Breed-Related Differential microRNA Expression and Analysis of Colostrum and Mature Milk Exosomes in Bamei and Landrace Pigs.

Breast milk, an indispensable source of immunological and nutrient components, is essential for the growth and development of newborn mammals. MicroRNAs (miRNAs) are present in various tissues and body fluids and are selectively packaged inside exosomes, a type of membrane vesicle. Milk exosomes have potential regulatory effects on the growth, development, and immunity of newborn piglets. To explore the differences in milk exosomes related to the breed and milk type, we isolated exosomes from colostrum and mature milk from domestic Bamei pigs and foreign Landrace pigs by using density gradient centrifugation and then characterized them by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Furthermore, the profiles and functions of miRNAs in the two types of pig milk exosomes were investigated using miRNA-seq and bioinformatics analysis. We identified a total of 1081 known and 2311 novel miRNAs in pig milk exosomes from Bamei and Landrace pigs. These differentially expressed miRNAs (DE-miRNAs) are closely associated with processes such as cell signaling, cell physiology, and immune system development. Functional enrichment analysis showed that DE-miRNA target genes were significantly enriched in endocytosis, the T cell receptor signaling pathway, and the Th17 cell differentiation signaling pathway. The exosomal miRNAs in both the colostrum and mature milk of the two pig species showed significant differences. Based on related signaling pathways, we found that the colostrum of local pig breeds contained more immune-system-development-related miRNAs. This study provides new insights into the possible function of milk exosomal miRNAs in the development of the piglet immune system.

Neutrophil membrane-engineered Panax ginseng root-derived exosomes loaded miRNA 182-5p targets NOX4/Drp-1/NLRP3 signal pathway to alleviate acute lung injury in sepsis: experimental studies.

BACKGROUND: The purpose of this study was to prepare neutrophil membrane-engineered Panax ginseng root-derived exosomes (N-exo) and investigate the effects of N-exo microRNA (miRNA) 182-5p (N-exo-miRNA 182-5p) on acute lung injury (ALI) in sepsis. METHODS: Panax ginseng root-derived exosomes were separated by differential centrifugation. Neutrophil membrane engineering was performed on exo to obtain N-exo. miRNA182-5p was transmitted into N-exo by electroporation technology to obtain N-exo-miRNA 182-5p. LPS was used to establish an in-vivo and in-vitro model of ALI of sepsis to evaluate the anti-inflammatory effect of N-exo-miRNA 182-5p. RESULTS: The results of transmission electron microscope showed that exo was a double-layer membrane structure like a saucer. Nanoparticle size analysis showed that the average particle size of exo was 129.7 nm. Further, compared with exo, the level of miRNA182-5p was significantly increased in N-exo. The experimental results showed that N-exo-miRNA 182-5p significantly improved ALI via target regulation of NOX4/Drp-1/NLRP3 signal pathway in vivo and in vitro . CONCLUSION: In conclusion, this study prepared a novel engineered exosome (N-exo and N-exo-miRNA 182-5p significantly improved ALI in sepsis via target regulation of NOX4/Drp-1/NLRP3 signal pathway, providing new ideas and methods for treatment of ALI in sepsis.

Human Breast Milk miRNAs: Their Diversity and Potential for Preventive Strategies in Nutritional Therapy.

The endogenous miRNAs of breast milk are the products of more than 1000 nonprotein-coding genes, giving rise to mature small regulatory molecules of 19-25 nucleotides. They are incorporated in macromolecular complexes, loaded on Argonaute proteins, sequestrated in exosomes and lipid complexes, or present in exfoliated cells of epithelial, endothelial, or immune origins. Their expression is dependent on the stage of lactation; however, their detection depends on progress in RNA sequencing and the reappraisal of the definition of small RNAs. Some miRNAs from plants are detected in breast milk, opening the possibility of the stimulation of immune cells from the allergy repertoire. Each miRNA harbors a seeding sequence, which targets mRNAs, gene promoters, or long noncoding RNAs. Their activities depend on their bioavailability. Efficient doses of miRNAs are estimated to be roughly 100 molecules in the cytoplasm of target cells from in vitro and in vivo experiments. Each miRNA is included in networks of stimulation/inhibition/sequestration, driving the expression of cellular phenotypes. Three types of stress applied during lactation to manipulate miRNA supply were explored using rodent offspring: a foster mother, a cafeteria diet, and early weaning. This review presents the main mature miRNAs described from current mothers' cohorts and their bioavailability in experimental models as well as studies assessing the potential of miR-26 or miR-320 miRNA families to alter offspring phenotypes.

