A Comprehensive Pan-Cancer Analysis of the Potential Biological Functions and Prognosis Values of RICTOR.

The importance of the network defined by phosphatidylinositol-3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) downstream of Receptor Tyrosine Kinase (RTK) has been recognized for many years. However, the central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently come to light. The function of RICTOR in pan-cancer still needs to be systematically elucidated. In this study, we examined RICTOR's molecular characteristics and clinical prognostic value by pan-cancer analysis. Our findings indicate that RICTOR was overexpressed in twelve cancer types, and a high RICTOR expression was linked to poor overall survival. Moreover, the CRISPR Achilles' knockout analysis revealed that RICTOR was a critical gene for the survival of many tumor cells. Function analysis revealed that RICTOR-related genes were mainly involved in TOR signaling and cell growth. We further demonstrated that the RICTOR expression was significantly influenced by genetic alteration and DNA-methylation in multiple cancer types. Additionally, we found a positive relationship between RICTOR expression and the immune infiltration of macrophages and cancer-associated fibroblasts in Colon adenocarcinoma and Head and Neck squamous cell carcinoma. Finally, we validated the ability of RICTOR in sustaining tumor growth and invasion in the Hela cell line using cell-cycle analysis, the cell proliferation assay, and wound-healing assay. Our pan-cancer analysis highlights the critical role of RICTOR in tumor progression and its potential as a prognostic marker for various cancer types.

Carboxypeptidase A6 suppresses the proliferation and invasion of colorectal cancer cells and is negatively regulated by miR-96-3p.

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumor, and this study aims to explore the role and the regulatory mechanism of carboxypeptidase A6 (CPA6) in CRC cells. METHODS: Specific shRNA targeting CPA6 mRNA was transfected into NCM460 and HT29 cells to down-regulate CPA expression, and expression plasmid was transfected into HCT116 cells to exogenously overexpress CPA6. The dual luciferase assay was used to detect the direct binding of miR-96-3p to CPA6 3'UTR. Phosphorylation and activation of Akt were detected using Western blot. Cells were treated with miR-96-3p mimics, Akt inhibitor (MK-2206) or agonist (SC79) for rescue experiments. The cell functions were evaluated using CCK-8, clone formation, transwell, and Western blot assays. Xenograft tumor assay was also used to analyze the effect of altered CPA6 expression on tumor growth. RESULTS: Knockdown of CPA6 promoted the proliferation, clone formation, migration, and invasion of NCM460 and HT29 cells in vitro, and the tumor growth of nude mouse xenograft tumor in vivo. Moreover, over-expression of CPA6 significantly inhibited the malignant proliferation and invasion of HCT116 cells in vitro, and the tumor growth of xenograft tumor in vivo. Furthermore, miR-96-3p could directly regulate CPA6 expression by targeting its 3'UTR, and miR-96-3p mimics rescued the inhibitory effects of CPA6 overexpression on the malignant proliferation and invasion of CRC cells. Finally, CPA6 knockdown enhanced Akt/mTOR phosphorylation and activation, while CPA6 overexpression inhibited Akt/mTOR activation. The regulatory effect of CPA6 on Akt/mTOR signaling was naturally regulated by miR-96-3p. Akt inhibitor or agonist rescued the effects of CPA6 knockdown or overexpression on proliferation and EMT of colon cancer cells. CONCLUSION: CPA6 has a significant tumor suppressive effect on CRC by inhibiting the activation of Akt/mTOR signaling, and miR-96-3p negatively regulates the expression of CPA6.

Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-Inflammatory Secretome Changes Induced by Oxidative Stress.

