Rs1894720 polymorphism in MIAT increased susceptibility to age-related hearing loss by modulating the activation of miR-29b/SIRT1/PGC-1α signaling.

BACKGROUND: MIAT may be implicated in the pathogenesis of age-related hearing loss (AHL). This study aimed to clarify the effect of a MIAT signaling pathway on the risk of AHL. METHODS: Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, auditory brainstem response (ABR) and quantitative hair cell counts were used to compare the hearing functions in different groups of mice. 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye method was used to establish the potential association between mitochondrial dysfunction and aging. Real-time polymerase chain reaction, Western blot analysis, computational analysis, and luciferase assay were conducted to establish a myocardial infarction associated transcript (MIAT) signaling pathway, whose role in the pathogenesis of AHL was further validated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and flow cytometry. RESULTS: Aged C57BL/6 mice were associated with a more severe level of hair cell loss, while exhibiting a higher ABR threshold at various frequencies as well as a lower percentage of inner/outer hair cells. A reduced mitochondrial membrane potential in the cochleae of aged C57BL/6 mice indicated the presence of mitochondrial dysfunction in these mice. Relative expression of MIAT, Sirtuin1 (SIRT1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) was downregulated in aged mice, with microRNA-29b (miR-29b) being highly expressed. Also, MIAT binds to miR-29b, an inhibitor of SIRT1 expression. The regulatory relationship among MIAT, miR-29b, and SIRT1 was further validated by comparing the differentiated expression of these factors in cells treated with phosphate-buffered saline + H2 O2, a negative control + H2 O2, MIAT + H2 O2 , or H2 O2 + anti-miR-29b. CONCLUSION: MIAT could elevate the expression of SIRT1/PGC-1α via downregulating miR-29b. And the downregulated SIRT/PGC-1α increased the incidence of AHL via promoting the apoptosis of cochlear hair cells.

Mutation screening of Chinese Treacher Collins syndrome patients identified novel TCOF1 mutations.

Treacher Collins syndrome (TCS) (OMIM 154500) is a rare congenital craniofacial disorder with an autosomal dominant manner of inheritance in most cases. To date, three pathogenic genes (TCOF1, POLR1D and POLR1C) have been identified. In this study, we conducted mutational analysis on Chinese TCS patients to reveal a mutational spectrum of known causative genes and show phenotype-genotype data to provide more information for gene counselling and future studies on the pathogenesis of TCS. Twenty-two TCS patients were recruited from two tertiary referral centres, and Sanger sequencing for the coding exons and exon-intron boundaries of TCOF1, POLR1D and POLR1C was performed. For patients without small variants, further copy number variations (CNVs) analysis was conducted using high-density SNP array platforms. The Sanger sequencing overall mutation detection rate was as high as 86.3% (19/22) for our cohort. Fifteen TCOF1 pathogenic variants, including ten novel mutations, were identified in nineteen patients. No causative mutations in POLR1D and POLR1C genes and no CNVs mutations were detected. A suspected autosomal dominant inheritance case that implies germinal mosaicism was described. Our study confirmed that TCOF1 was the main disease-causing gene for the Chinese TCS population and revealed its mutation spectrum. We also addressed the need for more studies of mosaicism in TCS cases, which could explain the mechanism of autosomal dominant inheritance in TCS cases and benefit the prevention of TCS.

Mutational Analysis of TCOF1, GSC, and HOXA2 in Patients With Treacher Collins Syndrome.

Treacher Collins syndrome is an autosomal dominant craniofacial malformation mainly caused by mutations in the TCOF1 gene. Few cases have been observed in the Chinese population. Herein, the authors report the mutational analysis of TCOF1, GSC, and HOXA2 to determine the mutational features of the 3 genes in Chinese patients with Treacher Collins syndrome. Genomic DNA of the patients and their parents was extracted from peripheral blood following a standard protocol. DNA sequencing analysis was performed on all exons and the exon-intron borders of TCOF1, GSC, and HOXA2 in addition to the 1200-bp upstream of TCOF1. Four novel single nucleotide polymorphisms were detected in TCOF1, one of which was in the promoter region. Mutations in GSC and HOXA2 were not found in the 3 patients. Our results suggest the possibility of genetic heterogeneity or different mechanisms leading to the disease. Further functional study of the alteration is necessary to obtain more definitive information.

Mitochondrial Regulation of Macrophages in Innate Immunity and Diverse Roles of Macrophages During Cochlear Inflammation.