Identifying the Potential of miRNAs in Houttuynia cordata-Derived Exosome-Like Nanoparticles Against Respiratory RNA Viruses.

Introduction: Pathogenic respiratory RNA viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), and SARS-CoV-2, are major causes of causes of acute respiratory infection globally. Plant-derived exosome-like nanoparticles containing miRNAs have shown substantial cross-kingdom regulatory effects on both viral and human transcripts. Houttuynia cordata (H. cordata), a traditional Chinese medicine frequently used to treat respiratory diseases. However, the role of H. cordata-derived exosome-like nanoparticles (HELNs) and the miRNA they encapsulated are unclear. Methods: HELNs were isolated from fresh underground roots (uHELNs) and above ground stems and leaves (aHELNs) using differential centrifugation. The HELNs were identified using transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Small RNA sequencing and RT-PCR were employed to determine the miRNA expression in uHELNs and aHELNs. All genomes were sourced from the NCBI database. Target prediction of viral genomes was performed using RNAhybrid, while human target prediction was conducted using both RNAhybrid and Miranda. Functional enrichment analysis was applied to the predicted human targets to explore the hub targets and their roles in antiviral effects. The accessibility of miRNA target sites was determined through the MFOLD web server, and customized dual-luciferase reporter assays were administered to validate the computational findings. Results: A total of 12 highly enriched miRNAs were identified in both uHELNs and aHELNs. Upon prediction and verification, miR858a and miR858b were shown to target the NP gene in H1N1, while miR166a-3p targeted the ORF1ab in SARS-CoV-2. However, no valid miRNA targets were found for RSV. Regarding human transcripts, miR168a-3p, miR168b-3p, and miR8175 were found to inhibit MAPK3 expression, and novel_mir2 could suppress both AKT1 and MAPK3 expression. Discussion: This study sheds light on the collaborative antiviral mechanism of miRNAs in HELNs across two species and explores the potential antiviral scopes of both H. cordata miRNAs and HELNs.

Exosomes and their miRNA/protein profile in keratoconus-derived corneal stromal cells.

Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles (30-150 nm) that mediate cellular communication via their protein, lipid, and nucleic acid content. We aimed to characterize the exosomes secreted by primary corneal fibroblasts from subjects with or without KC. Using human keratoconus stromal fibroblast cells (HKC, n = 4) and healthy stromal fibroblasts (HCF, n = 4), we collected and isolated exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView®, we compared the size and concentration of isolated exosomes. Different exosomal markers were identified and quantified using a transmission electron microscope (TEM) (CD81) and Western blot (CD9 and CD63). Exosomal miRNA profiles were determined by qRT-PCR using Exiqon Human panel I miRNA assays of 368 pre-selected miRNAs. Proteomic profiles were determined using a label-free spectral counting method with mass spectrometry. Differential expression analysis for miRNAs and proteins was done using student's t-test with a significance cutoff of p-value ≤0.05. We successfully characterized exosomes isolated from HCFs using several complementary techniques. We found no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs with or without KC. Expression of CD81 was confirmed by immuno-EM, and expression of CD63 and CD9 with western blots in all exosome samples. We detected the expression of 72-144 miRNAs (threshold cycle Ct < 36) in all exosome samples. In HKC-derived exosome samples, miR-328-3p, miR-532-5p, miR-345-5p, and miR-424-5p showed unique expression, while let-7c-5p and miR-665 have increased expression. Protein profiling identified 157 proteins in at least half of the exosome samples, with 38 known exosomal proteins. We identified 12 up- and 2 down-regulated proteins in HKC-derived exosomes. The proteins are involved in membrane-bounded vesicles, cytoskeletal, calcium binding, and nucleotide binding. These proteins are predicted to be regulated by NRF2, miR-205, and TGF-ß1, which are involved in KC pathogenesis. We successfully characterized the HKC-derived exosomes and profiled their miRNA and protein contents, suggesting their potential role in KC development. Further studies are necessary to determine if and how these exosomes with differential protein/miRNA profiles contribute to the pathogenesis of KC.