OBJECTIVES: Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sensorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely unknown, and no feasible in vitro cell culture model exists for the functional study of SV-ECs. METHODS: We isolated primary SV-ECs from the SV of neonatal mice. The apoptosis-reducing effects of fibronectin in SV-ECs cultured with serum-free medium were determined using ß-galactosidase staining and flow cytometry. SV-ECs incubated in serum-free medium were treated with various H2O2 concentrations to evaluate the effects of H2O2 on their viability. The secretome of SV-ECs treated with or without H2O2 (100 µM or 500 µM) was analyzed using high-resolution mass spectrometry. The function of the SV-EC secretome was evaluated by a macrophage assay. RESULTS: We successfully isolated and characterized the SV-ECs. Treatment with H2O2 at concentrations up to 500 µM for 2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significant effect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECs stimulated with 500 µM H2O2 was much higher than in those treated with 100 µM H2O2. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECs treated with 500 µM H2O2 were involved in the regulation of multiple signaling pathways and cellular processes. The secretome of H2O2-stimulated SV-ECs exhibited significant pro-inflammatory effects on macrophages. CONCLUSION: We successfully established an in vitro serum-free culture method, identified the differential proteins released by oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome of H2O2-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microenvironment of oxidative stress-induced cochlea, especially cochlear macrophages.

Knockdown of LRCH4 Remodels Tumor Microenvironment Through Inhibiting YAP and TGF-ß/Smad Signaling Pathway in Colorectal Cancer

BACKGROUND: Colorectal cancer is one of the most common gastrointestinal malignancies worldwide. LRCH4 is the top 1 gene associated with an unfavorable prognosis in colorectal cancer. METHODS: Here, we reported that the knockdown of LRCH4 inhibited the proliferation, migration and invasion in HT29 cells RESULTS: The activity of Yes-Associated Protein (YAP), a transcription factor in the Hppo-YAP signaling pathway, was significantly inhibited by LRCH4-siRNA. LRCH4 knockdown also reversed the EMT and regulated the expression of extracellular matrix (ECM) protein, Fibronectin and Collagen IV in HT29 cells. In addition, the TGF-ß/Smad signaling pathway, as the downstream pathway of Yap, was also inhibited by LRCH4 knockdown. CONCLUSION: Knockdown of LRCH4 involved in the regulation of ECM and EMT and inhibited YAP and the TGF-ß/Smad signaling pathway in colorectal cancer cells. Our study provided a mechanism of LRCH4 on colorectal cancer cells, and a new potential target for clinical tumor treatment.

Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKß.

BACKGROUND: The Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) are serine/threonine-directed protein kinases that are activated following increases in intracellular calcium, playing a critical role in neuronal signaling. Inner-ear-trauma-induced calcium overload in sensory hair cells has been well documented in the pathogenesis of traumatic noise-induced hair cell death and hearing loss, but there are no established pharmaceutical therapies available due to a lack of specific therapeutic targets. In this study, we investigated the activation of CaMKKß in the inner ear after traumatic noise exposure and assessed the prevention of noise-induced hearing loss (NIHL) with RNA silencing. RESULTS: Treatment with short hairpin RNA of CaMKKß (shCaMKKß) via adeno-associated virus transduction significantly knocked down CaMKKß expression in the inner ear. Knockdown of CaMKKß significantly attenuated noise-induced hair cell loss and hearing loss (NIHL). Additionally, pretreatment with naked CaMKKß small interfering RNA (siCaMKKß) attenuated noise-induced losses of inner hair cell synapses and OHCs and NIHL. Furthermore, traumatic noise exposure activates CaMKKß in OHCs as demonstrated by immunolabeling for p-CaMKI. CaMKKß mRNA assessed by fluorescence in-situ hybridization and immunolabeling for CaMKKß in OHCs also increased after the exposure. Finally, pretreatment with siCaMKKß diminished noise-induced activation of AMPKα in OHCs. CONCLUSIONS: These findings demonstrate that traumatic-noise-induced OHC loss and hearing loss occur primarily via activation of CaMKKß. Targeting CaMKKß is a key strategy for prevention of noise-induced hearing loss. Furthermore, our data suggest that noise-induced activation of AMPKα in OHCs occurs via the CaMKKß pathway.

Assessing Agreement between Frequency-Specific Chirp Auditory Steady-State Response and Pure Tone Audiometry in Adults by Intraclass Correlation Coefficient.