Macrophages are essential components of the innate immune system and constitute a non-specific first line of host defense against pathogens and inflammation. Mitochondria regulate macrophage activation and innate immune responses in various inflammatory diseases, including cochlear inflammation. The distribution, number, and morphological characteristics of cochlear macrophages change significantly across different inner ear regions under various pathological conditions, including noise exposure, ototoxicity, and age-related degeneration. However, the exact mechanism underlying the role of mitochondria in macrophages in auditory function remains unclear. Here, we summarize the major factors and mitochondrial signaling pathways (e.g., metabolism, mitochondrial reactive oxygen species, mitochondrial DNA, and the inflammasome) that influence macrophage activation in the innate immune response. In particular, we focus on the properties of cochlear macrophages, activated signaling pathways, and the secretion of inflammatory cytokines after acoustic injury. We hope this review will provide new perspectives and a basis for future research on cochlear inflammation.

Circular RNA ciRS-7 promotes laryngeal squamous cell carcinoma development by inducing TGM3 hypermethylation via miR-432-5p/DNMT3B axis.

OBJECTIVE: This work is to explore the mechanism by which circular RNA ciRS-7 affects laryngeal squamous cell carcinoma (LSCC). METHODS: ciRS-7 expression in LSCC tissues was detected by qRT-PCR, and the association between ciRS-7 with clinicopathological features of LSCC patients was evaluated. HN-4 and UM-SCC-10A cells were transfected or cotransfected with si-ciRS-7, miR-432-5p inhibitor, LV-DNMT3B or si-TGM3. Then, the viability and aggressive nature of the cells were tested. The binding site between ciRS-7 and miR-432-5p or between miR-432-5p and DNMT3B was predicted and the targeting relationship was identified. The specific binding between ciRS-7 and miR-432-5p was further verified by AGO2 RIP assay. HN-4 cells transfected with si-ciRS-7 was injected into nude mice to induce xenograft tumors. RESULTS: Higher ciRS-7 expression in LSCC tissues was closely associated with higher clinical stage, and exacerbated infiltration and lymph node metastasis in LSCC patients. Silencing ciRS-7 inhibited LSCC cell viability, epithelial-mesenchymal transition (EMT), and promoted the apoptosis. When miR-432-5p was inhibited or DNMT3B was overexpressed, the growth and EMT of LSCC cells were stimulated despite ciRS-7 silencing. Downregulation of ciRS-7 restrained the growth of xenograft tumors in vivo. CONCLUSION: ciRS-7 promotes the progression of LSCC through increasing TGM3 methylation via miR-432-5p/DNMT3B axis.

Clinical analysis based on 208 patients with microtia (especially reviewed oculo-auriculo-vertebral spectrum, hearing test, CT scan).

Microtia is a common birth defect and characteristic of abnormal auricle. It can be isolated or occur as a part of syndromes involving the first and second bronchial arch structures, such as oculo-auriculo-vertebral spectrum. We conducted a careful review of the literature regarding the clinical features of patients with microtia, but found few studies with respect to the Chinese population. In this study, we explored the clinical features of a single clinic population of 208 Chinese individuals with microtia. It showed that 15 cases (7.2%) had been afflicted with middle ear cholesteatoma, which would have brought about risky complications without an immediate removal; that 12 of 68 contralateral, normal-appearing ears had presented mild to moderate conductive or combined hearing loss (21-70 dB); that the degree of hearing loss deteriorated as the grade of microtia increased, with significant differences between grades I and III (p < 0.05); and that there was a male predominance, with the right side more likely to be affected.

LncRNA CCAT1 modulates the sensitivity of paclitaxel in nasopharynx cancers cells via miR-181a/CPEB2 axis.

Recent studies reported that long non-coding RNA (lncRNA) might play critical roles in regulating chemo-resistant of multiple types of cancer. This study aimed to investigate whether long non-coding RNA CCAT1 was involved in Paclitaxel resistance in nasopharyngeal carcinoma (NPC). qRT-PCR was used for testing the expression of CCAT1, miR-181a and CPEB2 in tumor tissues and NPC cancers. NPC cells were transfected with siRNAs to suppress the mRNA level of CCAT1 in NPC cells. MTT assays and flow cytometry analysis were used to assess the sensitivity of paclitaxel in NPC cells. Luciferase reporter assays were used to examine the interaction of CCAT1 or CPEB2 to miR-181a. Our findings revealed that the upregulated CCAT1 results in significantly enhancing paclitaxel resistance in nasopharyngeal cancer cells. Bioinformatics analysis and luciferase reporter assay indicated that the upregulated CCAT1 sponges miR-181a in NPC cells. Furthermore, RNA immuno-precipitation assays showed that miR-181a could directly bind to CCAT1 mRNA in NPC cells. We restored miR-181a in NPC cells, and found restoration of miR-181a re-sensitized the NPC cells to paclitaxel in vitro. In addition, our results also showed that miR-181a was a modulator of paclitaxel sensitivity due to its regulative effect on cell apoptosis via targeting CPEB2 in NPC cells. Taken together, lncRNA CCAT1 regulates the sensitivity of paclitaxel in NPC cells via miR-181a/CPEB2 axis.

