Human urine-derived stem cells protect against renal ischemia/reperfusion injury in a rat model via exosomal miR-146a-5p which targets IRAK1.
ABSTRACT: Rationale: Ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) that is associated with high morbidity and mortality, and for which specific treatments are lacking. In this study, we investigated the protective effect of human urine-derived stem cells (USCs) and their exosomes against IRI-induced AKI to explore the potential of these cells as a new therapeutic strategy. Methods: USCs were derived from fresh human urine. Cell surface marker expression was analyzed by flow cytometry to determine the characteristics of the stem cells. Adult male Sprague-Dawley rats were used to generate a lethal renal IRI model. One dose of USCs (2×106 cells/ml) or exosomes (20 µg/1 ml) in the experimental groups or saline (1 ml) in the control group was administered intravenously immediately after blood reperfusion. Blood was drawn every other day for measurement of serum creatinine (sCr) and blood urea nitrogen (BUN) levels. The kidneys were harvested for RNA and protein extraction to examine the levels of apoptosis and tubule injury. In vitro, the hypoxia-reoxygenation (H/R) model in human kidney cortex/proximal tubule cells (HK2) was used to analyze the protective ability of USC-derived exosomes (USC-Exo). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), western blotting, superoxide dismutase activity, and malonaldehyde content analyses were used to evaluate oxidative stress in HK2 cells treated with USC-Exo after H/R. Exosomal microRNA sequencing techniques and bioinformatics analysis were used to search for enriched miRNAs in the exosomes and interacting genes. The interaction between miRNAs and the 3' untranslated region of the target gene was detected using a dual luciferase reporting system. The miRNA mimic and inhibitor were used to regulate the miRNA level in HK2 cells. Results: Treatment with USCs led to reductions in the levels of sCr, BUN, and renal tubular cell apoptosis; inhibited the infiltration of inflammatory cells; and protected renal function in the rat IRI model. Additionally, USC-derived exosomes protected against IRI-induced renal damage. miR-146a-5p was the most abundant miRNA in exosomes obtained from the conditioned medium (CM) of USCs. miR-146a-5p targeted and degraded the 3'UTR of interleukin-1 receptor-associated kinase 1 (IRAK1) mRNA, subsequently inhibited the activation of nuclear factor (NF)-?B signaling, and protected HK2 cells from H/R injury. USC transplantation also upregulated miR-146a-5p expression, downregulated IRAK1 expression and inhibited nuclear translocation of NF-?B p65 in the kidney of the rat IRI model. Conclusions: According to our experimental results, USCs could protect against renal IRI via exosomal miR-146a-5p, which could target the 3'UTR of IRAK1 and subsequently inhibit the activation of NF-?B signaling and infiltration of inflammatory cells to protect renal function. As a novel cell source, USCs represent a promising non-invasive approach for the treatment of IRI.
Project description:Human urine-derived stem cells (USCs) protect rats against kidney ischemia/reperfusion (I/R) injury. Here we investigated the role of USCs exosomes (USCs-Exos) in protecting tubular endothelial cells and miRNA transfer in the kidney. Human USCs and USCs-Exos were isolated and verified by morphology and specific biomarkers. USC-Exos played a protective role in human proximal tubular epithelial cells (HK-2) exposed to hypoxia/reoxygenation (H/R). USCs-Exos were rich in miR-216a-5p, which targeted phosphatase and tensin homolog (PTEN) and regulated cell apoptosis through the Akt pathway. In HK-2 cells exposed to H/R, incubation with USC-Exos increased miR-216-5p, decreased PTEN levels, and stimulated Akt phosphorylation. Exposure of hypoxic HK-2 cells to USCs-Exos pretreated with anti-miR-216a-5p can prevent the increase of miR-216-5p and Akt phosphorylation levels, restore PTEN expression, and promote apoptosis. The dual-luciferase reported gene assay in HK-2 cells confirmed that miR-216a-5p targeted PTEN. In rats with I/R injury, intravenous infusion of USCs-Exos can effectively induce apoptosis suppression and functional protection, which is associated with decreased PTEN. Infusion of exosomes from anti-miR-216a-5p-transfected USCs weakened the protective effect in the I/R model. Therefore, USCs-Exos can reduce renal I/R injury by transferring miR-216a-5p targeting PTEN. Potentially, USCs-Exos rich in miR-216a-5p can serve as a promising therapeutic option for AKI.