INTRODUCTION: Chirp auditory steady-state response (ASSR) can be used to assess frequency-specific hearing thresholds. However, its reliability has not been confirmed yet. The purpose of this proposed study is to analyze the agreement of thresholds measured by chirp-ASSR and pure tone audiometry (PTA) to investigate the value of chirp-ASSR in hearing threshold evaluation. METHODS: Participants with normal hearing (age: 18-66, 108 ears) and patients with sensorineural hearing loss (age: 22-82, 75 ears) were tested using PTA and chirp-ASSR at 0.5, 1, 2, and 4 kHz, respectively. Intraclass correlation coefficient (ICC) and Bland-Altman plot were introduced to analyze the agreement between the 2 methods. RESULTS: One-hundred eight participants underwent both chirp-ASSR and PTA to estimate their thresholds. The ICCs yielded by these 2 methods are 0.757, 0.893, 0.883, and 0.921 (p < 0.001) at 0.5, 1, 2, and 4 kHz carrier frequency, respectively. However, there is a significant difference between the 2 methods at 2 kHz: the mean value of the ASSR thresholds is 5.27 dB HL higher than the value of PTA thresholds. Additionally, the 95% limits of agreement range from -27.48 to 26.66 dB HL at 0.5 kHz, from -18.19 to 17.87 dB HL at 1 kHz, from -12.01 to 22.55 dB HL at 2 kHz, and from -21.29 to 19.17 dB HL at 4 kHz, which are large enough to affect clinical decision-making. CONCLUSION: In this study, we have confirmed good to excellent correlation between chirp-ASSR and PTA in threshold estimation for adults with and without hearing loss. The degree of correlations is higher for participants with hearing loss and for measurements at high frequencies. However, significant systematic difference and large limits of agreement between the 2 methods have been found. These findings show that chirp-ASSR can be treated as a supplementary method to PTA when evaluating the hearing level, but the 2 methods are not interchangeable due to their systematic difference and large limits of agreement.

Utilizing True Wireless Stereo Earbuds in Automated Pure-Tone Audiometry.

True wireless stereo (TWS) earbuds have become popular and widespread in recent years, and numerous automated pure-tone audiometer applications have been developed for portable devices. However, most of these applications require specifically designed earphones to which the public may not have access. Therefore, the present study investigates the accuracy of automated pure-tone audiometry based on TWS earbuds (Honor FlyPods). The procedure for developing an automated pure-tone audiometer is reported. Calibration of the TWS earbuds was accomplished by electroacoustic measurements and establishing corrected reference equivalent threshold sound pressure levels. The developed audiometer was then compared with a clinical audiometer using 20 hearing-impaired participants. The average signed and absolute deviations between hearing thresholds measured using the two audiometers were 3.1 dB and 6.7 dB, respectively. The overall accuracy rate in determining the presence/absence of hearing loss was 81%. The results show that the proposed procedure for an automated air-conduction audiometer based on TWS earbuds is feasible, and the system gives accurate hearing level estimation using the reported calibration framework.

Cisplatin-induced ototoxicity: Updates on molecular mechanisms and otoprotective strategies.

Cisplatin is a highly effective antitumor drug generally used in the treatment of solid malignant tumors. However, cisplatin causes severe side effects such as bone marrow depression, nephrotoxicity, and ototoxicity, thus limiting its clinical application. The incidence of ototoxicity induced by cisplatin ranges from 20% to 70%, and it usually manifests as a progressive, bilateral and irreversible hearing loss. Although the etiology of cisplatin-induced ototoxicity remains unclear, an increasing body of evidence suggests that the ototoxicity of cisplatin is mainly related to the production of reactive oxygen species and activation of apoptotic pathway in cochlear tissues. Many drugs have been well proved to protect cisplatin-induced hearing loss in vitro and in vivo. However, the anti-tumor effect of cisplatin is also weakened by systemic administration of those drugs for hearing protection, especially antioxidants. Therefore, establishing a local administration strategy contributes to the otoprotection without affecting the effect of cisplatin. This review introduces the pathology of ototoxicity caused by cisplatin, and focuses on recent developments in the mechanisms and protective strategies of cisplatin-induced ototoxicity.

