Electro-Acupuncture Promotes the Differentiation of Endogenous Neural Stem Cells via Exosomal microRNA 146b After Ischemic Stroke.
ABSTRACT: Background: Evidences indicate that exosomes-mediated delivery of microRNAs (miRNAs or miRs) is involved in the neurogenesis of stroke. This study was to investigate the role of exosomal miRNAs in non-drug therapy of electro-acupuncture (EA) regulating endogenous neural stem cells for stroke recovery. Methods: The model of focal cerebral ischemia and reperfusion in rats were established by middle cerebral artery occlusion (MCAO) and treated by EA. The exosomes were extracted from peri-ischemic striatum and identified by exosomal biomarkers, and detected differentially expressed miRNAs with microarray chip. Primary stem cells were cultured, and oxygen-glucose deprivation and reperfusion (OGD/R) was used to mimic vitro ischemic injury. Results: The levels of exosomal biomarkers TSG101 and CD81 were increased in peri-ischemic striatum after EA treatment, and we revealed 25 differentially expressed miRNAs in isolated exosomes, of which miR-146b was selected for further analysis, and demonstrated that EA increased miR-146b expression and its inhibitors could block the effects. Subsequently, we confirmed that EA upregulated miR-146b expression to promote neural stem cells differentiation into neurons in peri-ischemic striatum. In vitro, it was verified that OGD/R hindered neural stem cells differentiation, and miR-146b inhibitors furtherly suppressed its differentiation, simultaneously NeuroD1 was involved in neural stem cells differentiation into neurons. Moreover, in vivo we found EA promoted NeuroD1-mediated neural stem cells differentiation via miR-146b. In addition, EA also could improve neurological deficits through miR-146b after ischemic stroke. Conclusion: EA promotes the differentiation of endogenous neural stem cells via exosomal miR-146b to improve neurological injury after ischemic stroke.
Project description:Pluripotent stem cells (PSCs) are regarded as potential sources that provide specific neural cells for cell therapy in some nervous system diseases. However, the mechanisms underlying the neural differentiation of PSCs remain largely unknown. MicroRNAs (miRNAs or miRs) are a class of small non‑pro-tein-coding RNAs that act as critical regulatory molecules in many cellular processes. In this study, we found that miR‑146b‑5p expression was markedly increased following the neural induction of mouse embryonic stem cells (ESCs) or induced PSCs (iPSCs). In this study, to further identify the role of miR‑146b‑5p, we generated stable miR‑146b‑5p-overexpressing ESC and iPSC cell lines, and induced the differentiation of these cells by the adherent monolayer culture method. In the miR‑146b‑5p-overexpressing ESC- or iPSC-derived cultures, RT-qPCR analysis revealed that the mRNA expression levels of neuroectoderm markers, such as Sox1, Nestin and Pax6, were markedly increased, and flow cytometric analysis verified that the number of Nestin‑positive cells was higher in the miR‑146b‑5p-overexpressing compared with the control cells. Mechanistically, the miR‑146b‑5p-overexpressing ESCs or iPSCs exhibited a significant reduction in Oct4 expression, which may be an explanation for these cells having a tendency to differentiate towards the neural lineage. Moreover, we confirmed that miR‑146b‑5p directly targeted Smad4 and negatively regulated the transforming growth factor (TGF)-β signaling pathway, which contributed to the neural commitment of PSCs. Collectively, our findings uncover the essential role of miR‑146b‑5p in the neural conversion of PSCs.
Project description:Gastric cancer (GC) remains a leading cause of cancer-related mortality in the United States and China, there is an urgent need to discover novel non-invasive biomarkers for the early diagnosis of GC to improve the prognosis of GC patients. Exosomal miRNAs are considered promising biomarkers for cancer diagnosis. Using next-generation sequencing (NGS), bioinformatics and further validation, we identified and evaluated exosomal miRNAs in serum as early diagnostic markers for GC. NGS revealed that the average mappable reads in the RNA libraries were about 6.5 million per patient including miRNAs (73.38%), rRNAs (17.10%), snRNAs (8.83%), snoRNAs (0.65%), and tRNAs (0.04%). A total of 66 up and 13 down-regulated exosomal miRNAs were found in the screened cohort. In the validation cohort, by comparing with healthy individuals, higher levels of serum exosomal miR-92b-3p, let-7g-5p, miR-146b-5p, and miR-9-5p were found to be significantly associated with early-stage GC (p < 0.05). Diagnostic power of the combined panels of the exosomal miRNAs or the combination of exosomal miRNAs and CEA outperformed that of single exosomal miRNA marker for establishing a diagnosis of early-stage GC. The combined diagnosis of exosomal miR-92b-3p + let-7g-5p + miR-146b-5p + miR-9-5p with CEA had the most powerful efficiency with an AUC up to 0.786. In addition, serum levels of exosomal miR-92b-3p were significantly associated with poor cohesiveness (p = 0.0021), let-7g-5p and miR-146b-5p were significantly correlated with nerve infiltration (p = 0.0234 and p = 0.0126, respectively), and miR146b-5p was statistically correlated with tumor invasion depth in early-stage GC (p = 0.0089). In conclusion, serum exosomal miR-92b-3p, -146b-5p, -9-5p, and let-7g-5p may serve as potential non-invasive biomarkers for early diagnosis of GC.