IL-27-induced, MSC-derived Exosomes Promote MMP3 Expression Through the miR-206/L3MBTL4 Axis in Synovial Fibroblasts.

Context: In rheumatoid arthritis (RA), hyperproliferative fibroblast-like synoviocytes (FLS) can secrete a variety of tissue hydrolases, such as matrix metalloproteinases (MMPs), causing the destruction of chondrocytes. Mesenchymal stem cells (MSCs) can directly affect FLS through extracellular vesicles (EVs). Interleukin-27 (IL-27) is a pleiotropic immune regulator frequently overexpressed in RA. Objective: The study intended to examine the effects of IL-27-induced exosomes from bone-marrow mesenchymal stem cells (BM-MSCs) and to determine if they promote the secretion of MMP3 in synovial cells. Design: The research team performed a genetic study. Setting: The study took place at the First Affiliated Hospital of Hainan Medical University in Haikou City, Hainan, China. Outcome Measures: The research team: (1) determined if IL-27 expression had occurred in the synovial fluid; (2) co-cultured IL-27-induced MSCs with FLS to detect the expression of MMP3 in the FLS; (3) Under IL-27 induction, MSC-derived exosomes with IL-27R knockdown were collected to detect the expression of microRNAs(miRNAs) associated with RA; (4) screened the miRNAs to determine the most significant differences in expression; (5) determined the miRNA target genes in arthritis, using Western blot (WB) and qRT-PCR; and (6) Dual luciferase and ChIP experiments confirm regulation of MMP3 by L3MBTL4. Results: IL-27 was highly expressed in RA, and the IL-27-induced, MSC-derived exosomes promoted the expression of MMP3 in FLS. The IL-27-induced MSC-derived exosomes significantly upregulated the expression of miR-206-3p, and the miR-206-3p target, miR-206/ lethal(3) malignant brain tumor-like protein 4 (L3MBTL4), regulated the MMP3 transcription. The IL-27-induced, MSC-derived exosomes promoted MMP3 expression in the FLS through the miR-206-3p/L3MBTL4 axis, thereby promoting chondrocyte degradation and aggravating RA. Conclusions: IL-27 can induce the expression of miR-206 in MSCs, and miR-206 can be transported into FLS through MSC-EVs to promote FLS migration and MMP3 expression and aggravate articular cartilage damage. Patients with RA who have a high IL-27 expression may not be suitable to receive treatment with MSCs, and clinicians can use MSCs that knock down or delete IL-27R to treat RA patients who have a high IL-27 expression.

Exosomes derived from hepatitis B virus-infected hepatocytes promote liver fibrosis via miR-222/TFRC axis.

Exosomal miRNAs activates hepatic stellate cell (HSC) and promote fibrosis. miR-222 was found to be increased in hepatitis B virus (HBV)-infected hepatocytes, and ferroptosis was reported to ameliorate liver fibrosis (LF). Although miR-222 and ferroptosis have been implicated in LF, the association between miR-222 and ferroptosis and how they coordinate to regulate LF are still not explicit. This study investigates the roles of miR-222 and transferrin receptor (TFRC) in LF. Lipid reactive oxygen species (ROS) level was analyzed by flow cytometry. FerroOrange staining was used to measure intracellular iron level. Luciferase reporter assay was adopted to confirm the binding of miR-222 and TFRC. Real-time quantitative PCR and immunoblots were applied to analyze gene and protein expression. The results showed that supplementation of exosomes derived from HBV-infected LO2 cells remarkably enhanced LX-2 cell activation, evidenced by elevated hydroxyprolin (Hyp) secretion and α-SMA and COL1A2 expression. miR-222 was significantly increased in HBV-Exo. Overexpressing miR-222 upregulated cell viability, secretion of Hpy, and expression of α-SMA and COL1A2, which were all blocked by overexpression of TFRC. Further study showed that TFRC was a target of miR-222, and miR-222 promoted LX-2 cell activation through suppressing TFRC-induced ferroptosis in LX-2 cells. Exosomal miR-222 derived from HBV-infected hepatocytes promoted LF through inhibiting TFRC and TFRC-induced ferroptosis. This study emphasizes the significance of miR-222/TFRC axis in LF and suggests new insights in clinical decision making while treating LF. Exosomes derived from HBV-infected LO2 cells promote LX-2 cell activation and liver fibrosis in mouse Exosomal miR-222 derived from HBV-infected LO2 cells promotes LX-2 cell activation TFRC is a target of miR-222 and inhibits LX-2 cell activation induced by miR-222 miR-222 promotes LX-2 cell activation through inhibiting TFRC-induced ferroptosis.