Notched Sound Alleviates Tinnitus by Reorganization Emotional Center.

BACKGROUND: Tinnitus is a common disease, and sound therapy is an effective method to alleviate it. Previous studies have shown that notched sound not only changes levels of cortical blood oxygen, but affects blood oxygen in specific cerebral cortical areas, such as Brodmann area 46 (BA46), which is associated with emotion. Extensive evidence has confirmed that tinnitus is closely related to emotion. Whether notched sound plays a role in regulating the emotional center is still unclear. METHODS: This study included 29 patients with newly diagnosed chronic tinnitus who were treated with notched sound. Functional near-infrared spectroscopy (fNIRS) was conducted before and after treatment to observe bilateral changes in cortical blood oxygen in the cerebral hemispheres. We compared the changes in connectivity between the two regions of interest (the superior temporal gyrus and BA46), as wells as other cortical regions before and after treatment. RESULTS: The results showed (1) That global connectivity between the bilateral auditory cortex of the superior temporal sulcus and the ipsilateral cortex did not change significantly between baseline and the completion of treatment, and (2) That the connectivity between channel 14 and the right superior temporal sulcus decreased after treatment. The overall connectivity between the right BA46 region and the right cortex decreased after treatment, and decreases in connectivity after treatment were specifically found for channels 10 and 14 in the right parietal lobe and channels 16, 20, 21, and 22 in the frontal lobe, while there was no significant change on the left side. There were no significant changes in the questionnaire measures of tinnitus, anxiety, or depression before and after treatment. CONCLUSION: The results of the study indicate that cerebral cortex reorganization occurs in tinnitus patients after submitted to treatment with notched sound for 1 month, and that notched sound decreases the connectivity between the auditory cortex and specific brain regions. SIGNIFICANCE: Notched sound not only regulates the auditory center through lateral inhibition, but also alleviates tinnitus by reorganizing the emotional control center.

Meta-Analysis Comparing Steroids and Diuretics in the Treatment of Acute Low-Tone Sensorineural Hearing Loss.

OBJECTIVE: Our objective was to perform a meta-analysis to compare the effectiveness of steroids and diuretics in the treatment of acute low-tone sensorineural hearing loss (ALHL). METHODS: PubMed, Google Scholar, and Sci databases were searched for randomized controlled trials (RCTs) examining the treatment of ALHL with steroids and diuretics. The Cochrane Reviewer's Handbook 5.0 evaluation criteria were used to evaluate the quality of the included RCTs. Meta-analysis was performed using Revman 5.3 software to compare the recovery rate of low-tone hearing levels between patients treated with steroids and diuretics. RESULTS: A total of 3 RCTs were included. There was no heterogeneity between the 3 studies (χ2 = 2.61, P = .27, I2 = 23%); thus, a fixed-effects model of analysis was used. Meta-analysis showed there was no significant difference in the recovery rate of patients treated with steroids and those treated with diuretics (odds ratio = 1.48, 95% confidence interval: 0.64-3.40, P = .36). CONCLUSION: Steroids and diuretics are equally effective for the treatment of ALHL.

Combined application of single-energy metal artifact reduction and reconstruction techniques in patients with Cochlear implants.

BACKGROUND: The purpose of this study was to develop an effective method of reducing metal artifacts in cochlear implant (CI) electrodes. METHODS: The temporal bones of 30 patients (34 ears) after CI were examined with 320-detector row computed tomography, which was evaluated by two senior radiologists using a double-blind method. Noise, artifact index, signal-to-noise ratio, and the subjective image quality score were compared before versus after using single-energy metal artifact reduction (SEMAR). The electrode position, single electrode visibility, and electrode count were evaluated using SEMAR combined with either multi-planar reconstruction (MPR) or maximum intensity projection. RESULTS: The two radiologists' measurements had good consistency. SEMAR significantly reduced the image noise and artifacts index and significantly improved the signal-to-noise ratio and subjective image quality score (P < 0.01). The combination of SEMAR with MPR was conducive to accurate assessment of electrode position and single-electrode visibility. The combination of SEMAR with MIP facilitated accurate and intuitive matching of the assessed electrode count with the number of electrodes implanted during the operation (P = 0.062). CONCLUSION: SEMAR significantly reduces metal artifacts generated by CI electrodes and improves the quality of computed tomography images. The combination of SEMAR with MPR and maximum intensity projection is beneficial for evaluating the position and number of CI electrodes.