Project description:Chronic non-healing wounds represent one of the most common complications of diabetes and need advanced treatment strategies. Exosomes are key mediators of cell paracrine action and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of exosomes from human urine-derived stem cells (USC-Exos) on diabetic wound healing and the underlying mechanism. Methods: USCs were characterized by flow cytometry and multipotent differentiation potential analyses. USC-Exos were isolated from the conditioned media of USCs and identified by transmission electron microscopy and flow cytometry. A series of functional assays in vitro were performed to assess the effects of USC-Exos on the activities of wound healing-related cells. Protein profiles in USC-Exos and USCs were examined to screen the candidate molecules that mediate USC-Exos function. The effects of USC-Exos on wound healing in streptozotocin-induced diabetic mice were tested by measuring wound closure rates, histological and immunofluorescence analyses. Meanwhile, the role of the candidate protein in USC-Exos-induced regulation of angiogenic activities of endothelial cells and diabetic wound healing was assessed. Results: USCs were positive for CD29, CD44, CD73 and CD90, but negative for CD34 and CD45. USCs were able to differentiate into osteoblasts, adipocytes and chondrocytes. USC-Exos exhibited a cup- or sphere-shaped morphology with a mean diameter of 51.57 ± 2.93 nm and positive for CD63 and TSG101. USC-Exos could augment the functional properties of wound healing-related cells including the angiogenic activities of endothelial cells. USC-Exos were enriched in the proteins that are involved in regulation of wound healing-related biological processes. Particularly, a pro-angiogenic protein called deleted in malignant brain tumors 1 (DMBT1) was highly expressed in USC-Exos. Further functional assays showed that DMBT1 protein was required for USC-Exos-induced promotion of angiogenic responses of cultured endothelial cells, as well as angiogenesis and wound healing in diabetic mice. Conclusion: Our findings suggest that USC-Exos may represent a promising strategy for diabetic soft tissue wound healing by promoting angiogenesis via transferring DMBT1 protein.
Project description:Long non-coding RNAs (lncRNAs) are considered as crucial regulatory factors in cancer biology. However, the biological function of long intergenic non-protein coding RNA 960 (LINC00960) in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC) is still unknown. The goal of this study is to investigate the role of LINC00960 in PDAC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine the expression levels of LINC00960 in PDAC tissues and cell lines. After transfection, the loss-of-function models of LINC00960 or interleukin 1 receptor-associated kinase 1 (IRAK1) were established with BxPC-3 cells and Colo357 cells, and the malignant phenotypes of BxPC-3 cells and Colo357 cells were detected by CCK-8 assay, BrdU assay and Transwell assay, respectively. The interactions among LINC00960, miR-146a-5p and IRAK1 were predicted by bioinformatics analysis, and verified by luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. The regulatory functions of LINC00960 and miR-146a-5p on IRAK1 were detected by Western blot. We demonstrated that the LINC00960 expression was increased in PDAC tissues and cell lines. Knocking down LINC00960 or IRAK1 could repress the viability, migration, and invasion of BxPC-3 and Colo357 cells. LINC00960 functioned as a molecular sponge for miR-146a-5p, and IRAK1 was verified as a target gene of miR-146a-5p. Additionally, LINC00960 could up-regulate IRAK1 expression via repressing miR-146a-5p, and the oncogenic properties of LINC00960 were partly reversed by miR-146a-5p. Our findings reveal that LINC00960 is a promoter of PDAC progression through regulating miR-146a-5p/IRAK1axis.