Mutational analysis of GSC, HOXA2 and PRKRA in 106 Chinese patients with microtia.

OBJECTIVE: Microtia is defined as a developmental malformation characterized by a small, abnormal shaped auricle, with atresia or stenosis of the auditory canal. Genes responsible for nonsyndromic microtia have remained elusive. We therefore report a mutational analysis of GSC, HOXA2 and PRKRA in 106 congenital microtia patients without any combined malformation to explore the relationship between GSC, HOXA2, PRKRA and nonsyndromic microtia. METHODS: A total of 106 patients with a clinical diagnosis of congenital microtia and a control group (100 unaffected controls) were recruited through the Eye and ENT Hospital of Fudan University in China. Genomic DNA was extracted following a standard protocol. DNA sequencing analysis was performed in all exons and the exon-intron borders of GSC, HOXA2 and PRKRA. RESULTS: We identified 5 genomic variants in GSC, HOXA2 and PRKRA. As to the GSC, we obtained a reported variant g.994C > T in exon 2, which resulted in no change of protein. Our results revealed that g.994C > T was also detected in 10 control cases. We also detected 2 novel variants, g.90G > A and g.114A > C, in the 5'UTR of HOXA2. No class 5 or 4 genomic variant of PRKRA was identified in our microtia patients. Additionally, two previously reported SNVs in GSC and PRKRA were also presented. CONCLUSIONS: We suggest that g.994C > T is a new SNV, which is different from the previous report. Further study is needed to prove the function of 2 novel variants in the 5'UTR of HOXA2, and to explore the possible mechanism of these variants in the occurrence of microtia.

[Incidence of facial nerve dehiscence in mastoidectomy].

OBJECTIVE: To describe the incidence and location of the facial nerve dehiscence (FND) in chronic suppurative otitis media patients with and without cholesteatoma. METHODS: 360 patients (370 ears) who received canal wall down tympanomastoidectomy due to otitis media(145 ears without cholesteatoma and 225 ears with cholesteatoma) were analyzed retrospectively, in which the incidence and locations of FND was studied, and the relevance for FND, clinical features (age, disease duration, preoperative facial paralysis) and intraoperative findings (state of FND and lateral semicircular canal fistula), were analyzed. RESULTS: The presence of FND was 31.6% of total surgical procedures and the locations of FND were the tympanic segment. The dehiscence was detected 28.4% (94/334) in adults, but 61.1% (22/36) in the patients 18 years and younger, the differences were statistical significance (P < 0.05). The dehiscence rate was 37.1% (104/280) and 14.4% (13/90) respectively, in the cases of disease duration more than and less than 5 years, with significant difference (P < 0.05). Facial nerve dehiscence was detected in 29 patients (20.0%) and 89(39.1%) in cases without and with cholesteatoma respectively (P < 0.05). Facial nerve prolapse over the oval window was 11.4% (42/370), with FND of 83.3% (35/42). The incidence of lateral semicircular canal fistula was 7.8% (29/370), with FND of 65.5% (19/29). The presence of preoperative facial paralysis with FND was 75.0% (18/24), and that without FND was 28.6% (99/346), the differences were statistical significance (P < 0.05). CONCLUSIONS: The incidence of FND most commonly located at the tympanic segment. The facial nerves should be taken much care in mastoidectomy for patients with cholesteatoma, preoperative facial paralysis and lateral semicircular canal fistula, as well as long disease duration.

Phenotypic characterization and risk factors for microtia in East China, a case-control study.