Chinese validation and clinical application of the tinnitus functional index.

OBJECTIVE: The Tinnitus Functional Index (TFI) is a new diagnostic measure of the functional impact of tinnitus that is also a sensitive measure of treatment-related changes. However, the TFI has not been translated into Chinese and fully validated in China. The aim of the present study was to evaluate the validity of a Chinese version of the TFI as a diagnostic measure of tinnitus severity in a sample of Chinese patients and to verify the value of its clinical application in China. DESIGN: A sample of 206 patients whose primary complaint was tinnitus was used to analyze the reliability and validity of the TFI. In addition, patients were asked to fill out the Tinnitus Handicap Inventory (THI) and the Center for Epidemiologic Studies-Depression Scale (CES-D), the Beck Anxiety Inventory (BAI), and the Satisfaction With Life Scale (SWLS) to compare TFI with their association. The internal consistency of the TFI was assessed with Cronbach's alpha coefficient. The factor structure of the TFI was assessed by Exploratory Factor Analysis (EFA). The extracted factors were compared to those of the original TFI scale. RESULTS: The reliability of the Chinese version of the TFI (Cronbach' s α = .969) showed high internal consistency. The exploratory factor analysis (EFA) of the TFI showed that six factors with one main factor could be extracted instead of eight factors as described in the original version. Nevertheless, relations to the original eight subscales could be demonstrated. A high correlation between the TFI and the THI (r = .865, p < 0.01) and lower correlations between the TFI and the CES-D (r = .334, p < 0.01), BAI (r = .559, p < 0.01), and SWLS (r = - 0.324, p < 0.01) confirmed the satisfactory convergent and discriminant validity of the TFI. CONCLUSION: After translated and validated a Chinese version of the TFI and found that the TFI had high reliability and validity, which means both instruments are reliable instruments to assess the severity of tinnitus in clinical applications in China.

Cortical Activation Patterns of Different Masking Noises and Correlation With Their Masking Efficacy, Determined by Functional Near-Infrared Spectroscopy.

Acoustic therapy in tinnitus treatment is poorly characterized, and efficacy assessment depends on subjective descriptions. Narrow-band noise, notched sound, and white noise have positive therapeutic effects on monotonous tinnitus. Considering the tonotopic characteristics of the auditory system and the spectral characteristics of these three masking sounds, the activation pattern of the auditory cortex and the mechanism of inhibiting tinnitus may be different. This study aimed to compare the activation patterns of three spectrally different masking noises and study the correlation between the masking effects and variational amplitude of oxygenated hemoglobin (HbO) in the corresponding cortical regions. We also assessed near-infrared spectroscopy brain function imaging (NIRS) as an objective assessment tool in acoustic therapy. Patients with persistent non-pulsatile tinnitus and control volunteers without tinnitus were enrolled in this study. The subjects were seated in a sound-proof room, with two optode arrays covering the bilateral temporal lobe. Auditory stimuli were presented; stimulation sequences followed the block design: different noises appeared randomly and repeated in five cycles. Tinnitus match and residual inhibition were performed in the tinnitus group. The data analyses were conducted using the NIRS_SPM toolbox. The group analysis results showed that the narrow-band noise caused a marginally significant decrease in HbO signal in the Brodmann 21 region (BA21), while white noise caused a significant increase in HbO signal in BA21. Notched sound did not cause significant changes in the HbO signal in the temporal cortex. And none of the three masking noises caused significant changes in the HbR signal in the temporal cortex. The depth of residual inhibition induced by the narrow-band noise and white noise significantly correlated with ΔHbO in the region of interest (ROI). However, neither the depth nor duration of the residual inhibition induced by notched sound correlated with the ΔHbO. Thus, NIRS showed three cortical activation patterns induced by three different masking noises, and correlations between residual inhibition effects and change of HbO amplitude were found. NIRS could therefore be applied in objective assessment of acoustic therapy.