Coffee-Derived Exosome-Like Nanoparticles: Are They the Secret Heroes?

BACKGROUND: Regular coffee consumption has beneficial and preventative effects on liver and chronic neurodegenerative diseases. However, the studies performed with the ingredients found in coffee beverages have not clarified the responsible mechanisms. Exosomes are small, membrane-coated cargo packages secreted by prokaryote and eukaryote cells. Exosomes regulate intercellular communication and affect cellular metabolic activities even among different species. In this study, we aimed to isolate and characterize the edible plant-derived exosome-like nanoparticles from roasted hot coffee beverages, hypothesizing that the edible plant-derived exosome-like nanoparticles were responsible for the beneficial effects of coffee. METHODS: Size exclusion chromatography and commercial kits were used for the isolation process. Efficient coffee edible plant-derived exosome-like nanoparticle fractions were determined by an ultraviolet-visible spectrophotometer. Harvested coffee edible plant-derived exosome-like nanoparticles were characterized by transmission electron microscopy. The quantification procedure was performed using a commercial kit. Coffee edible plant-derived exosome-like nanoparticles' proliferative effects on human hepatic stellate cells and human hepatocellular carcinoma cells were studied using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Whole-exosome RNA sequencing was performed. RESULTS: Transmission electron microscopy scanning analysis indicated round-shaped nanoparticles with sizes ranging from 40 to 100 nm. Both size exclusion chromatography and kit-isolated edible plant-derived exosome-like nanoparticle samples showed maximum absorbance at 227.5 nm in ultraviolet-visible spectrophotometer analysis. Regarding the quantitation results, kit isolation was more efficient than the size exclusion chromatography method when the harvested particle numbers were compared. An important MTT assay finding confirmed the observed beneficial effects of coffee beverages: coffee edible plant-derived exosome-like nanoparticles significantly suppressed hepatocellular carcinoma cell proliferation. As a result of sequencing, we identified 15 mature miRNAs. A MapReduce-based MicroRNA Target Prediction Method (The DIANA tools' MR-microT algorithm) highlighted 2 genes specifically associated with the miRNAs that we obtained: KMT2C and ZNF773. CONCLUSION: For the first time in the literature, coffee edible plant-derived exosome-like nanoparticles were identified. These nanoparticles may have therapeutic effects on chronic liver diseases. Experimental studies, therefore, should be performed on disease models to demonstrate their efficacy.

Post-prostatic-massage urine exosomes of men with chronic prostatitis/chronic pelvic pain syndrome carry prostate-cancer-typical microRNAs and activate proto-oncogenes.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a high prevalence of up to 15% and accounts for 90-95% of prostatitis diagnoses, and yet its etiopathogenesis and link to prostate cancer (PCa) are still unclear. Here, we investigated microRNAs in exosomes isolated from blood and post-prostatic-massage urine of CP/CPPS type IIIb patients and healthy men. THP-1 monocytes (human leukemia monocytic cell line) were treated with exosomes and subjected to mRNA arrays "Cancer Inflammation and Immunity Crosstalk" and "Transcription Factors." Using The Cancer Genome Atlas, the expression of CP/CPPS-associated microRNAs was analyzed in PCa and normal prostate tissue. In silico functional studies were carried out to explore the disease ontology of CP/CPPS. In CP/CPPS, urine exosomes exhibited significant upregulation of eight PCa-specific microRNAs (e.g., hsa-miR-501, hsa-miR-20a, and hsa-miR-106), whose target genes were significantly enriched for GO terms, hallmark gene sets, and pathways specific for carcinogenesis. In THP-1 monocytes, CP/CPPS-derived urine exosomes induced upregulation of PCa-associated proinflammatory genes (e.g., CCR2 and TLR2) and proto-oncogene transcription factors (e.g., MYB and JUNB). In contrast, CP/CPPS-derived blood exosomes exhibited molecular properties similar to those of healthy men. Thus, CP/CPPS exhibits molecular changes that constitute a risk for PCa and should be considered in the development of PCa biomarkers and cancer screening programs.

