Islet ß cell: An endocrine cell secreting miRNAs.

The pancreas is an important endocrine organ. Pancreatic beta (ß) cells secrete insulin to regulate the metabolism of glucose and maintain the stability of blood glucose. MicroRNAs (miRNAs), small (20-25 nt) non-coding RNA molecules, function in target gene silencing through post-transcriptional patterns, and multiple miRNAs regulate insulin secretion, insulin signaling pathways in pancreatic ß cells and islet activity. In recent years, it has been found that many types of cells can secrete regulatory miRNA, but whether islet cells can secrete miRNA has not been clarified yet. In this study, we detected miRNA expressions in cell culture medium of primary mouse islet cells and islet cell line MIN6. And we found that islet cells can selectively and actively secrete miRNAs, mainly through the way of exosome package, and that miRNA secretion profiling patterns vary according to different insulin secretion stimulating conditions (high Glucose, high Kcl, high Arginine and high Free fat acid (FFA)). Additionally, we found that these miRNAs secreted by islet cells can be transported and have gene-regulating functions on recipient tissue cells, such as liver and muscles. In conclusion, we find another secretory function of the islet ß cells: not only insulin, but also miRNAs.

Microvesicle-mediated transfer of microRNA-150 from monocytes to endothelial cells promotes angiogenesis.

Recent studies by our group and others show that microRNAs can be actively secreted into the extracellular environment through microvesicles (MVs) and function as secretory signaling molecules that influence the recipient cell phenotypes. Here we investigate the role of monocyte-secreted miR-150 in promoting the capillary tube formation of endothelial cells and in enhancing angiogenesis. In vitro capillary tube formation and in vivo angiogenesis assays showed that monocyte-derived MVs have strong pro-angiogenic activities. By depleting miR-150 from monocytic MVs and increasing miR-150 in MVs derived from cells that normally contain low levels of miR-150, we further demonstrated that the miR-150 content accounted for the pro-angiogenic activity of monocytic MVs in these assays. Using tumor-implanted mice and ob/ob mice as models, we revealed that miR-150 secretion, which is increased for diseases such as cancers and diabetes, significantly promotes angiogenesis. The delivery of anti-miR-150 antisense oligonucleotides into tumor-implanted mice and ob/ob mice via MVs, however, strongly reduced angiogenesis in both types of mice. Our results collectively demonstrate that secretion of miR-150 via MVs can promote angiogenesis in vitro and in vivo, and we also present a novel microRNA-based therapeutic approach for disease treatment.

High-fat diet promotes prostate cancer metastasis via RPS27.

BACKGROUND: Metastasis is the leading cause of death among prostate cancer (PCa) patients. Obesity is associated with both PCa-specific and all-cause mortality. High-fat diet (HFD) is a risk factor contributing to obesity. However, the association of HFD with PCa metastasis and its underlying mechanisms are unclear. METHODS: Tumor xenografts were conducted by intrasplenic injections. The ability of migration or invasion was detected by transwell assay. The expression levels of RPS27 were detected by QRT-PCR and western blot. RESULTS: The present study verified the increase in PCa metastasis caused by HFD in mice. Bioinformatics analysis demonstrated increased RPS27 in the experimentally induced PCa in HFD mice, indicating that it is an unfavorable prognostic factor. Intrasplenic injections were used to demonstrate that RPS27 overexpression promotes, while RPS27 knockdown significantly reduces, PCa liver metastasis. Moreover, RPS27 inhibition suppresses the effects of HFD on PCa metastasis. Further mRNA sequencing analysis revealed that RPS27 promotes PCa metastasis by selectively enhancing the expression of various genes. CONCLUSION: Our findings indicate that HFD increases the risk of PCa metastasis by elevating RPS27 expression and, subsequently, the expression of genes involved in PRAD progression. Therefore, RPS27 may serve as a novel target for the diagnosis and treatment of metastatic PCa.