Electrophysiologic Characteristics of Various Degrees of Selective Outer Hair Cell Loss in Rats.

HYPOTHESIS: The characteristics of auditory brainstem response (ABR), electrocochleogram (ECochG), and distortion product otoacoustic emissions (DPOAE) of different degrees of selective outer hair cells (OHCs) loss may be helpful for clinicians to evaluate the pathogeny, diagnosis, and rehabilitation of individuals' hearing loss. BACKGROUND: How many OHCs are necessary to maintain cochlear amplifier function remains unknown. The electrophysiologic characteristics may indicate different degrees of OHCs loss. METHODS: Electrophysiological characteristics were tested using 8-kHz pure-tone stimulus and OHCs counted specifically in the region of the cochlea corresponding to 8-kHz. Rat models of selective OHCs loss were established by injecting kanamycin (KM) at various dosages, and the region of 8-kHz was obtained by 8-kHz pure-tone exposure. RESULTS: The ABR thresholds were affected slightly with OHCs loss < 30%, and were increased dramatically with OHCs loss ranging from 30 to 70%, but the thresholds did not increase further when OHCs loss exceeded 70%. As OHCs loss increased, the compound action potential (CAP) amplitude decreased. The CAP amplitude and OHCs loss were negatively correlated. Moreover, the summating potential (SP)/action potential (AP) increased as OHCs loss increased. DPOAE and cochlear microphonics (CM) exhibited reduced amplitudes when OHCs loss < 30%. CONCLUSIONS: Electrophysiologic characteristics may indicate different degrees of OHCs loss. While OHCs loss > 70%, the cochlear amplification may lose completely, but it is difficult to detect OHCs loss < 30%, because the ABR or DPOAE may reveal "normal" at this level. Moreover, the decreased CAP amplitude or increased SP/AP may be indicators for OHCs loss.

Time-compression thresholds for Mandarin sentences in normal-hearing and cochlear implant listeners.

Faster speech may facilitate more efficient communication, but if speech is too fast it becomes unintelligible. The maximum speeds at which Mandarin words were intelligible in a sentence context were quantified for normal hearing (NH) and cochlear implant (CI) listeners by measuring time-compression thresholds (TCTs) in an adaptive staircase procedure. In Experiment 1, both original and CI-vocoded time-compressed speech from the MSP (Mandarin speech perception) and MHINT (Mandarin hearing in noise test) corpora was presented to 10 NH subjects over headphones. In Experiment 2, original time-compressed speech was presented to 10 CI subjects and another 10 NH subjects through a loudspeaker in a soundproof room. Sentences were time-compressed without changing their spectral profile, and were presented up to three times within a single trial. At the end of each trial, the number of correctly identified words in the sentence was scored. A 50%-word recognition threshold was tracked in the psychophysical procedure. The observed median TCTs were very similar for MSP and MHINT speech. For NH listeners, median TCTs were around 16.7 syllables/s for normal speech, and 11.8 and 8.6 syllables/s respectively for 8 and 4 channel tone-carrier vocoded speech. For CI listeners, TCTs were only around 6.8 syllables/s. The interquartile range of the TCTs within each cohort was smaller than 3.0 syllables/s. Speech reception thresholds in noise were also measured in Experiment 2, and were found to be strongly correlated with TCTs for CI listeners. In conclusion, the Mandarin sentence TCTs were around 16.7 syllables/s for most NH subjects, but rarely faster than 10.0 syllables/s for CI listeners, which quantitatively illustrated upper limits of fast speech information processing with CIs.

Inhibition of Histone Methyltransferase G9a Attenuates Noise-Induced Cochlear Synaptopathy and Hearing Loss.