Identification of Candidate Immune System MicroRNAs Differentially Found in Colostrum and Milk Exosomes.

BACKGROUND: The fetus grows in a sterile womb environment. After birth, the newborn immune system has two immediate hurdles to clear. First immediate suppression of the womb compatible immune system and turn on the immune system of the newborn that can counter the antigenic world. The underlying mechanism of immune fluctuation by milk microRNAs (miRNAs) can be crucial for the treatment of critical or premature newborn. METHODS: We collected fourteen samples of each colostrum and mature milk from lactating mothers, four samples of each were used for microarray analysis, and the other ten were used for miRNA expression profiling by real-time PCR. RESULTS: From the microarray, 154 differentially expressed miRNAs were identified, whereas 49 miRNAs were revealed as immune-related miRNAs based on a literature study. Among the 49 miRNAs, 33 were already shown as strongly validated immune-related miRNAs (validated by qPCR, Western Blot, and Luciferase assay) and were considered for further analysis. Twenty-two miRNA expressions were analysed by real-time PCR as their Ct values were within considerable limits. Twelve numbers of miRNAs were significantly downregulated in mature milk compared to colostrum, which were again subjected to bioinformatics analysis to predict the biological mechanisms behind the differentially expressed miRNAs. CONCLUSION: This study shed light on the human milk exosome miRNA expression dynamics during lactation and their possible role in the gradual skewing of the newborns' immune system. The information is crucial for the development and onset of sepsis in premature newborns in the NICU.

Identification and functional characterization of multiple inositol polyphosphate phosphatase1 (Minpp1) isoform-2 in exosomes with potential to modulate tumor microenvironment.

Inositol polyphosphates (InsPs) play key signaling roles in diverse cellular functions, including calcium homeostasis, cell survival and death. Multiple inositol polyphosphate phosphatase 1 (Minpp1) affects the cellular levels of InsPs and cell functions. The Minpp1 is an endoplasmic reticulum (ER) resident but localizes away from its cytosolic InsPs substrates. The current study examines the heterogeneity of Minpp1 and the potential physiologic impact of Minpp1 isoforms, distinct motifs, subcellular distribution, and enzymatic potential. The NCBI database was used to analyze the proteome diversity of Minpp1 using bioinformatics tools. The analysis revealed that translation of three different Minpp1 variants resulted in three isoforms of Minpp1 of varying molecular weights. A link between the minpp1 variant-2 gene and ER-stress, using real-time PCR, suggests a functional similarity between minpp1 variant-1 and variant-2. A detailed study on motifs revealed Minpp1 isoform-2 is the only other isoform, besides isoform-1, that carries a phosphatase motif for InsPs hydrolysis but no ER-retention signal. The confocal microscopy revealed that the Minpp1 isoform-1 predominantly localized near the nucleus with a GRP-78 ER marker, while Minpp1 isoform-2 was scattered more towards the cell periphery where it co-localizes with the plasma membrane-destined multivesicular bodies biomarker CD63. MCF-7 cells were used to establish that Minpp1 isoform-2 is secreted into exosomes. Brefeldin A treatment resulted in overexpression of the exosome-associated Minpp1 isoform-2, suggesting its secretion via an unconventional route involving endocytic-generated vesicles and a link to ER stress. Results further demonstrated that the exosome-associated Minpp1 isoform-2 was enzymatically active. Overall, the data support the possibility that an extracellular form of enzymatically active Minpp1 isoform-2 mitigates any anti-proliferative actions of extracellular InsPs, thereby also impacting the makeup of the tumor microenvironment.

Hyperthermia promotes exosome secretion by regulating Rab7b while increasing drug sensitivity in adriamycin-resistant breast cancer.

