experienced, Virologically Suppressed People Living with HIV in the United States.

With obesity on the rise among people living with HIV (PLWH), there is growing concern that weight gain may result as an undesired effect of antiretroviral therapy (ART). This analysis sought to assess the association between ART regimens and changes in body mass index (BMI) among ART-experienced, virologically suppressed PLWH. ART-experienced, virologically suppressed PLWH ≥18 years of age in the Observational Pharmacoepidemiology Research and Analysis (OPERA) cohort were included for analysis if prescribed a new regimen containing one of the following core agents: dolutegravir (DTG), elvitegravir/cobicistat (EVG/c), raltegravir (RAL), rilpivirine (RPV), or boosted darunavir (bDRV), for the first time between August 1, 2013 and December 31, 2017. Multivariable linear regression was used to assess the association between regimen and mean changes in BMI at 6, 12, and 24 months after switch. In unadjusted analyses, BMI increases ranged from 0.30 kg/m2 (bDRV) to 0.83 kg/m2 (RPV) at 24 months following switch, but gains were observed with every regimen. In adjusted analyses, compared to DTG, only bDRV was associated with a smaller increase in BMI at all time points, while EVG/c and RAL were associated with smaller increases in BMI at 6 months only. Overall, results were consistent in analyses stratified by baseline BMI category. BMI increases were relatively small but followed an upward trend over time in this cohort of treatment-experienced, suppressed PLWH. Gains were attenuated with a longer period of follow-up. BMI gains did not differ by regimens, except for bDRV regimens, which were consistently associated with smaller BMI increases than DTG.

Lyn kinase-dependent regulation of miR181 and myeloid cell leukemia-1 expression: implications for drug resistance in myelogenous leukemia.

Imatinib, a BCR-Abl inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). Despite the success of imatinib, multiple mechanisms of resistance remain a problem, including overexpression of Lyn kinase (Lyn) and Bcl-2 family antiapoptotic proteins. Profiling micro-RNA (miRNA) expression in a model of Lyn-mediated imatinib-resistant CML (MYL-R) identified approximately 30 miRNAs whose expression differed >2-fold compared with drug-sensitive MYL cells. In particular, the expression of the miR181 family (a-d) was significantly reduced (∼11- to 25-fold) in MYL-R cells. Incubation of MYL-R cells with a Lyn inhibitor (dasatinib) or nucleofection with Lyn-targeting short interfering RNA increased miR181b and miR181d expression. A similar Lyn-dependent regulation of miR181b and miR181d was observed in imatinib-resistant K562 CML cells. Sequence analysis of potential targets for miR181 regulation predicted myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member whose expression is increased in MYL-R cells and drug-resistant leukemias. Inhibition of Lyn or rescue of miR181b expression reduced Mcl-1 expression in the MYL-R cells. To further investigate the mechanism of Mcl-1 repression by miR181, a luciferase reporter construct incorporating the Mcl-1 3'-untranslated region was tested. Overexpression of miR181b reduced luciferase activity, whereas these effects were ablated by the mutation of the seed region of the miR181 target site. Finally, stimulation of Lyn expression by 1,25-dihydroxyvitamin D(3) treatment in HL-60 cells, a cell model of acute myelogenous leukemia, decreased miR181b expression and increased Mcl-1 expression. In summary, our results suggest that Lyn-dependent regulation of miR181 is a novel mechanism of regulating Mcl-1 expression and cell survival.

Essential Role for Integrin-Linked Kinase in Melanoblast Colonization of the Skin.

Melanocytes are pigment-producing cells found in the skin and other tissues. Alterations in the melanocyte lineage give rise to a plethora of human diseases, from neurocristopathies and pigmentation disorders to melanoma. During embryogenesis, neural crest cell subsets give rise to two waves of melanoblasts, which migrate dorsolaterally, hone to the skin, and differentiate into melanocytes. However, the mechanisms that govern colonization of the skin by the first wave of melanoblasts are poorly understood. Here we report that targeted inactivation of the integrin-linked kinase gene in first wave melanoblasts causes defects in the ability of these cells to form long pseudopods, to migrate, and to proliferate in vivo. As a result, integrin-linked kinase-deficient melanoblasts fail to populate normally the developing epidermis and hair follicles. We also show that defects in motility and dendricity occur upon integrin-linked kinase gene inactivation in mature melanocytes, causing abnormalities in cell responses to the extracellular matrix substrates collagen I and laminin 332. Significantly, the ability to form long protrusions in mutant cells in response to collagen is restored in the presence of constitutively active Rac1, suggesting that an integrin-linked kinase-Rac1 nexus is likely implicated in melanocytic cell establishment, dendricity, and functions in the skin.

Integrin-linked kinase regulates melanosome trafficking and melanin transfer in melanocytes.