Posttranslational modification of histones alters their interaction with DNA and nuclear proteins, influencing gene expression and cell fate. In this study, we investigated the effect of G9a (KMT1C, EHMT2), a major histone lysine methyltransferase encoded by the human EHMT2 gene and responsible for histone H3 lysine 9 dimethylation (H3K9me2) on noise-induced permanent hearing loss (NIHL) in adult CBA/J mice. The conditions of noise exposure used in this study led to losses of cochlear synapses and outer hair cells (OHCs) and permanent auditory threshold shifts. Inhibition of G9a with its specific inhibitor BIX 01294 or with siRNA significantly attenuated these pathological features. Treatment with BIX 01294 also prevented the noise-induced decrease of KCNQ4 immunolabeling in OHCs. Additionally, G9a was increased in cochlear cells, including both outer and inner sensory hair cells, some spiral ganglion neurons (SGNs), and marginal cells, 1 h after the completion of the noise exposure. Also subsequent to noise exposure, immunoreactivity for H3K9me2 appeared in some nuclei of OHCs following a high-to-low frequency gradient with more labeled OHCs in the 45-kHz than the 32-kHz region, as well as in the marginal cells and in some SGNs of the basal turn. These findings suggest that epigenetic modifications of H3K9me2 are involved in NIHL and that pharmacological targeting of G9a may offer a strategy for protection against cochlear synaptopathy and NIHL.

Long Non-Coding RNA TTN-AS1 Promotes the Proliferation and Invasion of Colorectal Cancer Cells by Activating miR-497-Mediated PI3K/Akt/mTOR Signaling.

INTRODUCTION: Long non-coding RNAs (lncRNAs) have obtained increasing attention due to their regulatory functions in many cancers. This work aimed to investigate the functional roles of lncRNA TTN-AS1 in colorectal cancer (CRC) and to explore the underlying mechanisms. METHODS: The expression profiles of TTN-AS1 and miR-497 in CRC tissues and cell lines were determined by RT-qPCR analysis. MTT assay, transwell assay, western blot analysis, and xenograft tumors in nude mice were employed to analyze the effects of TTN-AS1 on the proliferation, migration, invasion, EMT, and in vivo tumorigenesis of CRC cells. Bioinformatics analysis and dual-luciferase reporter assay determined the direct binding relation between TTN-AS1 and miR-497 in CRC. RESULTS: We observed a significant increase of TTN-AS1 expression level in CRC tissues and cell lines compared with normal counterparts. High expression of TTN-AS1 predicted a poor prognosis and was correlated with aggressive clinicopathological characteristics in CRC patients. Functionally, gain- and loss-of-function studies indicated that TTN-AS1 knockdown suppressed the proliferation, migration, invasion and epithelial-mesenchymal transition of CRC cells in vitro, whereas TTN-AS1 overexpression showed the complete opposite effects. Mechanistically, we found that TTN-AS1 could directly interact with miR-497, and co-transfection with miR-497 mimics blocked the activation of PI3K/Akt/mTOR signaling, and reversed the effects of TTN-AS1 overexpression in CRC cells. CONCLUSION: To conclude, our findings provide novel insight into CRC tumorigenesis and indicate that TTN-AS1 may serve as a potential therapeutic target for CRC treatment.

microRNA­183 is involved in the differentiation and regeneration of Notch signaling­prohibited hair cells from mouse cochlea.