PURPOSE: To investigate the mechanism through which hyperthermia promotes exosome secretion and drug sensitivity in adriamycin-resistant breast cancer. MATERIALS AND METHODS: We first evaluated the effect of hyperthermia on adriamycin-resistant breast cancer viability and used transmission electron microscopy, nanoparticle tracking analysis, and a bicinchoninic acid kit to validate the effect of hyperthermia on exosome secretion. The effective targeting molecules and pathways changed by hyperthermia were explored by RNA microarray and verified in vitro. The adriamycin-resistant MCF-7/ADR cells co-incubated with the exosomes produced by MCF-7/ADR cells after hyperthermia were assessed. The uptake of exosomes by MCF-7/ADR cells after hyperthermia treatment was evaluated by confocal microscopy. Finally, the mechanism through which hyperthermia promotes exosome secretion by hyperthermia was determined. RESULTS: Hyperthermia significantly suppressed the growth of adriamycin-resistant breast cancer cells and increased drug sensitivity by upregulating FOS and CREB5, genes related to longer overall survival in breast cancer patients. Moreover, hyperthermia promoted exosome secretion through Rab7b, a small GTPase that controls endosome transport. The upregulated FOS and CREB5 antioncogenes can be transferred to MCF-7/ADR cells by hyperthermia-treated MCF-7/ADR cell-secreted exosomes. CONCLUSIONS: Our results demonstrated a novel function of hyperthermia in promoting exosome secretion in adriamycin-resistant breast cancer cells and revealed the effects of hyperthermia on tumor cell biology. These hyperthermia-triggered exosomes can carry antitumor genes to the residual tumor and tumor microenvironment, which may be more beneficial to the effects of hyperthermia. These results represent an exploration of the relationship between therapeutic strategies and exosome biology.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Exploring the pathogenesis of type 2 diabetes mellitus intestinal damp-heat syndrome and the therapeutic effect of Gegen Qinlian Decoction from the perspective of exosomal miRNA.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetes is a common, complex, chronic metabolic disease. A randomized, double-blind, placebo-parallel controlled clinical study has shown that Gegen Qinlian Decoction (GQD) can reduce glycosylated hemoglobin in type 2 diabetes mellitus (T2DM) intestinal damp-heat syndrome patients in a dose-dependent manner. AIM: To explore the pathogenesis of T2DM intestinal damp-heat syndrome and the therapeutic effect of GQD from the perspective of exosomal microRNA (miRNA). METHODS: Eligible patients were selected and treated with GQD for 3 months to evaluate their clinical efficacy. Effective cases were matched with healthy volunteers, and saliva samples were collected. Exosomal miRNA was extracted from saliva and analyzed by chip sequencing. Subsequently, the function of the differential gene and the signal transduction pathway were analyzed using bioinformatics technology. Finally, three target miRNAs were randomly selected from the T2DM group/healthy group, and two target miRNAs in the T2DM before treatment/after treatment group were randomly selected for qPCR verification. Finally, we conducted a correlation analysis of the miRNAs and clinical indicators. The registration number for this research is ChiCTR-IOR-15006626. RESULTS: (1) The expression of exosomal miRNA chips showed that there were 14 differentially expressed miRNAs in the T2DM group/healthy group, and 26 differentially expressed miRNAs in the T2DM before treatment/after treatment group. (2) Enrichment results showed that in the T2DM group/healthy group, it was primarily related to cell development, body metabolism, TGF-ß, and ErbB signaling pathways. In the T2DM before treatment/after treatment group, it was mainly related to cellular metabolic regulation processes, and insulin, Wnt, and AMPK signaling pathways. (3) The qPCR verification showed that the expressions of hsa-miR-9-5p, hsa-miR-150-5p, and hsa-miR-216b-5p in the T2DM group was higher (P<0.05). Following GQD treatment, hsa-miR-342-3p and hsa-miR-221-3p were significantly downregulated (P<0.05). (4) hsa-miR-9-5p was positively correlated with BMI (P<0.05), and hsa-miR-150-5p was positively correlated with total cholesterol and triglycerides (P<0.05). The GQD efficacy-related gene hsa-miR-342-3p was positively correlated with the patient's initial blood glucose level (P<0.05), and hsa-miR-221-3p was positively correlated with total cholesterol and triglycerides (P<0.05). CONCLUSION: The exosomal miRNA expression profile and signaling pathways related to T2DM intestinal damp-heat syndrome and the efficacy of GQD were established, which provides an alternative strategy for precision traditional Chinese medicine treatment.

