miR-130b-3p involved in the pathogenesis of age-related hearing loss via targeting PPARγ and autophagy.

The study focuses on the underlying regulatory mechanism of age-related hearing loss (ARHL), which results from autophagy dysregulation mediated by miR-130b-3p targeting PPARγ. We constructed miR-130b-3p knockout (antagomir) and PPARγ over-expression (OE-PPARγ) mice model by injecting mmu-miR-130b-3p antagomir and HBAAV2/Anc80-m-Pparg-T2A-mCHerry into the right ear' round window of each mouse, respectively. In vitro, we introduced oxidative stress within HEI-OC1 cells by H2O2 and exogenously changed the miR-130b-3p and PPARγ levels. MiRNA level was detected by RT-qPCR, proteins by western blotting and immunohistochemistry. Morphology of autophagosomes was observed by electron microscopy. In vivo, the cochlea of aged mice showed higher miR-130b-3p expression and lower PPARγ expression, while exogenous inhibition of miR-130b-3p up-regulated PPARγ expression. Autophagy-related biomarkers expression (ATG5, Beclin-1 and LC3B II/I) decreased in aged mice, which reversely increased after the inhibition of miR-130b-3p. The elevation of PPARγ demonstrated similar effects. Contrarily, exogenous overexpression of miR-130b-3p resulted in the decrease of ATG5, Beclin-1 and LC3B II/I. We created oxidative stress within HEI-OC1 by H2O2, subsequently observed the formation of autophagosomes under electron microscope, so as the elevated cell apoptosis rate and weakened cell viability. MiR-130b-3p/PPARγ contributed to the premature senescence of these H2O2-induced HEI-OC1 cells. MiR-130b-3p regulated HEI-OC1 cell growth by targeting PPARγ, thus leading to ARHL.

Differentially expressed miRNA profiles of serum-derived exosomes in patients with sudden sensorineural hearing loss.

Objectives: This study aimed to compare the expressed microRNA (miRNA) profiles of serum-derived exosomes of patients with sudden sensorineural hearing loss (SSNHL) and normal hearing controls to identify exosomal miRNAs that may be associated with SSNHL or serve as biomarkers for SSNHL. Methods: Peripheral venous blood of patients with SSNHL and healthy controls was collected to isolate exosomes. Nanoparticle tracking analysis, transmission electron microscopy, and Western blotting were used to identify the isolated exosomes, after which total RNA was extracted and used for miRNA transcriptome sequencing. Differentially expressed miRNAs (DE-miRNAs) were identified based on the thresholds of P < 0.05 and |log2fold change| > 1 and subjected to functional analyses. Finally, four exosomal DE-miRNAs, including PC-5p-38556_39, PC-5p-29163_54, PC-5p-31742_49, and hsa-miR-93-3p_R+1, were chosen for validation using quantitative real-time polymerase chain reaction (RT-qPCR). Results: Exosomes were isolated from serum and identified based on particle size, morphological examination, and expression of exosome-marker proteins. A total of 18 exosomal DE-miRNAs, including three upregulated and 15 downregulated miRNAs, were found in SSNHL cases. Gene ontology (GO) functional annotation analysis revealed that target genes in the top 20 terms were mainly related to "protein binding," "metal ion binding," "ATP binding," and "intracellular signal transduction." Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these target genes were functionally enriched in the "Ras," "Hippo," "cGMP-PKG," and "AMPK signaling pathways." The expression levels of PC-5p-38556_39 and PC-5p-29163_54 were significantly downregulated and that of miR-93-3p_R+1 was highly upregulated in SSNHL. Consequently, the consistency rate between sequencing and RT-qPCR was 75% and sequencing results were highly reliable. Conclusion: This study identified 18 exosomal DE-miRNAs, including PC-5p-38556_39, PC-5p-29163_54, and miR-93-3p, which may be closely related to SSNHL pathogenesis or serve as biomarkers for SSNHL.

3D-real IR MRI detects serendipity of inner ear in enlarged vestibular aqueduct syndrome.