Auditory hair cell regeneration following injury is critical to hearing restoration. The Notch signaling pathway participates in the regulation of inner ear development and cell differentiation. Recent evidence suggests that microRNA (miR)­183 has a similar role in the inner ear. However, it is unclear how Notch signaling functions in hair cell regeneration in mammals and if there is cross­talk between Notch signaling and miR­183. The present study used a gentamicin­induced cochlear injury mouse model. Gentamicin­induced damage of the hair cells activated the Notch signaling pathway and downregulated miR­183 expression. Notch signaling inhibition by the γ­secretase inhibitor, 24­diamino­5­phenylthiazole (DAPT), attenuated gentamicin­induced hair cell loss and reversed the downregulation of miR­183 expression. Further investigation revealed that the novel hair cells produced, induced by DAPT, were derived from transdifferentiated supporting cells. Additionally, myosin VI­positive hair cell numbers were increased by Notch signaling inhibition in in vitro experiments with cultured neonatal mouse inner ear precursor cells. This effect was reversed by miR­183 inhibition. These findings indicate that the Notch signaling pathway served a repressing role during the regeneration of hair cells. Inhibiting this signal improved hair cell regeneration in the gentamicin­damaged cochlear model. miR­183 was demonstrated to be involved in hair cell differentiation and regeneration, and was required for the differentiation of the Notch­inhibited hair cells.

The regulatory role of Fos related antigen­1 in inflammatory bowel disease.

The etiology of inflammatory bowel disease (IBD) remains unclear. The ratio of Fos related antigen­1 (Fra­1)­positive intestinal mucosa epithelial cells is significantly increased in active IBD. This study intends to explore the regulatory role of Fra­1 in IBD. The Fra­1 eukaryotic expression vector was constructed and stably transfected to establish the Fra­1 overexpression HCT­116 (116­Fra­1) intestinal epithelial cell line. The impact of Fra­1 overexpression on intestinal mucosal epithelial cell damage repair function was tested using a scratch assay. The role of Fra­1 overexpression on intestinal mucosal epithelial cell proliferation was evaluated using a Cell Counting Kit-8 assay. Apoptosis related proteins, B­cell lymphoma 2 (Bcl­2), c­Myc, Survivin and Bcl­extra large (Bcl­xL), expression levels were detected by western blotting. Fra­1 suppressed intestinal mucosal epithelial cell damage repair and proliferation. Fra­1 inhibited the protein levels of Bcl­2, c­Myc, Survivin, and Bcl­xL. Fra­1 overexpression in intestinal mucosal epithelial cells may restrain damage repair after intestinal mucosal injury in IBD remittent period through weakening the protective effect of intestinal mucosa, thus increasing the risk of recurrence. Therefore, suppressing Fra­1 expression in intestinal mucosal epithelial cells may contribute to IBD remittent maintenance and recurrence delay.

DNER modulates the length, polarity and synaptogenesis of spiral ganglion neurons via the Notch signaling pathway.

The Delta/Notch­like epidermal growth factor­related receptor (DNER) serves an important role in the developing central nervous system. However, the actions of DNER in the development of the spiral ganglion in the inner ear have yet to be elucidated. Wild­type C57BL/6 mice were housed and time­mated for use in the present study. Primary neuronal cultures were prepared using spiral ganglion progenitors isolated from the modiolus of postnatal day 1 (P1) mice. DNER recombinant lentiviral vectors were constructed and transfected into the cultured primary neurons. The relative proportion of differentiated neurons and the length of their neurites were evaluated using microscopy. The results of the present study demonstrated that DNER was expressed in spiral ganglion neurons (SGNs) that exhibited significant polarity in the early differentiation stages; DNER expression gradually decreased until the polarity was lost on week 35. The in vitro expression of DNER was revealed to be similar to that in vivo. When DNER expression was silenced using RNA interference, the polarity of the differentiated neurons was altered and they exhibited significantly reduced dendritic length. In addition, the proportion of bipolar neurons was decreased compared with the control group. Furthermore, the expression of α­synuclein and the GluR2/3 subunits of the α­amin­o­3­hydroxy­5­methyl­4-isoxazolepropionic acid glutamate receptor were also reduced in cultured neurons in which DNER was silenced. Notch1 was co­expressed with DNER in SGNs isolated from P1 mice. The indirect Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]­S­phenylglycine t­butyl ester also affected the polarity and the formation of protrusions, and reduced the expression of DNER and glial fibrillary acidic protein in SGNs. In conclusion, the present study demonstrated that DNER was expressed in SGNs and appeared to be involved in the mechanisms underlying neuronal polarity and neuritogenesis, via a Notch­dependent signaling pathway.