Project description:Articular cartilage injury can result in chondrocyte loss and diminishment of specialised extracellular matrix, which can progress to an osteoarthritic (OA) phenotype. Stem cells have emerged as a favourable approach for articular cartilage regeneration. Identification of miRNAs which influence stem cell fate offers new approaches for application of miRNAs to regenerate articular cartilage. Skeletal stem cells (SSCs) isolated from human bone marrow were cultured as high density micromass' using TGF-?3 to induce chondrogenesis. qPCR and TaqMan qPCR were used to assess chondrogenic gene and miRNA expression. Target prediction algorithms identified potential targets of miR-146b. Transient transfection with miR-146b mimic and western blotting was used to analyse SOX5. Human OA articular chondrocytes were examined for miR-146b expression. Chondrogenic differentiation of human bone marrow derived SSCs resulted in significant down-regulation of miR-146b. Gain of miR-146b function resulted in down-regulation of SOX5. MiR-146b expression was up-regulated in OA chondrocytes. These findings demonstrate the functional role of miR-146b in the chondrogenic differentiation of human bone marrow derived SSCs. MiR-146b may play a role in the pathophysiology of OA. Application of miR-146b combined with stem cell therapy could enhance regeneration of cartilaginous tissue and serve as a potential therapeutic target in the treatment of OA.
Project description:The physiological role of microRNAs (miRNAs) in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs) culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (?8.84%) could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05) when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221) were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation.
Project description:Background: A liquid biopsy using circulating exosomal genetic materials provides new insights for thyroid cancer diagnosis. This study aimed to identify plasma-derived exosomal biomarkers that could be used for early detection of papillary thyroid carcinoma (PTC). Method: Exosomal miRNAs in plasma were isolated from patients with benign thyroid nodules and patients with PTC. Profiling of exosomal miRNA was performed using RNA sequencing (RNA-seq) to identify miRNA candidates and differentiate the benign from malignant. The validation cohort consisted of 30 patients with benign thyroid nodules, 35 PTC patients, and 31 healthy individuals. Real-time PCR was used to quantify the expression of miRNA candidates. The diagnostic potential of the candidates was evaluated by receiver operating characteristic (ROC) curves. Results: After RNA-seq, eight plasma exosomal miRNAs were selected as candidates. Further validation indicated that the levels of exosomal miR-16-2-3p, miR-223-5p, miR-34c-5p, miR-182-5p, miR-223-3p, and miR-146b-5p were significantly lower in nodules compared to healthy controls (p < 0.0001), whereas miR-16-2-3p and miR-223-5p were significantly higher in the PTC cases than in those with benign nodules (p < 0.05). ROC analyses revealed that the above six miRNAs were potent indicators for detection of thyroid nodules. Meanwhile, miR-16-2-3p and miR-223-5p can be utilized for detecting PTC from benign nodules. Additionally, combined miRNA panels showed increased diagnostic sensitivities and specificities compared to single miRNA markers. Conclusion: Six aberrantly expressed plasma exosomal miRNAs may be used as diagnostic biomarkers to differentiate thyroid nodules from healthy individuals. The panel consisting of miR-16-2-3p, miR-223-5p, miR-101-3p, and miR-34c-5p are eligible for discriminating benign from malignant thyroid nodules.
Project description:Accumulating studies report that microRNAs (miRNAs) are actively involved in skeletal myogenesis. Previously, our study revealed that miR-146b-3p was related to the growth of skeletal muscle. Here, we further report that miR-146b-3p is essential for the proliferation, differentiation, and apoptosis of chicken myoblast. Elevated expression of miR-146b-3p can dramatically suppress proliferation and differentiation, and facilitate apoptosis of chicken myoblast. Besides, we identified two target genes of miR-146b-3p, AKT1 and MDFIC, and found that miR-146b-3p can inhibit the PI3K/AKT pathway. Our study also showed that both AKT1 and MDFIC can promote the proliferation and differentiation while inhibit the apoptosis of myoblast in chicken. Overall, our results demonstrate that miR-146b-3p, directly suppressing PI3K/AKT pathway and MDFIC, acts in the proliferation, differentiation, and apoptosis of myoblast in chicken.
Project description:Transplanted neural stem cells promote neural tissue regeneration and functional recovery primarily by releasing paracrine factors. Exosomes act as important secreted paracrine molecules to deliver therapeutic agents involved in cellular functions. Here, we focused on the role of exosomes (hNSC-Exo) derived from human neural stem cells (hNSCs). We utilized the pro-inflammatory factor interferon gamma (IFN-?) to induce the generation of altered exosomes (IFN-?-hNSC-Exo), and compared their roles with those of hNSC-Exo and explored the potential mechanism. Importantly, IFN-? preconditioning did not affect the secretion, but significantly altered the ability of exosomes derived from hNSCs. Moreover, IFN-?-hNSC-Exo was functionally superior to hNSC-Exo; showed increased cell proliferation and cell survival and decreased cell apoptosis in vitro. Furthermore, IFN-?-hNSC-Exo further exerted therapeutic effects (showed better behavioral and structural outcomes) compared to those of hNSCs-Exo in an ischemic stroke rat model. Next-generation sequencing (NGS) revealed specific exosomal miRNAs (hsa-miR-206, hsa-miR-133a-3p and hsa-miR-3656) in IFN-?-hNSC-Exo with important roles in cell survival. Thus, our findings demonstrate that the inflammatory factor IFN-? can regulate the functions of exosomes and highlight its role in regulating the application of neural stem cell-derived exosomes.
