The mucosal epithelium of the head and neck region (including the oral cavity, nasal cavity, pharynx, nasopharynx, and larynx) is the primary site exposed to tobacco smoke, and its presence of nicotinic acetylcholine receptors (nAChRs) has been observed in the mucosal epithelial cells of this area. It remains unclear whether HNSC cells can migrate and invade through nAChR signaling. A model of HNSC cells exposed to nicotine is established. Cell proliferation following nicotine exposure is assessed using the CCK-8 assay, while migration and invasion are evaluated through wound healing and Transwell assays. The effects of CHRNA5 knockdown and overexpression are also investigated. Immunofluorescence staining is used to analyze CHRNA5 expression and localization, and clonogenic assays are performed to measure colony proliferation after CHRNA5 knockdown and overexpression. The interaction between CHRNA5 and CES1 is examined using molecular docking, co-immunoprecipitation, and immunofluorescence. Differentially expressed genes are subjected to pathway enrichment analysis, and MEK/ERK protein expression and phosphorylation are validated via western blot. Tumor formation assays are performed in nude mice using sh-CHRNA5 Cal27 cells, followed by western blot and immunohistochemical staining. Additionally, laryngeal and hypopharyngeal cancer tissues are analyzed through immunohistochemistry. Nicotine significantly enhanced the proliferation, migration, and invasion capabilities of head and neck tumor cells, including Cal27, Fadu, HN6, and Tu686 cells, through the expression of CHRNA5. Knockdown of CHRNA5 can reduce cell migration, invasion, and proliferation, whereas nicotine exposure can reverse this trend. Additionally, the mRNA and protein expression of CES1 decreases with the knockdown of CHRNA5, indicating a regulatory relationship between the two. Transcriptomics revealed that the knockdown of CHRNA5 is associated with the MEK/ERK signaling pathway. Further cellular- and tissue-level evidence confirmed that the levels of p-MEK/MEK, p-ERK/ERK, and CES1 decreased following knockdown of CHRNA5, a trend that nicotine can reverse. Nicotine promotes the proliferation, migration, and invasion of HNSC by upregulating CHRNA5 expression. Knockdown of CHRNA5 reduces these effects, which can be reversed by nicotine. Nicotine exposure activates CHRNA5, regulating CES1 expression via the MEK/ERK pathway, contributing to the recurrence and metastasis of head and neck squamous carcinoma.

Peripheral nerve injury (PNI) is often resulting from trauma, which leads to severe and permanently disability. Schwann cells are critical for facilitating the regeneration process after PNI. Adipose-derived mesenchymal stem cells (ADSCs) exosomes have been used as a novel treatment for peripheral nerve injury. However, the underlying mechanism remains unclear. In this study, we isolated ADSCs and extracted exosomes, which were verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot (WB). Cocultured with Dorsal Root Ganglion (DRG) and Schwann cells (SCs) to evaluate the effect of exosomes on the growth of DRG axons by immunofluorescence, and the proliferation and migration of SCs by CCK8 and Transwell assays, respectively. Through exosomal miRNA sequencing and bioinformatic analysis, the related miRNAs and target gene were predicted and identified by dual luciferase assay. Related miRNAs were overexpressed and inhibited, respectively, to clarify their effects; the downstream pathway through the target gene was determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and WB. Results found that ADSC-exosomes could promote the proliferation and migration of SCs and the growth of DRG axons, respectively. Exosomal miRNA-22-3p from ADSCs directly inhibited the expression of Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), activated phosphorylation of the AKT/mTOR axis, and enhanced SCs proliferation and migration. In conclusion, our findings suggest that ADSC-exosomes could promote SCs function through exosomal miRNA-22-3p, which could be used as a therapeutic target for peripheral nerve injury.

Reduced tolerance to sound stimuli (hyperacusis) is commonly seen in tinnitus patients. Dysfunction of limbic systems, such as the nucleus accumbens (NAc), may be involved in emotional reactions to the sound stimuli in tinnitus patients. To study the functional changes in the NAc in hyperacusis, we have examined the neural activity changes of the NAc using c-Fos staining in an animal model of hyperacusis. The c-Fos staining was also examined in the medial geniculate nucleus (MGN), a central auditory pathway which has neural projections to the NAc. Postnatal rats (14 days) were exposed to loud noise (115 dB SPL, 4 hours for two consecutive days) to induce hyperacusis ( ). Rats without noise exposure were used as the controls ( ). After P35, rats in both groups were put in a behavioral training for sound detection. After they were trained to detect sound stimuli, their reaction time to noise bursts centered at 2 kHz (40-110 dB SPL) was measured. Rats in the noise group showed a significantly shorter reaction time than those in the control group to the noise bursts at high intensities, suggesting the noise exposure induced hyperacusis behavior. The c-Fos expressions in the NAc and the MGNs of the noise group were significantly higher than those of the control group. Our results suggested that early-age noise exposure caused hyperactivity in the NAc and the MGNs which may induce the loudness increase in these rats.

Background: The microenvironment of head and neck squamous cell carcinoma (HNSC) is made up of cancer and non-cancerous cells, and their interactions have profound effects on anti-tumor immunity. However, a thorough understanding of the genetic and cellular-level intercellular communication networks involved in tumor progression remains a significant obstacle. Material/Methods: 460 HNSC patients from various cohorts were included. To identify the marker genes, we analyzed single-cell RNA-sequencing (scRNA-seq) data from GEO database. An analysis of immunological infiltrating cell density was carried out using cell-type identification by calculating relative subsets of RNA transcripts (CIBERSORT). The bulk RNA-seq dataset from TCGA database was used to construct signature, and the GSE 65858 were used for validation. And the expression of related proteins were verified using HPA database and western blotting. Results: A three-gene signature (CES1, ELF3 and SERPINE1) was developed for prognostic prediction in the TCGA dataset, which divided patients into high-risk and low-risk categories based on overall survival. The prognostic potential of the signature was confirmed by GSE 65858. The signature protein expression was validated by HPA database and western blotting. Furthermore, the riskScore was identified as a significant prognostic factor in the multivariate analysis, indicating that the signature had high predictive ability. In addition, patients with high-risk scores obtained fewer benefits from immunotherapy. Conclusions: Our study identified a distinctive predictive signature for HNSC patients based on CES1, ELF3, and SERPINE1. The signature may be used as a predictor for immunotherapy and as an indicator of survival in patients with HNSC.