METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression.

BACKGROUND: N6-methyladenosine (m6A) RNA methylation and its methyltransferase METTL3 have been widely reported to be involved in different cancers by regulating RNA metabolism and function. Here, we aimed to explore the biological function and clinical significance of m6A modification and METTL3 in head and neck squamous cell carcinoma (HNSCC). METHODS: The prognostic value of METTL3 expression was evaluated using tissue microarray and immunohistochemical staining analyses in a human HNSCC cohort. The biological role and mechanism of METTL3 in HNSCC tumour growth, metastasis and angiogenesis were determined in vitro and in vivo. RESULTS: M6A levels and METTL3 expressions in HNSCC tissues were significantly increased compared with paired adjacent tissues. Meanwhile, METTL3 was an independent risk factor for the prognosis of HNSCC patients. Moreover, METTL3 overexpression promoted HNSCC cell proliferation, migration, invasion, and angiogenesis, while knockdown of METTL3 had an opposite effect in vivo and in vitro. Mechanistically, METTL3 enhanced the m6A modification of CDC25B mRNA, which maintained its stability and upregulated its expression, thereby activating G2/M phase of cell cycle and leading to HNSCC malignant progression. CONCLUSIONS: METTL3 may be a potential prognostic biomarker and therapeutic target for HNSCC.

The correlation between FeNO and nNO in allergic rhinitis and bronchial asthma.

ABSTRACT: This study aimed to evaluate the correlation between fractional exhaled nitric oxide (FeNO) and nasal nitric oxide (nNO) in allergic rhinitis (AR) and patients with or without bronchial asthma (BA).A total of 90 patients who were diagnosed with persistent AR (AR group, n = 30), BA (BA group, n = 30), or allergic rhinitis with bronchial asthma (AR-BA) (AR-BA group, n = 30), were enrolled in this study, along with 30 healthy adult volunteers (control group, n = 30). The participants were further divided into 2 groups based on the results of a skin-prick test (SPT): a highly atopic group (SPT = 3+ and above) and a moderately atopic group (SPT = 2+ and below). All participants underwent FeNO and nNO measurement, an absolute blood eosinophil count, total serum immunoglobulin measurement, and horizontal baseline lung capacity determination.The results showed that the FeNO levels in the 3 observation groups were significantly higher than those in the control group (P < .01), and in the BA group they were significantly higher than in the AR-BA group (P < .01). The levels of nNO in both the AR group and the AR-BA group were higher than those in the control group and the BA group (P < .01), but there was no significant difference between the AR group and the AR-BA group (P > .05). The levels of nNO in the BA group were also significantly different from those in the control group (P < .01).FeNO and nNO are positively correlated with the degree of AR in patients with BA; therefore, nNO levels can be used as an inflammatory marker of AR in patients with BA. FeNO can also be used as an inflammatory marker of AR in patients complicated with BA as a warning indicator of asthma.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

Prodigiosin Inhibits Proliferation, Migration, and Invasion of Nasopharyngeal Cancer Cells.

BACKGROUND/AIMS: Nasopharyngeal carcinoma remains a devastating and difficult disease to treat. This study explores the antineoplastic effect of prodigiosin on nasopharyngeal cancer cells. METHODS: Human nasopharyngeal carcinoma CNE2 cells and human normal nasopharyngeal epithelial NP69 cells were obtained and treated with prodigiosin or fluorouracil (5-FU). Colony formation assay was performed to screen for the optimal experimental concentrations of prodigiosin and 5-FU, and MTT assay was used to examine cell proliferative ability. Flow cytometry was used to examine cell cycle distribution, the scratch test was employed to examine cell migration, and Transwell migration assay (Boyden chamber) was used to study cell invasion. RESULTS: The optimal concentrations of prodigiosin and 5-FU for treatment were 4 mg/L and 0.35 mg/L, respectively. Both prodigiosin and 5-FU inhibited tumor cell proliferation. The percentage of cells in G0/G1 phase was higher and the percentage of cells in S phase was lower in the prodigiosin and 5-FU groups than in the untreated groups. Both prodigiosin and 5-FU inhibited tumor cell migration and tumor cell invasion. CONCLUSIONS: Our results suggest that prodigiosin can inhibit proliferation, migration, and invasion of nasopharyngeal carcinoma cells.