OBJECTIVE: Although congenital microtia has been reported in various studies, little is known about the etiology of isolated and sporadic cases. The aim was to analyze potential risk factors for isolated and sporadic microtia using case-control study in East China. METHODS: The study analyzed data from the hospital-based recruitment for deliveries between 2007 and 2013. Nine hundred eleven patients with microtia enrolled in the phenotypic characterization analysis, and then were adjusted by sex, age, region, syndrome and family history to compare with 562 random normal controls for potential risk factors. RESULTS: Microtia is observed more often in males (69.7%), and the cases were typically unilateral (74.0%), right-sided (57.2%), sporadic (92.0%) and isolated (69.5%). Mothers of children with microtia were more likely to have suffered a periconceptional cold-like syndrome as well as to have had a history of previous spontaneous abortion. Inflammatory infection (aOR, 3.56; 95% CI, 2.07-6.13) and chemical exposure (aOR, 2.77; 95% CI, 1.78-4.32) was associated with a higher risk of microtia. However, threatened abortion was not the risk factor (aOR, 1.14; 95% CI, 0.78-1.67), using progesterone may increase the risk (aOR, 1.92; 95% CI, 1.03-3.59). CONCLUSION: The results of phenotypic characterization analysis were similar to other studies. By controlling the effects of potential confounders, some risk factors could be teratogens of isolated and sporadic microtia in East China.

MicroRNA expression profiling and target genes study in congenital microtia.

OBJECTIVE: Microtia is a complicated congenital anomaly with a genetic and environmental predisposition, and the molecular events underlying this disease are not fully understood. MicroRNAs (miRNAs) are a class of 20-22 nucleotide non-coding RNAs that function to control post-transcriptional gene expression. We want to find the miRNA expression profiling of microtia by using Affymetrix GeneChip(®) miRNA 2.0 Arrays. METHODS: We selected 9 microtia cartilages and 3 normal controls for GeneChip(®) miRNA 2.0 Arrays analysis. The altered miRNA were analyzed by poly (A) RT-PCR from 58 microtia samples and 16 normal controls. We predicted the target genes of miRNAs by bioinformatics and RT-PCT was used to confirm the target genes. RESULTS: We found 11 miRNAs with significantly altered expression in the microtic group compared to the normal controls, which included 6 up-regulated miRNAs and 5 down-regulated miRNAs. These miRNAs were further examined using poly (A) RT-PCR analysis, we found that miR-451 and miR-486-5p were significantly up-regulated and miR-200c was significantly down-regulated in the microtic group compared to the normal controls (p<0.05). Several complementary target messenger RNAs (mRNAs) had been predicted. OSR1, the target gene of miR-451 and miR-200c, was significantly up-regulated (p<0.01); TRPS1, the target gene of miR-200c, was significantly down-regulated in the microtic group compared to the controls (p<0.0001). CONCLUSION: The reduction in miR-200c expression and the accretion of miR-451 and miR-486-5p expression in microtic samples could be possible causes of the abnormal development of the external ear. OSR1 and TRPS1, as the complementary target mRNAs, may play important roles during the development of the external ear. Further studies are still needed to identify the miRNA target genes and to determine their function in the pathogenesis of microtia. This is the first report of a relationship between miRNAs and microtia.

[Analysis of correlation between congenital auricular deformities and middle ear malformations].

OBJECTIVE: To explore the potential value of knowing the relationship between congenital auricular deformities and middle ear malformations. METHODS: A total of 86 patients with congenital auricular deformities and middle ear malformations, including 51 males and 35 females, were admitted from January 2008 to December 2009 to the Eye Ear Nose and Throat Hospital of Fudan University. Fifty-eight patients had unilateral deformities (R:L = 34:24), while 28 were bilateral. One hundred and fourteen ears with congenital auricular deformities were included. High-resolution CT (HRCT) data was obtained from each patient. The auricular deformities were classified into three grades using the Marx H classification system. The modified Jahrsdoerfer grading system was used to score the malformations using HRCT data. The correlation between the grades of auricular deformities and scores of middle ear malformations was analyzed using Spearman rank correlation analysis. RESULTS: The Marx H grades of congenital auricular deformities were 12 patients with grade I, 25 patients with grade II and 77 patients with grade III, while their corresponding Jahrsdoerfer scores were 7.8 ± 2.4, 6.8 ± 2.6 and 6.0 ± 2.8, respectively. The statistical analysis suggested a trend of negative correlation between the Marx H grades of auricular deformities and the Jahrsdoerfer scores of middle ear malformations (r = -0.2386, P = 0.0106). CONCLUSION: There was a trend to a negative correlation between congenital auricular deformities and middle ear malformations.