Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT

Abstract Background Metastasis is the main cause of lung cancer mortality. Bone marrow-derived mesenchymal stem cells (BMSCs) are a component of the cancer microenvironment and contribute to cancer progression. Intratumoral hypoxia affects both cancer and stromal cells. Exosomes are recognized as mediators of intercellular communication. Here, we aim to further elucidate the communication between BMSC-derived exosomes and cancer cells in the hypoxic niche. Methods Exosomal miRNA profiling was performed using a microRNA array. Lung cancer cells and an in vivo mouse syngeneic tumor model were used to evaluate the effects of select exosomal microRNAs. Hypoxic BMSC-derived plasma exosomal miRNAs were assessed for their capacity to discriminate between cancer patients and non-cancerous controls and between cancer patients with or without metastasis. Results We demonstrate that exosomes derived from hypoxic BMSCs are taken by neighboring cancer cells and promote cancer cell invasion and EMT. Exosome-mediated transfer of select microRNAs, including miR-193a-3p, miR-210-3p and miR-5100, from BMSCs to epithelial cancer cells activates STAT3 signaling and increases the expression of mesenchymal related molecules. The diagnostic accuracy of individual microRNA showed that plasma exosomal miR-193a-3p can discriminate cancer patients from non-cancerous controls. A panel of these three plasma exosomal microRNAs showed a better diagnostic accuracy to discriminate lung cancer patients with or without metastasis than individual exosomal microRNA. Conclusions Exosome-mediated transfer of miR-193a-3p, miR-210-3p and miR-5100, could promote invasion of lung cancer cells by activating STAT3 signalling-induced EMT. These exosomal miRNAs may be promising noninvasive biomarkers for cancer progression

Genome sequencing of the perciform fish Larimichthys crocea provides insights into molecular and genetic mechanisms of stress adaptation.

The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea

Hypoxia stress exerts responses involving the HPA and HPT axes.

<p>Under hypoxia, the potential neuro-endocrine-immune/metabolism networks contribute to the regulation of moderate inflammation and the maintenance of energy balance. Hypoxia may inhibit the hypothalamic-pituitary-adrenal (HPA) axis and induce the expression of ET-1/ADM, and the latter then promote the expression of IL-6/TNF-α and form a positive feedback loop with them. On the other hand, the ET-1/ADM and IL-6/TNF-α may in turn activate the HPA axis, and the latter subsequently induces glucocorticoids and generates a negative feedback to inhibit <i>ET-1/ADM</i> and <i>IL-6/TNF-α</i> expression to reduce the inflammatory responses in brain. Therefore a HPA axis-ET-1/ADM-IL-6/TNF-α feedback regulatory loop is outlined here. Besides, both SOCS-1 and SOCS-3 may have complementary roles in down-regulating <i>IL-6</i> and <i>TNF-α</i>, and both IL-6 and TNF-α have reciprocal functions to induce <i>SOCS-1</i> and <i>SOCS-3</i> expression, thus constituting a SOCS-1/3-dependent feedback regulation to modify cerebral inflammation. The hypothalamic-pituitary-thyroid (HPT) axis was inhibited in <i>L</i>. <i>crocea</i> brains during the early period of hypoxia, thus leading to a decrease in thyroid hormone (T3 and T4) production. Decrease of HPT axis-thyroid hormones subsequently may inhibit protein synthesis by the PI3K-Akt-mTOR-S6K signaling pathway, which contributes to the reduction in energy consumption during hypoxia stress. Meanwhile, down-regulation of HPT axis-thyroid hormones also represses the tricarboxylic acid (TCA) cycle and accelerates the anaerobic glycolytic pathway, which facilitates O₂-independent ATP production under hypoxia. Therefore the HPT axis-mediated effects may play roles in response to hypoxia by reorganizing energy consumption and generation. Genes related to the neuro-endocrine system (orange), immunity (red), and metabolic system and protein synthesis (blue) are indicated. The outer border indicates the brain of <i>L</i>. <i>crocea</i>. The arrow represents promotion, and the interrupted line represents inhibition. Solid lines indicate direct relationships between genes. Dashed lines indicate that more than one step is involved in the process.</p

Characterisation of the T-cell lineages in <i>L</i>. <i>crocea</i> adaptive immunity and the expanded genes in antiviral immunity.

<p>(<b>A</b>) A schematic diagram summarising genes related to different T-cell lineages in <i>L</i>. <i>crocea</i> is shown. The inducible factors, the main regulatory transcriptional factors, and the immune effectors of T cells are present in green, blue, and orange backgrounds, respectively. The genes that have been annotated by genome survey are shown in black, and the unannotated genes are shown in red. IL-2R represents all three subunit genes of <i>L</i>. <i>crocea</i> IL-2 receptor, IL-2RB, IL-2RG and IL-2RA/ IL-15RA genes. The majority of the CD4⁺ T-helper type 1 (Th1), CD4⁺ T-helper type 2 (Th2) and CD8⁺ T cell-related genes have been found in the <i>L</i>. <i>crocea</i> genome. The genes related to Th17 cell- and γδ-T cell-mediated mucosal immunity are also conserved in <i>L</i>. <i>crocea</i>. (<b>B</b>) Several key genes are expanded in the antiviral immunity pathways in <i>L</i>. <i>crocea</i>. The genes that have been identified in the <i>L</i>. <i>crocea</i> genome are shown in orange boxes, and the lost gene (<i>RIG-I</i>) is shown in the grey box. LGP2 is able to bind to double-stranded RNA (dsRNA) to trigger interferon production, but the adaptor molecule of LGP2 is still unknown in fish. The red boxes indicate gene families (<i>TRIM25</i>, <i>cGAS</i>, <i>DDX41</i>, and <i>NLRC3</i>) that are expanded in <i>L</i>. <i>crocea</i>. The arrow represents induction, and the interrupted line represents inhibition.</p

Summary of the <i>Larimichthys crocea</i> genome

<p>BAC = bacterial artificial chromosome; WGS = whole genome shotgun.</p><p>Summary of the <i>Larimichthys crocea</i> genome</p

Phylogenetic tree of and orthologous genes in <i>L</i>. <i>crocea</i> and other vertebrates.

<p>(<b>A</b>) The phylogenetic tree was constructed from 2,257 single-copy genes with 3.18 M reliable sites by maximum likelihood methods. The red points on six of the internal nodes indicate fossil calibration times in the analysis. Blue numbers indicate the divergence time (Myr, million years ago), and the green and red numbers represent the expanded and extracted gene families, respectively, in <i>L</i>. <i>crocea</i>. (<b>B</b>) The different types of orthologous relationships are shown. “1:1:1” = universal single-copy genes; “N:N:N” = orthologues exist in all genomes; “Fish” = fish-specific genes; “SD” = genes that have undergone species-specific duplication; “Homology” = genes with an e-value less than 1e-5 by BLAST but do not cluster to a gene family; “ND” = species-specific genes; and “Others” = orthologues that do not fit into the other categories. (<b>C</b>) The shared and unique gene families in five teleost fish are shown in the Venn diagram. (<b>D</b>) Distribution of the identity values of orthologous genes is compared among <i>L</i>. <i>crocea</i> and other teleosts.</p