Nicotine downregulates miR-375-3p via neurotrophic tyrosine receptor kinase 2 to enhance the malignant behaviors of laryngopharyngeal squamous epithelial cells.

Nicotine exposure from smoking constitutes a significant global public health concern. Furthermore, smoking represents a pivotal risk factor for head and neck squamous cell carcinoma (HNSCC). However, the influence of nicotine on HNSCC remains relatively underexplored. Our aim was to unravel the molecular mechanisms that underlie the effect of nicotine on the metastatic cascade of HNSCC. In this study, we discovered a significant association between smoking and HNSCC metastasis and prognosis. Nicotine significantly enhanced HNSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Analysis of TCGA-HNSCC and FDEENT-HNSCC cohorts revealed reduced miR-375-3p levels in HNSCC tumor tissues, particularly among current smokers. Additionally, miR-375-3p level was strongly correlated with both lymph node metastasis and tumor stage. By downregulating miR-375-3p, nicotine promotes HNSCC cell metastasis in vitro and hematogenous metastatic capacity in vivo. Utilizing transcriptomic sequencing, molecular docking, dual-luciferase reporter assay, and fluorescence in situ hybridization (FISH), we demonstrated that miR-375-3p specifically binds to 3' untranslated region (3'UTR) of NTRK2 mRNA. Thus, this study uncovers a novel nicotine-induced mechanism involving miR-375-3p-mediated NTRK2 targeting, which promotes HNSCC metastasis. These findings have implications for improving the prognosis of patients with HNSCC, especially in smokers.

Correlation Between the NLRP3 Inflammasome and the Prognosis of Patients With LSCC.

Background: NLRP3 inflammasome is an inflammatory mediator. The expression of NLRP3 inflammasome is associated with the development of various tumors and is closely related to the prognosis of tumors. However, the role of NLRP3 inflammasome in laryngeal squamous cell carcinoma (LSCC) remains unclear. This study aim to investigate the influence of NLPR3 inflammasome expression in LSCC, and especially the NLRP3 inflammasome expression level and the prognosis of LSCC after surgery in a Chinese population. Methods: We used quantitative real-time PCR and immunohistochemical (IHC) staining to calculate the mRNA (20 patients, fresh tissue) and protein expression (104 patients, paraffin tissue microarray) levels of the NLRP3 inflammasome (NLRP3/IL-18/IL-1ß/ASC/caspase-1), respectively. We also analyzed the relationship between NLRP3 inflammasome expression levels and LSCC cancer tissues compared with adjacent normal tissues and the clinical features of LSCC. Kaplan-Meier survival curves of overall survival (OS) and disease-free survival (DFS) in LSCC patients were compared and analyzed under different expression levels of the NLRP3 inflammasome. Results: Our results indicated that the mRNA expression of the NLRP3 inflammasome was higher in LSCC cancer tissues compared with adjacent normal tissues (p < 0.001). The IHC staining score also demonstrated that the expression of the NLRP3 inflammasome was higher than in the adjacent normal tissues (p < 0.001). The NLRP3 inflammasome expression also exhibited a close relationship with the clinicopathological characteristics (especially the stage of LSCC) of LSCC. Univariate Cox regression analysis and multivariate Cox regression analysis revealed that both NLRP3 and IL-1ß had an increased risk of LSCC progression (p < 0.05). The Kaplan-Meier log rank test (OS and DFS) demonstrated that high expression of NLRP3/IL-18/IL-1ß/ASC was statistically different than the low expression group (p < 0.05) of LSCC patients after surgery. Conclusion: The high expression group of the NLRP3 inflammasome (NLRP3/IL-18/IL-1ß/ASC) had a poorer prognosis (OS and DFS) than the low expression group of LSCC patients 5 years after surgery. The NLRP3 inflammasome (NLRP3/IL-18/IL-1ß/ASC) may be used as an auxiliary indicator to predict LSCC patient prognosis after surgery.

Hypoxia promotes stem-like properties of laryngeal cancer cell lines by increasing the CD133+ stem cell fraction.