Melanosomes are melanin-containing organelles that provide pigmentation and protection from solar UV radiation to the skin. In melanocytes, melanosomes mature and traffic to dendritic tips, where they are transferred to adjacent epidermal keratinocytes through pathways that involve microtubule networks and the actin cytoskeleton. However, the role of scaffold proteins in these processes is poorly understood. Integrin-linked kinase (ILK) is a scaffold protein that regulates microtubule stability and F-actin dynamics. Here we show that ILK is necessary for normal trafficking of melanosomes along microtubule tracks. In the absence of ILK, immature melanosomes are not retained in perinuclear regions, and mature melanosome trafficking along microtubule tracks is impaired. These deficits can be attenuated by microtubule stabilization. Microtubules are also necessary for the formation of dendrites in melanocytes, and Ilk inactivation reduces melanocyte dendricity. Activation of glycogen synthase kinase-3 (GSK-3) interferes with microtubule assembly. Significantly, inhibition of GSK-3 activity or exogenous expression of the GSK-3 substrate collapsin response mediator protein 2 (CRMP2) in ILK-deficient melanocytes restored dendricity. ILK is also required for normal melanin transfer, and GSK-3 inhibition in melanocytes partially restored melanin transfer to neighboring keratinocytes. Thus, our work shows that ILK is a central modulator of melanosome movements in primary epidermal melanocytes and identifies ILK and GSK-3 as important modulators of melanin transfer to keratinocytes, a key process for epidermal UV photoprotection.

MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer.

Lung cancer is the most frequent cause of cancer-related death in this country for men and women. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25nt long) capable of targeting genes for either degradation of mRNA or inhibition of translation. We identified aberrant expression of 41 miRNAs in lung tumor versus uninvolved tissue. MiR-133B had the lowest expression of miRNA in lung tumor tissue (28-fold reduction) compared to adjacent uninvolved tissue. We identified two members of the BCL-2 family of pro-survival molecules (MCL-1 and BCL2L2 (BCLw)) as predicted targets of miR-133B. Selective over-expression of miR-133B in adenocarcinoma (H2009) cell lines resulted in reduced expression of both MCL-1 and BCL2L2. We then confirmed that miR-133B directly targets the 3'UTRs of both MCL-1 and BCL2L2. Lastly, over-expression of miR-133B induced apoptosis following gemcitabine exposure in these tumor cells. To our knowledge, this represents the first observation of decreased expression of miR-133B in lung cancer and that it functionally targets members of the BCL-2 family.

Isolation, Culture, and Motility Measurements of Epidermal Melanocytes from GFP-Expressing Reporter Mice.

In this article, we provide a method to isolate primary epidermal melanocytes from reporter mice, which also allow targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26mT/mG reporter background, which results in GFP expression in the targeted melanocytic cell population. These cells are isolated and cultured to >95% purity. The cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as capacity for migration. Melanocytes are slow moving cells, and we also provide a method to measure motility using individual cell tracking and data analysis.

Publisher Correction: Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

In the originally published version of this Article, the positions of the final two authors in the author list were inadvertently inverted during the production process. This error has now been corrected in both the PDF and HTML versions of the Article.

Scaffolding proteins in the development and maintenance of the epidermal permeability barrier.

The skin of mammals and other terrestrial vertebrates protects the organism against the external environment, preventing heat, water and electrolyte loss, as well as entry of chemicals and pathogens. Impairments in the epidermal permeability barrier function are associated with the genesis and/or progression of a variety of pathological conditions, including genetic inflammatory diseases, microbial and viral infections, and photodamage induced by UV radiation. In mammals, the outside-in epidermal permeability barrier is provided by the joint action of the outermost cornified layer, together with assembled tight junctions in granular keratinocytes found in the layers underneath. Tight junctions serve as both outside-in and inside-out barriers, and impede paracellular movements of ions, water, macromolecules and microorganisms. At the molecular level, tight junctions consist of integral membrane proteins that form an extracellular seal between adjacent cells, and associate with cytoplasmic scaffold proteins that serve as links with the actin cytoskeleton. In this review, we address the roles that scaffold proteins play specifically in the establishment and maintenance of the epidermal permeability barrier, and how various pathologies alter or impair their functions.

A reporter mouse model for in vivo tracing and in vitro molecular studies of melanocytic lineage cells and their diseases.

Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSAmT/mG and Tyr::CreERT2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage.

Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas.

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases. Nearly all primary melanomas within the epidermis lacked Cx43. In contrast, nodal metastases expressed low levels of Cx43 which was markedly higher in distant metastases that had invaded vital organs. Importantly, in all stages of melanoma progression, Cx43 could be detected in intracellular compartments but was rarely assembled into gap junctions indicative of functional gap junction channels. Overall, these studies suggest that melanocytes do not form Cx43 homocellular gap junctions and even though Cx43 levels increase during melanoma progression, this connexin rarely assembles into gap junction structures.