Role of epithelial-mesenchymal transition markers E-cadherin, N-cadherin, ß-catenin and ZEB2 in laryngeal squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) allows neoplastic cells to gain the invasive phenotype and become migratory, which is required for cancer progression and metastasis. In the present study, the expression of EMT-associated biomarkers and their association with clinicopathological parameters in laryngeal squamous cell carcinoma (LSCC) was investigated. E-cadherin, N-cadherin, ß-catenin and zinc finger E-box binding homeobox 2 (ZEB2) protein expression was evaluated with immunohistochemistry in a cohort of 76 patients with operable LSCC. The association between these transition markers, clinicopathological parameters and their prognostic impact in LSCC was analyzed. Immunohistochemical analysis revealed that EMT-associated proteins were differentially expressed between LSCC and adjacent non-neoplastic laryngeal tissue. Negative E-cadherin expression and positive N-cadherin, ß-catenin and ZEB2 expression were associated with a later tumor (T) stage, decreasing tumor differentiation and a reduced overall survival (OS) time (OS: E-cadherin, P=0.016; N-cadherin, P=0.003; ß-catenin, P=0.002; ZEB2, P=0.0003). E-cadherin/ß-catenin co-expression was significantly associated with the majority of clinicopathological parameters assessed, including lymph node metastases, T stage and tumor cell differentiation (P=0.004, P=0.005, and P<0.001, respectively). Multivariate analysis indicated that T stage and the positive expression of ß-catenin and ZEB2 were independent risk factors for OS in LSCC (P=0.014, P=0.025 and P=0.003, respectively). It was concluded that EMT mediates tumor progression, and reduces OS time in patients with LSCC. E-cadherin/ß-catenin co-expression may be associated with clinicopathological parameters. T stage, and the positive co-expression of ß-catenin and ZEB2 may be independent predictors of prognosis in LSCC.

Experimental study of local inner ear gene therapy for controlling autoimmune sensorineural hearing loss.

This study aimed to investigate the efficacy of gene therapy for treating autoimmune sensorineural hearing loss (ASHL) via local administration of a recombinant adenovirus vector containing the Fas ligand or interleukin IL-10 gene. Guinea pigs were divided into four groups, with different microinjections in the scala tympani. Group A were injected with FasL-EGFP, B with IL-10-EGFP, C with EGFP, and D with artificial perilymph. Seven days later, auditory brain-stem response (ABR) was tested, and the temporal bone was stained and observed by light microscopy. The spiral ligament and basement membrane were observed using transmission electron microscopy. FasL and IL-10 expression were examined using immunofluorescence histochemistry. Immunohistochemical analysis showed that the recombinant adenovirus vector in Groups A, B, and C can transfect the stria vascularis, the spiral ligament, the organ of Corti, the spiral ganglion, the region surrounding the small blood vessel in the modiolus, and the cochlear bone wall. Compared with those in Groups C and D, the ABR wave III mean thresholds were significantly lower and the inner ear immunoinflammatory responses in Groups A and B were significantly alleviated. Inhibition of immunoinflammatory response alleviated immunoinflammatory injury and auditory dysfunction. This technique shows potential as a novel therapy for ASHL.

Inhibition of Myo6 gene expression by co­expression of a mutant of transcription factor POU4F3 (BRN­3C) in hair cells.

An eight­base pair (bp) deletion in the Pou4f3 gene in hair cells is associated with DFNA15, a hereditary form of hearing loss. To explore the pathological mechanisms underlying the development of DFNA15, the effect of the mutation in Pou4f3 on the activity of the myosin VI (Myo6) promoter, was investigated. The upstream regulatory sequence of Myo6 (2625 bp), consisting of an 1899 bp upstream sequence and a 727 bp intron 1 sequence, was amplified using polymerase chain reaction and subcloned into the pGL3­Basic vector expressing firefly luciferase. For verification of inserted fragments, plasmids were subjected to restriction analysis and then sequenced. HEK293T human embryonic kidney cells were transiently transfected with renilla luciferase­thymidine kinase vectors expressing Renilla luciferase and the Myo6 promoter­driven firefly luciferase expressing vectors along with pIRES2­enhanced green fluorescent protein (EGFP)­Pou4f3 (expressing wild­type Pou4f3) or pIRES2­EGFP­Pou4f3 (expressing the truncation mutant of Pou4f3). The relative luciferase activities were measured to determine the activity of the Myo6 promoter. The Myo6 promoter activity was not affected by co­expression of wild­type Pou4f3, as indicated by the comparable relative luciferase activities in the presence of the pIRES2­EGFP­Pou4f3 and the empty control vectors. However, co­expression of mutated Pou4f3 significantly inhibited the activity of the Myo6 promoter to almost half of that of the control (P<0.001). The data suggests that mutated Pou4f3 has a negative role in the promoter activity of Myo6, and by extension, the expression of myosin VI, and this may be an underlying mechanism of DFNA15 hearing loss.