Project description:BACKGROUND:Organoids play an important role in basic research, drug screening, and regenerative medicine. Here, we aimed to develop a novel kind of three-dimensional (3D) organoids generated from urine-derived stem cells (USCs) and to explore whether kidney-specific extracellular matrix (kECM) could enable such organoids for renal function in vitro. METHODS:USCs were isolated from human urine samples and cultured with kECM extraction to generate 3D organoids in vitro. Eight densities from 1000 to 8000 cells per organoids were prepared, and both ATP assay and Live/Dead staining were used to determine the optimal USC density in forming organoids and kECM additive concentration. The morphology and histology of as-made organoids were evaluated by hematoxylin and eosin (H.E.) staining, immunofluorescence staining and whole mount staining. Additionally, RT-qPCR was implemented to detect renal-related gene expression. Drug toxicity test was conducted to evaluate the potential application for drug screening. The renal organoids generated from whole adult kidney cells were used as a positive control in multiple assessments. RESULTS:The optimized cell density to generate ideal USC-derived organoids (USC-organoids) was 5000 cells/well, which was set as applying density in the following experiments. Besides, the optimal concentration of kECM was revealed to be 10%. On this condition, Live/Dead staining showed that USC-organoids were well self-organized without significant cell death. Moreover, H.E. staining showed that compact and viable organoids were generated without obvious necrosis inside organoids, which were very close to renal organoids morphologically. Furthermore, specific proximal tubule marker Aquaporin-1 (AQP1), kidney endocrine product erythropoietin (EPO), kidney glomerular markers Podocin and Synaptopodin were detected positively in USC-organoids with kECM. Nephrotoxicity testing showed that aspirin, penicillin G, and cisplatin could exert drug-induced toxicity on USC-organoids with kECM. CONCLUSIONS:USC-organoids could be developed from USCs via an optimal procedure. Combining culture with kECM, USC-organoid properties including morphology, histology, and specific gene expression were identified to be similar with real renal organoids. Additionally, USC-organoids posed kECM in vitro showed the potential to be a drug screening tool which might take the place of renal organoids to some extent in the future.
Project description:BACKGROUND:microRNA-146a-5p (miRNA-146a-5p) is a key molecule in the negative regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain. METHODS:The sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quantitative real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain-related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia. RESULTS:We found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mechanical and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mechanical and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further. CONCLUSIONS:miRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.
Project description:The availability of autologous adult stem cells is one of the essential prerequisites for human stem cell therapy. Urine-derived stem cells (USCs) are considered as desirable cell sources for cell therapy because donor-specific USCs are easily and non-invasively obtained from urine. Efficient isolation, expansion, and differentiation methods of USCs are necessary to increase their availability. Here, we developed a method for efficient isolation and expansion of USCs using Matrigel, and the rho-associated protein kinase (ROCK) inhibitor, Y-27632. The prepared USCs showed significantly enhanced migration, colony forming capacity, and differentiation into osteogenic or chondrogenic lineage. The USCs were successfully reprogramed into induced pluripotent stem cells (USC-iPSCs) and further differentiated into kidney organoid and hematopoietic progenitor cells (HPCs). Using flavonoid molecules, the isolation efficiency of USCs and the production of HPCs from the USC-iPSCs was increased. Taken together, we present an improved isolation method of USCs utilizing Matrigel, a ROCK inhibitor and flavonoids, and enhanced differentiation of USC-iPSC to HPC by flavonoids. These novel findings could significantly enhance the use of USCs and USC-iPSCs for stem cell research and further application in regenerative stem cell-based therapies.