LncRNA HOTTIP from synovial fibroblast-derived exosomes: A novel molecular target for rheumatoid arthritis through the miR-1908-5p/STAT3 axis.

Rheumatoid arthritis (RA) is a chronic inflammation mediated by autoimmune responses. HOTTIP, a long noncoding RNA (lncRNA), participates in cell proliferation and invasion. However, the correlation between HOTTIP and RA remains unclear. Therefore, this study aimed to clarify how HOTTIP works in RA and to investigate its role in the development of RA. Flow cytometry was used to analyze cell cycle progression. Binding between HOTTIP, signal transducer and activator of transcription 3 (STAT3) and miR-1908-5p was demonstrated by dual-luciferase assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression of T cell differentiation-related proteins. We found that HOTTIP was upregulated in rheumatoid arthritis synovial fibroblasts (RASFs). HOTTIP directly bound to miR-1908-5p and negatively modulated miR-1908-5p expression while positively regulating STAT3. The effects of HOTTIP overexpression on regulating the balance of the Th17/Treg cell ratio were partly reversed by miR-1908-5p overexpression. In addition, in vivo experiments demonstrated that overexpression of HOTTIP aggravated inflammation in RA mice, which was demonstrated by hematoxylin and eosin (HE) staining and the increased expression levels of CD4+ interleukin (IL)-17+, forkhead Box P3 (FOXP3) and retinoid-related orphan receptor gamma-t (RORγt). In summary, our study suggests that HOTTIP plays a damaging role in RA by promoting inflammation, which may be related to the regulation of miR-1908-5p expression and the STAT3 signaling pathway. These results suggest that the regulation of HOTTIP may be a promising therapeutic strategy for RA.

Tumor-derived exosomes encapsulating miR-34a promote apoptosis and inhibit migration and tumor progression of colorectal cancer cells under in vitro condition.

BACKGROUND: MicroRNA (miR)-34a, as a master tumor suppressor in colorectal cancer (CRC), could regulate multiple genes participating in tumor proliferation, invasion, immune evasion, and inflammation-induced progression. Exosomes, as novel nano-carriers, were found to be capable of shuttling crucial mediators to various cells. Since the conventional CRC therapeutics currently are a matter of debate, implication of microRNAs in malignancy remedies have been addressed illustrating promising outlooks. OBJECTIVES: In this study, we aimed to investigate the delivery of miR-34a to CRC cell line CT-26 by encapsulating into tumor-derived exosomes (TEXs), in order to evaluate the anti-proliferative and progressive effects of the novel nano-carrier complex under in vitro condition. METHODS: Exosomes were purified from the starved CT-26 cells and then enriched by miR-34a using the calcium chloride (Cacl2) modified solution. Following the detection of miR-34a expression in the enriched TEXs, the viability of CT-26 cells treated by multiplicity concentrations of either TEXs or TEX-miR-34a was examined. Moreover, the apoptosis rate of the cells was evaluated, and the migration of CT-26 cells subjected to both TEX-miR-34a and TEX was also measured. Thereafter, the expressions of miR-34a target genes, as IL-6R, STAT3, PD-L1, and VEGF-A, which play roles in tumor progression, were determined in the treated CT-26 cells. RESULTS: The viability of CT-26 cells was harnessed following the treatment with TEX-miR-34a and the apoptosis levels of the cells were also observed to be enhanced dose-dependently. TEX-miR-34a was able to diminish the migration rate of the TEX-miR-34a treated cells and the expressions of IL-6R, STAT3, PD-L1, and VEGF-A were significantly restricted. Moreover, TEXs alone increased the apoptosis rate of tumor cells and repressed the proliferation and migration of these cells which were boosted by enrichment of TEXs with miR-34a. CONCLUSION: Exosomes isolated from the starved CT-26 cells were found to have a potential to deliver miR-34a into tumor cells properly with high functionality maintenance for miR-34a in case of regulating genes related to tumor progression and TEXs which showed no positive effect favoring cancer cells, presumably act as a favorable adjuvant in the CRC therapy.

["Acupuncture network drug"--transformation and application strategies of exosomes based on characteristics of network regulation of acupuncture therapy].

