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Overexpression of miR-210 and its significance in ischemic tissue damage.

ABSTRACT: Hypoxia-induced miR-210 displays a pro-survival, cytoprotective and pro-angiogenic role in several in vitro systems. In vivo, we previously found that miR-210 inhibition increases ischemic damage. Here we describe the generation of a versatile transgenic mouse model allowing the evaluation of miR-210 therapeutic potential in ischemic cardiovascular diseases. We generated a Tet-On miR-210 transgenic mouse strain (TG-210) by targeted transgenesis in the ROSA26 locus. To functionally validate miR-210 transgenic mice, hindlimb ischemia was induced by femoral artery dissection. Blood perfusion was evaluated by power Doppler while tissue damage and inflammation were assessed by histological evaluation. We found that miR-210 levels were rapidly increased in TG-210 mice upon doxycycline administration. miR-210 overexpression was maintained over time and remained within physiological levels in multiple tissues. When hindlimb ischemia was induced, miR-210 overexpression protected from both muscular and vascular ischemic damage, decreased inflammatory cells density and allowed to maintain a better calf perfusion. In conclusion, we generated and functionally validated a miR-210 transgenic mouse model. Albeit validated in the context of a specific cardiovascular ischemic disease, miR-210 transgenic mice may also represent a useful model to assess the function of miR-210 in other physio-pathological conditions.

SUBMITTER: Zaccagnini G

PROVIDER: S-EPMC5573334 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Overexpression of miR-210 and its significance in ischemic tissue damage.

Zaccagnini G G Maimone B B Fuschi P P Maselli D D Spinetti G G Gaetano C C Martelli F F

Scientific reports 20170825 1

Hypoxia-induced miR-210 displays a pro-survival, cytoprotective and pro-angiogenic role in several in vitro systems. In vivo, we previously found that miR-210 inhibition increases ischemic damage. Here we describe the generation of a versatile transgenic mouse model allowing the evaluation of miR-210 therapeutic potential in ischemic cardiovascular diseases. We generated a Tet-On miR-210 transgenic mouse strain (TG-210) by targeted transgenesis in the ROSA26 locus. To functionally validate miR-2 ...[more]

PMID: 28842599

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Project description:Background and aimHypoxic-ischemic encephalopathy (HIE) is a disorder featured by hypoxic and ischemic damages during the perinatal period and its high mortality (i.e., 15%-20%) could be partly attributed to late diagnosis. Therefore, miR-210 and miR-374a were investigated to find if they could improve the diagnostic values of S100B protein and neuron-specific enolase (NSE) for HIE.MethodsAltogether 167 HIE newborns and 82 healthy newborns were recruited, and their blood were sampled for determining the levels of biomarkers. Specifically, S100B protein and NSE levels were detected based on the enzyme-linked immunosorbent assay (ELISA) kit, while the expressions of miR-210 and miR-374a were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Moreover, the receiver operating characteristic (ROC) curves were established to assess the diagnostic values of the above biomarkers for HIE. Finally, the correlation analysis between miR-210/miR-374 and Neonatal Behavioral Neurological Assessment (NBNA) scoring or Gesell intellectual development were also conducted.ResultsThe levels of miR-210, miR-374a, S100B protein, and NSE were significantly distinct between HIE patients and healthy newborns (p < .05). Besides, miR-210 (rs = .573), miR-374a (rs = .651), NSE level (rs = -.622), and S100B level (rs = -.55) were all, respectively, correlated with NBNA scoring with statistical significance (p < .05). Furthermore, it was revealed that the combined diagnosis of miR-210, miR-374a, S100B protein, and NSE could obtain the highest accuracy regarding pairs of mild HIE versus moderate HIE (AUC = 0.898), moderate HIE versus severe HIE (AUC = 0.922), mild HIE versus severe HIE (AUC = 0.996), and HIE versus control (AUC = 0.960). More than that, the four molecules were also remarkably associated with Gesell intellectual development (p < .05).ConclusionMiR-210 and miR-374a could help to elevate the diagnostic value and prognostic prediction of S100B protein and NSE for HIE.

Project description:Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.

Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3’UTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia.

Project description:BackgroundNickel nanoparticles (Nano-Ni) are increasingly used in industry and biomedicine with the development of nanotechnology. However, the genotoxic and carcinogenic effects of Nano-Ni and the underlying mechanisms are still unclear.MethodsAt first, dose-response (0, 10, 20, and 30 μg/mL) and time-response (0, 3, 6, 12, and 24 h) studies were performed in immortalized normal human bronchial epithelial cells BEAS-2B to observe the effects of Nano-Ni on DNA damage response (DDR)-associated proteins and the HIF-1α/miR-210/Rad52 pathway by real-time PCR or Western blot. Then, a Hsp90 inhibitor (1 µM of 17-AAG, an indirect HIF-1α inhibitor), HIF-1α knock-out (KO) cells, and a miR-210 inhibitor (20 nM) were used to determine whether Nano-Ni-induced Rad52 down-regulation was through HIF-1α nuclear accumulation and miR-210 up-regulation. In the long-term experiments, cells were treated with 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles (~ 150 days), and the level of anchorage-independent growth was determined by plating the cells in soft agar. Transduction of lentiviral particles containing human Rad52 ORF into BEAS-2B cells was used to observe the role of Rad52 in Nano-Ni-induced cell transformation. Nano-Ni-induced DNA damage and dysregulation of HIF-1α/miR-210/Rad52 pathway were also investigated in vivo by intratracheal instillation of 50 µg per mouse of Nano-Ni. gpt delta transgenic mice were used to analyze mutant frequency and mutation spectrum in mouse lungs after Nano-Ni exposure.ResultsNano-Ni exposure caused DNA damage at both in vitro and in vivo settings, which was reflected by increased phosphorylation of DDR-associated proteins such as ATM at Ser1981, p53 at Ser15, and H2AX. Nano-Ni exposure also induced HIF-1α nuclear accumulation, miR-210 up-regulation, and down-regulation of homologous recombination repair (HRR) gene Rad52. Inhibition of or knocking-out HIF-1α or miR-210 ameliorated Nano-Ni-induced Rad52 down-regulation. Long-term low-dose Nano-Ni exposure led to cell malignant transformation, and augmentation of Rad52 expression significantly reduced Nano-Ni-induced cell transformation. In addition, increased immunostaining of cell proliferation markers, Ki-67 and PCNA, was observed in bronchiolar epithelial cells and hyperplastic pneumocytes in mouse lungs at day 7 and day 42 after Nano-Ni exposure. Finally, using gpt delta transgenic mice revealed that Nano-Ni exposure did not cause increased gpt mutant frequency and certain DNA mutations, such as base substitution and small base insertions/deletions, are not the main types of Nano-Ni-induced DNA damage.ConclusionsThis study unraveled the mechanisms underlying Nano-Ni-induced cell malignant transformation; the combined effects of Nano-Ni-induced DNA damage and DNA repair defects through HIF-1α/miR-210/Rad52 pathway likely contribute to Nano-Ni-induced genomic instability and ultimately cell transformation. Our findings will provide information to further elucidate the molecular mechanisms of Nano-Ni-induced genotoxicity and carcinogenicity.

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Overexpression of miR-210 and its significance in ischemic tissue damage.

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Overexpression of miR-210 and its significance in ischemic tissue damage.

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