Project description:Elevated toll-like receptor 4 (TLR4) expression is associated with a high risk of radiation-induced liver disease (RILD). MicroRNA (miR)-146a-5p is a key regulator of lipopolysaccharide (LPS)/TLR4 signaling, but its role in modulation of RILD remains unclear. Here, we found that irradiation and LPS stimulation induced TLR4 and miR-146a-5p expression in the human hepatic stellate cell (HSC) line LX2. Ectopic expression of miR-146a-5p in LX2 inhibited irradiation-induced and LPS-induced pro-inflammatory cytokine secretion and cell proliferation, and promoted cell apoptosis by down-regulating the expression levels of TLR4, interleukin-1 receptor associated kinase 1 (IRAK1), tumor necrosis factor receptor associated factor 6 (TRAF6) and phosphorylation of nuclear factor-kappa B. In addition, the culture medium from the irradiated and LPS-stimulated HSCs transfected with miR-146a-5p significantly attenuated apoptosis in irradiated hepatocytes. Overexpression of miR-146a-5p reduced ?-smooth muscle actin production in irradiated and LPS-stimulated LX2 cells, which was associated with inhibition of TRAF6-mediated JNK and Smad2 phosphorylation. Knockdown of TRAF6 or IRAK1 mimicked the effects of miR-146a-5p on HSC function. Furthermore, miR-146a-5p treatment alleviated irradiation-induced and endotoxin-induced hepatic inflammatory response and fibrogenesis in mice through inhibition of the TLR4 signaling pathway. Collectively, this study reveals the anti-pro-inflammatory and anti-fibrotic effects of miR-146a-5p on liver injury, and suggests a potential application of miR-146a-5p in the therapeutic prevention of RILD.
Project description:<h4>Objective</h4>(1) To examine usual source of care (USC) trends across four categories (No USC, Person USC, Person, in Facility USC, and Facility USC), and (2) to determine whether USC types are associated with emergency department (ED) visits and hospital admissions.<h4>Data source</h4>1996-2014 Medical Expenditure Panel Surveys.<h4>Study design</h4>We stratified each USC category, by age, region, gender, poverty, insurance, race/ethnicity, and education and used regression to determine the characteristics associated with USC types, ED visits, and hospital admissions.<h4>Principal findings</h4>Those with No USC and Facility USCs increased 10 and 18 percent, respectively, while those with Person USCs decreased by 43 percent. Compared to those in the lowest income bracket, those in the highest income bracket were less likely to have a Facility USC. Among those with low incomes, individuals with No USC, Person, in Facility, and Facility USCs were more likely to have ED visits than those with Person USCs.<h4>Conclusions</h4>A growing number are reporting facilities as their USCs or none at all. The impact of these trends is uncertain, although we found that some USC types are associated with ED visits and hospital admissions. Tracking USCs will be crucial to measuring progress toward enhanced care efficiency.
Project description:<h4>Background</h4>Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets.<h4>Methods</h4>Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs.<h4>Results</h4>We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs.<h4>Conclusions</h4>Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.
Project description:Lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway is demonstrated to be involved in the hepatic fibrosis. MicroRNA (miR)-146a-5p is a key regulator of the innate immune response. The functional significance of miR-146a-5p during the LPS/TLR4 mediated hepatic fibrosis process remains unclear. In this study, we found that TLR4 and ?-smooth muscle actin (?-SMA) were up-regulated and miR-146a-5p was down-regulated in human hepatic stellate cell (HSC) line LX2 after LPS stimulation. Overexpression of miR-146a-5p inhibited LPS induced pro-inflammatory cytokines secretion through down-regulating the expression levels of TLR-4, IL-1 receptor-associated kinase 1 (IRAK1), TNF receptor associated factor-6 (TRAF6) and phosphorylation of nuclear factor-kappa B (NF-?B). Knockdown of IRAK1 and TRAF6 also suppressed pro-inflammatory cytokine production by inhibiting NF-?B phosphorylation. In addition, miR-146a-5p mimic blocked LPS induced TRAF6 dependent c-Jun N-terminal kinase (JNK) and Smad2 activation as well as ?-SMA production. Taken together, these results suggest that miR-146a-5p suppresses pro-inflammatory cytokine secretion and cell activation of HSC through inhibition of TLR4/NF-?B and TLR4/TRAF6/JNK pathway.