Although the basic mechanism of acupuncture-moxibustion has been revealed from many aspects, there are still many shackles in the transformation of the related research achievements. The transformation of academic achievements of experimental acupunctology is an urgent issue to be solved at present. Network regulation is the basic action mode of acupuncture therapy. In the present paper, we proposed that the "acupuncture network drug" could carry a variety of effective active ingredients which may be the core component of network regulation of acupuncture therapy. The "exosomes", polyvesicle derived from the intracellular lysosomal microparticles invagination, contain complex RNAs and proteins and exist in the body fluids and function in secreting abundant activate substances to participate in intercellular communication, which is the research hotspot in the field of frontier life science in the world. They play an important role in the diagnosis and treatment of diseases, and drug development, etc.. Our studies using rats with adjuvant arthritis and mice with sepsis displayed that after intraperitoneal administration of serum exosomes derived from normal animals receiving acupuncture intervention, an acupuncture-like analgesic effect and an anti-inflammatory effect were achieved, respectively. It is thus possible that acupuncture network drugs could be developed from serum exosomes secreted by exosome autogenous living cells after acupuncture intervention by virtue of the characteristics of low immunogenicity and may have great advantages in drug development and modification. It is also expected to provide new ideas for the transformation of experimental research results and to in depth give explanations about the underlying mechanisms of acupuncture therapy.

The emerging role of miR-128 in musculoskeletal diseases.

MicroRNA-128 (miR-128) is associated with cell proliferation, differentiation, migration, apoptosis, and survival. Genetic analysis studies have demonstrated that miR-128 participates in bone metabolism, which involves bone marrow-derived mesenchymal stem cells, osteoblasts, osteoclasts, and adipocytes. miR-128 also participates in regeneration of skeletal muscles by targeting myoblast-associated proteins. The deregulation of miR-128 could lead to a series of musculoskeletal diseases. In this review, we discuss recent findings of miR-128 in relation to bone metabolism and muscle regeneration to determine its potential therapeutic effects in musculoskeletal diseases, and to propose directions for future research in this significant field.

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

[The miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance].

OBJECTIVE: To study the miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance. METHODS: Between 2015 and 2019, we collected 285 seminal plasma samples from 97 azoospermia (AS) and 96 asthenospermia (AZS) patients and 92 age-matched normal fertile controls in Jiangsu Provincial Hospital of Traditional Chinese Medicine, General Hospital of Eastern Theater Command and the First Hospital Affiliated to Wenzhou Medical University, identified the isolated seminal plasma exosomes by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot, and detected the miR-184 level in the seminal plasma exosomes by quantitative real-time PCR (qRT-PCR). We determined the clinical value of the miR-184 level and its correlation with semen parameters by multiple statistics, predicted the target genes and involved pathways of miR-184 by bioinformatic algorithms, and analyzed their relationship with male infertility. RESULTS: NTA, TEM and Western blot exhibited plenty of exosomes in the seminal plasma of the patients. The results of qRT-PCR showed that the miR-184 level in the seminal plasma exosome was dramatically decreased in the AS patients compared with that in the normal fertile controls (0.227 ［0.092, 0.790］ vs 0.650 ［0.408, 1.061］, P < 0.01), but increased in AZS males in comparison with that in the control group (1.176 ［0.661, 1.946］ vs 0.650 ［0.408, 1.061］, P < 0.01). The areas under the ROC curve (AUC) for differentiating the AS and AZS patients from the controls were 0.866 (95% CI: 0.815－0.916) and 0.724 (95% CI: 0.653－0.795), respectively, and that for differentiating the AS from the AZS group was 0.964 (95% CI: 0.943－0.985). The miR-184 level in the seminal plasma exosome of the AZS patients was correlated positively with the sperm count (r = 0.243, P = 0.017) but negatively with the percentage of progressively motile sperm (r = －0.407, P = 0.006). Bioinformatics analysis indicated that the downstream target genes of miR-184 were significantly enriched in the protein regulatory pathways closely related to male reproduction and spermatogenesis. CONCLUSIONS: The miR-184 level in the seminal plasma exosome of infertility patients is significantly different from that of normal fertile males, which may serve as a potential auxiliary marker for the diagnosis of and participate in the development and progression of male infertility.