Prostate cancer cells synergistically defend against CD8+ T cells by secreting exosomal PD-L1.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) remains fatal and incurable, despite a variety of treatments that can delay disease progression and prolong life. Immune checkpoint therapy is a promising treatment. However, emerging evidence suggests that exosomal programmed necrosis ligand 1 (PD-L1) directly binds to PD-1 on the surface of T cells in the drain lineage lymph nodes or neutralizes administered PD-L1 antibodies, resulting in poor response to anti-PD-L1 therapy in mCRPC. MATERIALS AND METHODS: Western blotting and immunofluorescence were performed to compare PD-L1 levels in exosomes derived from different prostate cancer cells. PC3 cells were subcutaneously injected into nude mice, and then ELISA assay was used to detect human specific PD-L1 in exosomes purified from mouse serum. The function of CD8+ T cells was detected by T cell mediated tumor cell killing assay and FACS analysis. A subcutaneous xenograft model was established using mouse prostate cancer cell RM1, exosomes with or without PD-L1 were injected every 3 days, and then tumor size and weight were analyzed to evaluate the effect of exosomal PD-L1. RESULTS: Herein, we found that exosomal-PD-L1 was taken up by tumor cells expressing low levels of PD-L1, thereby protecting them from T-cell killing. Higher levels of PD-L1 were detected in exosomes derived from the highly malignant prostate cancer PC3 and DU145 cell lines. Moreover, exosomal PD-L1 was taken up by the PD-L1-low-expressing LNCaP cell line and inhibited the killing function of CD8-T cells on tumor cells. The growth rate of RM1-derived subcutaneous tumors was decreased after knockdown of PD-L1 in tumor cells, whereas the growth rate recovered following exosomal PD-L1 tail vein injection. Furthermore, in the serum of mice with PCa subcutaneous tumors, PD-L1 was mainly present on exosomes. CONCLUSION: In summary, tumor cells share PD-L1 synergistically against T cells through exosomes. Inhibition of exosome secretion or prevention of PD-L1 sorting into exosomes may improve the therapeutic response of prostate tumors to anti-PD-L1 therapy.

Pancreatic ß cells control glucose homeostasis via the secretion of exosomal miR-29 family.

Secreted microRNAs (miRNAs) are novel endocrine factors that play essential pathological and physiological roles. Here, we report that pancreatic ß cell-released exosomal miR-29 family members (miR-29s) regulate hepatic insulin sensitivity and control glucose homeostasis. Cultured pancreatic islets were shown to secrete miR-29s in response to high levels of free fatty acids (FFAs) in vitro. In vivo, high levels of FFAs, promoted by either high-fat diet (HFD) feeding (physiopathological) or fasting (physiological), increased the secretion of miR-29s into plasma. Intravenous administration of exosomal miR-29s attenuated insulin sensitivity. The overexpression of miR-29s in the ß cells of transgenic (TG) mice promoted the secretion of miR-29s and inhibited the insulin-mediated suppression of glucose output in the liver. We used selective overexpression of traceable heterogenous mutant miR-29s in ß cells to confirm that islet-derived exosomal miR-29s target insulin signalling in the liver and blunt hepatic insulin sensitivity. Moreover, in vivo disruption of miR-29s expression in ß cells reversed HFD-induced insulin resistance. In vitro experiments demonstrated that isolated exosomes enriched in miR-29s inhibited insulin signalling in the liver and increased hepatic glucose production. These results unveil a novel ß cell-derived secretory signal-exosomal miR-29s-and provide insight into the roles of miR-29s in manipulating glucose homeostasis.

Gonadal white adipose tissue-derived exosomal MiR-222 promotes obesity-associated insulin resistance.