Project description:BACKGROUND:Urinary exosomal miRNAs are gaining increasing attention for their potential as ideal non-invasive biomarkers for kidney diseases; however, little is known about their diagnostic ability for paediatric nephrotic syndrome (NS). This study explored the clinical value of urinary exosomal miRNAs for paediatric idiopathic NS. METHODS:Urine samples were collected from 129 NS children and 126 age-/sex-matched healthy controls. The miRNA profile of urinary exosomes was analysed by high-throughput Illumina sequencing via synthesis (SBS) technology followed by verification with a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay arranged in two independent cohorts. Additionally, paired urine samples from 65 of these patients were collected before and after treatment. FINDINGS:The Illumina SBS identified 30 markedly increased urinary exosomal miRNAs in NS children compared with controls (? 5-fold, P?<?.05). Fifteen miRNAs were selected for further investigation, of which 5 (miR-194-5p, miR-146b-5p, miR-378a-3p, miR-23b-3p and miR-30a-5p) were verified by RT-qPCR to be significantly and steadily increased in NS (> 3-fold, P?<?.01) and markedly reduced during the clinical remission period (P?<?.001). Moreover, the concentrations of miR-194-5p and miR-23b-3p were significantly positively correlated with the urine protein content and were markedly higher in the high urine protein group than in the low urine protein group (P?<?.001 and P?<?.01, respectively). INTERPRETATIONS:We identified 5 altered urinary exosomal miRNAs in NS children with disease progression and treatment. These urinary exosomal miRNAs could be promising and non-invasive potential biomarker candidates for diagnosing, monitoring and stratifying paediatric NS. FUND: National Natural Science Foundation of China; Fund of State Key Laboratory of Analytical Chemistry for Life Science; National Basic Research Programme of China; Foundation of Jiangsu Provincial Medical Youth Talent.
Project description:Blood-stasis syndrome (BSS) is one of the Traditional Chinese medicine (TCM) syndrome differentiations that are commonly seen in stroke and ischemic heart diseases; however, the BSS differentiation criterion is not standardized. More objective biomarkers for BSS diagnosis are needed.Acute ischemic stroke (AIS) or unstable angina (UA) patients with BSS and healthy controls were enrolled. The miRNA and mRNA expression profiles of UA patients and AIS patients were compared to those of healthy controls to identify the differentially expressed miRNA and mRNA of BSS. Bioinformatics analysis was used to identify significantly deregulated miRNAs and mRNAs correlated to BSS. QRT-PCR was performed to validate the bioinformatics analysis results.Approximately 401 mRNAs and 11 miRNAs were differentially expressed in both UA and AIS patients compared to healthy controls. Gene ontology (GO) functional analysis was performed, and multiple GO terms were enriched. Among the overlapping DE miRNAs and mRNAs, miR-146b-5p, -199a-5p and 23 targeted mRNAs were pivotal genes in the BSS genomic characteristics. These 2 miRNAs and 23 mRNAs formed network-type biomarkers for BSS.The genomic characteristics of BSS were shown in this study. miR-146b-5p, -199a-5p and the 23 targeted mRNAs formed a diagnostic network for BSS. Further improvement and validation of this diagnostic network might lead to more objective diagnostic criteria for BSS.
Project description:High-grade gliomas (HGGs) are very aggressive brain tumors with a cancer stem cell component. Cells, including cancer stem cells, release vesicles called exosomes which contain small non-coding RNAs such as microRNAs (miRNAs). These are thought to play an important role in cell-cell communication. However, we have limited knowledge of the types of exosomal miRNAs released by pediatric HGG stem cells; a prerequisite for exploring their potential roles in HGG biology. Here we isolated exosomes released by pediatric glioma stem cells (GSCs) and compared their repertoire of miRNAs to genetically normal neural stem cells (NSCs) exosomes, as well as their respective cellular miRNA content. Whereas cellular miRNAs are similar, we find that the exosomal miRNA profiles differ between normal and tumor cells, and identify several differentially expressed miRNAs. Of particular interest is miR-1290 and miR-1246, which have previously been linked to 'stemness' and invasion in other cancers. We demonstrate that GSC-secreted exosomes influence the gene expression of receiving NSCs, particularly targeting genes with a role in cell fate and tumorigenesis. Thus, our study shows that GSCs and NSCs have similar cellular miRNA profiles, yet differ significantly in the repertoire of exosomal miRNAs and these could influence malignant features of HGG.