Myosin light chain kinase regulates hearing in mice by influencing the F-actin cytoskeleton of outer hair cells and cochleae.

Myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chains to facilitate its interaction with actin filaments and produce contractile activity. The outer hair cells (OHCs) in the ear contain large amounts of actin and a variety myosins. The stereociliary and somatic motility of OHCs are closely related to hearing. It appears likely that MLCK may play an important role in acoustic trans-duction. In this study, we analyzed, both in vivo and in vitro, the OHCs of mice bearing a specific deletion of the MLCK gene and the OHCs of control mice. The phenotype was assessed by auditory function [acoustic brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs)], inner ear morphology and histology. MLCK-deficient mice aged 6-7 months showed impaired hearing, a 5- to 10-dB sound pressure level (SPL) increase in the ABR thresholds, when responding to clicks and tones of different frequencies (8 and 16 kHz) (P<0.05). The DPOAE amplitudes of 3-month-old MLCK-deficient mice decreased significantly (>10 dB SPL) at low frequencies (4, 5 and 6 kHz). The OHCs in the MLCK-deficient mice increased with abnormal stereocilia. The staining of F-actin and the phosphorylation of the regulatory light chain in MLCK-deficient OHCs was weak. Our results indicate that MLCK may regulate the structure and the motility of stereocilia through F-actin polymerization.

[Study on conditional myosin light chain kinase gene knockout mice resulting in hearing loss].

OBJECTIVE: To investigate the function of myosin light chain kinase (MLCK) in hearing in mouse by generating inner hair cell-specific Mlck knockout mice and analyze the effect on their hearing. METHODS: Cross Mlck floxed mice with IHC-Cre mice, the genotype and knockout efficiency were confirmed by PCR. We used auditory brain stem response (ABR) to evaluate mice hearing function at different frequencies. RESULTS: Mlck knockout mice were selected by mice tail DNA genotyping and confirmed the deletion of the target gene by isolated inner hair cell DNA genotyping. Mlck-deficient mice showed impaired hearing with a rise in ABR threshold response to click and three different pure tones (8 kHz, 16 kHz, 32 kHz), and the rise was over 20 dB at high-frequency(32 kHz). Further analyses of waveforms showed that wave-I amplitudes on 60 dB SPL, 50 dB SPL and 40 dBSPL in response to tone (16 kHz) were less than control group(P < 0.05) on average, but the ratio of wave I/II and I/III were not difference (P > 0.05). CONCLUSIONS: Mlck is successfully deleted in inner hair cell-specific Mlck knockout mice. Mlck knockout mice display a significantly higher threshold in response to click and tones, especially in high-frequencies.

[Expression of E-cadherin and uPA and their prognostic value in carcinoma of human larynx.].

OBJECTIVE: To investigate the expression of E-cadherin (ECD) and uPA in laryngeal cancer and evaluate their clinical value in prognosis. METHODS: ECD and uPA were determined by immunohistochemistry of Envision methods in carcinoma tissues of 51 patients of laryngeal carcinoma. All patients were followed and the prognostic factors were analyzed. RESULTS: Among 51 patients' tumor tissues, 24 (47.1%) were negative expression of ECD, and 26 (51.0%) were positive uPA immunoreaction was observed. There were four subgroups of patterns of ECD and uPA expression: 14 (27.1%) ECD-positive/uPA-negative, 13 (25.5%) ECD-negative/uPA-negative, 11 (21.6%) ECD-positive/uPA-positive, and 13 (25.5%) ECD-negative/uPA-positive. The tumor tissues with ECD-negative and uPA-positive expression were significant associated with lymph nodes metastasis (chi(2) = 5.545, 5.79, P = 0.019, 0.016 respectively). Patients with ECD-negative expression had a shorter survival than the patients with ECD positive expression but no statistic difference (chi(2) = 2.534, P > 0.05). Patients with uPA-positive expression had a significantly shorter survival time than those with uPA-negative expression (chi(2) = 6.259, P < 0.05). There was a difference for the median survival time between the patients with uPA-negative/ECD-positive and the patients with uPA-positive/ECD-negative in laryngeal cancer tissue (chi(2) = 6.559, P = 0.01), and the survival curves between these two groups was also statistically significant difference. Multivariate analysis of Cox revealed that clinical stage and ECD/uPA (P = 0.009, 0.007 respectively) were two independent prognostic factors. CONCLUSIONS: The combination analysis of uPA and ECD immunohistochemical expression in the laryngeal cancer tissue may be useful for predicting tumor metastatic risks and patient's prognosis.