In this study, we investigated the role of serum exosomal miR-222 in obesity-related insulin resistance. Bioinformatics analyses showed that miR-222 levels were significantly upregulated in the white adipose tissue of obese patients with insulin resistance (GSE25402 dataset) and in serum samples from type 2 diabetes mellitus (T2DM) patients (GSE90028 dataset). Moreover, analysis of miRNA expression in adipose tissue-specific Dicer knockout mice (GitHub dataset) and diabetic model mice (GSE81976 and GSE85101 datasets), gonadal white adipose tissue (gWAT) was the main source of serum exosomal miR-222. MiR-222 levels were significantly elevated in the serum, serum exosomes and gWAT of mice fed a high-fat diet (HFD), and there was a corresponding downregulation of IRS1 and phospho-AKT levels in their liver and skeletal muscle tissues, which correlated with impaired insulin sensitivity and glucose intolerance. These effects were abrogated by surgically removing the gWAT from the HFD-fed mice. Thus, gWAT-derived serum exosomal miR-222 appears to promote insulin resistance in the liver and skeletal muscle of HFD-fed obese mice by suppressing IRS1 expression.

Proteomic profiling of MIN6 cell-derived exosomes.

Exosomes have been widely used in research on the early clinical diagnosis, prognosis and treatment of various cancers due to their features of small size (30-120 nm), non-immunogenicity and ability to cross biological barriers. However, few studies have investigated exosomes involved in metabolic diseases. Early studies have found that adipose tissue can be a source of exosomes regulating metabolism, but the related functions of exosomes secreted by other tissues in the regulation of metabolic diseases have not been determined. In addition, islets were found to be able to secrete miRNA via exosomes, suggesting that islet exosomes may be among the sources of exosomes involved in the regulation of metabolic diseases and that the relevant protein profiles have not been characterized to date. Therefore, identifying the protein contents of pancreatic ß cell-derived exosomes would benefit further research investigating the protein functions and mechanisms associated with diabetes-related metabolic diseases. SIGNIFICANCE: Exosomes are emerging tools for investigating metabolic diseases in recent years, but little research has been done. In our work, functional identification of MIN6 cell-derived exosomal proteins and comparative analysis of islet ß cell exosomal protein data from different cell lines or from different species revealed that exosomes secreted by islet ß cells may be involved in the regulation of glucose metabolism. These results may suggest that intercellular communication induced by exosome transfer among tissues may account for the major reason of diabetic metabolic disorder. In addition, these results may provide a theoretical basis for the study of the physiological and pathological functions of islet ß cell exosomes for the future studies.

Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components.

Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects.

Targeted exosome-mediated delivery of opioid receptor Mu siRNA for the treatment of morphine relapse.

Cell-derived exosomes have been demonstrated to be efficient carriers of small RNAs to neighbouring or distant cells, highlighting the preponderance of exosomes as carriers for gene therapy over other artificial delivery tools. In the present study, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in exosomes. These exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. Surprisingly, MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. In conclusion, our results demonstrate that targeted RVG exosomes can efficiently transfer siRNA to the central nervous system and mediate the treatment of morphine relapse by down-regulating MOR expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system.

Microvesicle-delivery miR-150 promotes tumorigenesis by up-regulating VEGF, and the neutralization of miR-150 attenuate tumor development.

Tumor-associated macrophages (TAMs) mostly exhibit M2-like (alternatively activated) properties and play positive roles in angiogenesis and tumorigenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic factor. During tumor development, TAMs secrete VEGF and other factors to promote angiogenesis; thus, anti-treatment against TAMs and VEGF can repress cancer development, which has been demonstrated in clinical trials and on an experimental level. In the present work, we show that miR-150 is an oncomir because of its promotional effect on VEGF. MiR-150 targets TAMs to up-regulate their secretion of VEGF in vitro. With the utilization of cell-derived vesicles, named microvesicles (MVs), we transferred antisense RNA targeted to miR-150 into mice and found that the neutralization of miR-150 down-regulates miR-150 and VEGF levels in vivo and attenuates angiogenesis. Therefore, we proposed the therapeutic potential of neutralizing miR-150 to treat cancer and demonstrated a novel, natural, microvesicle-based method for the transfer of nucleic acids.