BACKGROUND: Although, the diagnostic criteria for enlarged vestibular aqueduct syndrome (EVAS) were determined by years. On the shoulders of predecessors, we still detected some new discoveries about EVAS by using 3D-real IR MRI. AIMS/OBJECTIVES: To analyze the signal intensity of membranous and osseous labyrinths of vestibular aqueduct (VA) and endolymphatic sac (ES) in EVAS using three-dimensional real inversion recovery (3D-Real-IR) magnetic resonance imaging (MRI) after intratympanic injection of gadolinium. MATERIAL AND METHODS: The study is a prospective trial, diagnosed EVAS patients (n = 10) and none- patients (n = 10) were included. 3D-real-IR MRIs were performed to assess the endolymphatic hydrops (EH) and differentiated the endolymphatic and perilymphatic signal intensities of VA and ES. RESULTS: Compared to control group, EVAS group had VA osseous labyrinths middle diameter >1.5 mm different from membranous labyrinths. The cochlear EH was correlated with Mondini malformation and irrelation with the level of hearing loss (HL). CONCLUSIONS AND SIGNIFICANCE: Interspace of osseous labyrinths of VA and ES are much larger than their membranous labyrinths, which is not consistance with previous research. And cochlear Mondini malformation may cause endolymphatic fluid malabsorption, inducing cochlear EH. Osteal ampliative of VA and ES and cochlear EH, which are morphogenetic anomalies, may not the direct cause of HL in EVAS.

Targeted antiangiogenesis gene therapy using targeted cationic microbubbles conjugated with CD105 antibody compared with untargeted cationic and neutral microbubbles.

OBJECTIVE: This study aimed to develop targeted cationic microbubbles conjugated with a CD105 antibody (CMB105) for use in targeted vascular endothelial cell gene therapy and ultrasound imaging. We compared the results with untargeted cationic microbubbles (CMB) and neutral microbubbles (NMB). METHODS: CMB105 were prepared and compared with untargeted CMB and NMB. First, the microbubbles were characterized in terms of size, zeta-potential, antibody binding ability and plasmid DNA loading capacity. A tumor model of subcutaneous breast cancer in nude mice was used for our experiments. The ability of different types of microbubbles to target HUVECs in vitro and tumor neovascularization in vivo was measured. The endostatin gene was selected for its outstanding antiangiogenesis effect. For in vitro experiments, the transfection efficiency and cell cycle were analyzed using flow cytometry, and the transcription and expression of endostatin were measured by qPCR and Western blotting, respectively. Vascular tube cavity formation and tumor cell invasion were used to evaluate the antiangiogenesis gene therapy efficiency in vitro. Tumors were exposed to ultrasound irradiation with different types of microbubbles, and the gene therapy effects were investigated by detecting apoptosis induction and changes in tumor volume. RESULTS: CMB105 and CMB differed significantly from NMB in terms of zeta-potential, and the DNA loading capacities were 16.76±1.75 µg, 18.21±1.22 µg, and 0.48±0.04 µg per 5×10(8) microbubbles, respectively. The charge coupling of plasmid DNA to CMB105 was not affected by the presence of the CD105 antibody. Both CMB105 and CMB could target to HUVECs in vitro, whereas only CMB105 could target to tumor neovascularization in vivo. In in vitro experiments, the transfection efficiency of CMB105 was 24.7-fold higher than the transfection efficiency of NMB and 1.47-fold higher than the transfection efficiency of CMB (P<0.05). With ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were the highest in the CMB105 group (P<0.001); the antiangiogenesis effect and inhibition of tumor cells invasion was better with CMB105 than CMB or NMB in vitro (P<0.01). After gene therapy, the tumor volumes of CMB105 group were significantly smaller than that of CMB and NMB, and many tumor cells had begun apoptosis in the CMB105 group, which had the highest apoptosis index (P<0.001). CONCLUSIONS: As a contrast agent and plasmid carrier, CMB105 can be used not only for targeted ultrasound imaging but also for targeted gene therapy both in vitro and in vivo. The plasmid DNA binding ability of the CMB was not affected by conjugation of the CMB with the CD105 antibody, and because of its targeting ability, the gene transfection efficiency and therapeutic effect were better compared with the untargeted CMB and NMB. The advantages of targeted gene therapy with CMB105 in vivo were more prominent than with CMB or NMB because neither can target the endothelia in vivo.