Evidence indicates that a hypoxic micro-environment plays an essential role in the regulation of cancer stem cells (CSCs). However, whether hypoxia is able to regulate the stem-like biological properties of laryngeal cancer cells remains unknown. In this study, we investigated the influence of hypoxia on the stemness of two laryngeal cancer cell lines, Hep-2 and AMC-HN-8. We cultured the two cell lines under hypoxia and normoxia and examined the influence of hypoxia on the expression of hypoxia-inducible factors (HIFs) and the cancer stem-like properties of these cells, including cell cycle distribution, expression of stem cell genes (OCT4, SOX2 and NANOG) and laryngeal CSC surface marker (CD133), proliferation, invasion, colony formation and sphere formation capacity. We determined that both of these cell lines, when maintained under hypoxic conditions, showed expanded cells in the G0/G1 phase, exhibited preferential expression of stem cell genes and CD133, and manifested upregulation of HIFs. When treated with hypoxia followed by normoxia exposure, the two cell lines exhibited enhanced capacities for proliferation, invasion, and sphere and colony formation compared with cells maintained consistently under normoxia. Our findings indicate that a hypoxic microenvironment may upgrade the stem-like biological properties of laryngeal cancer cell lines by the expansion of the CD133(+) stem cell fraction.

Establishment and characterization of a novel HPV-negative laryngeal squamous cell carcinoma cell line, FD-LSC-1, with missense and nonsense mutations of TP53 in the DNA-binding domain.

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy in China; however, publically available LSCC cell lines are few and not established from Chinese populations. Hence, novel and well-characterized LSCC cell lines of Chinese origin are urgently needed to provide researchers with a comprehensive database for LSCC research. From 40 cases of LSCC, we established a novel cell line that was maintained for more than 100 passages in vitro and was found to have typical epithelial morphology and ultrastructure. In-depth characterization analysis revealed polyploidy in DNA content; a doubling time of some 24h; high tumorigenicity in immunodeficient mice; higher invasive potential and more sensitive to radiation and cisplatin compared with HeLa cell line; upregulated Ki67, Notch1, EGFR, and CK5 protein levels; negative infection of human papillomavirus (HPV) and mycoplasma; expression of head and neck squamous cell carcinoma (HNSCC) biomarkers; mutations of TP53 in exons 5 and 8; a near-triploid karyotype with complex structural aberrations; and dozens of dysregulated genes and miRNAs. Cell authentication testing by the American Type Culture Collection (ATCC) confirmed the human origin of this cell line. Our findings indicate that a novel and well-differentiated LSCC cell line recapitulating the primary tumor's malignant characteristics is established and well characterized. It does not match any cell lines within the ATCC database and helps to elucidate the molecular pathogenesis of LSCC.

[Seasonal variation of blood pressure and related factors in Chinese adults: a survey of over 50 000 people in Suzhou, China].

OBJECTIVE: Seasonal variation in blood pressure had been observed in several studies on Western populations, but uncertainty remains about the strength of the relationship in other populations and the extent to which it was modified by other factors. METHODS: This study was based on cross-sectional data from the China Kadoorie Biobank study with 53 260 men and women from the Suzhou area involved. Linear regression model was used to analyze the association of blood pressure with outdoor temperature-overall and in various subgroups. RESULTS: Blood pressure varied with the seasons, ascending in winter and descending in summer. The difference in systolic blood pressure (SBP) between summer and winter was 8.8 mm Hg in men and 7.0 mm Hg in women. SBP was inversely correlated with outdoor temperature, especially above 10°C, with every 10°C colder temperature causing 6.1 mm Hg increase of SBP. The seasonal variation in SBP was more obviously seen in older people and in those with lower body mass index. CONCLUSION: Blood pressure was strongly and inversely associated with outdoor temperature in the population in Suzhou area. Seasonal variation of blood pressure should be considered when the hypertension screening programs, clinical management and data management on hypertensive patients.

Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia.

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.

Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line.

CONCLUSIONS: Carcinoma-associated fibroblasts (CAFs) can influence the biological characteristics of a laryngeal carcinoma cell line. These results could lay the foundation for further studies on the role of CAFs in the laryngeal tumor-host microenvironment. OBJECTIVE: CAFs are important contributors to the microenvironment in determining the fate of tumors. The aim of this study was to separate, culture, and identify laryngeal CAFs and investigate their biological influence on the laryngeal carcinoma cell line. METHODS: The primary CAFs and normal fibroblasts (NFs) of the larynx were obtained by tissue culture. The cells were verified according to immunohistochemical and immunofluorescence staining of certain proteins. Conditioned medium (CM) from CAFs and NFs was obtained. Functional assays were performed to test the influence of each CM on laryngeal carcinoma cell lines. RESULTS: Third-passage purified laryngeal CAFs and NFs were successfully attained. The CAFs showed positive staining for vimentin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The migration ability of the CAFs increased significantly compared with that of NFs (p < 0.05). CM from CAFs (compared with CM from NFs) stimulated proliferation, migration, and invasion to a greater extent.

[Relationship between behavior of weekly alcohol drinking and the prevalence of hypertension in adults of Suzhou city].

OBJECTIVE: To explore the relationship between weekly alcohol drinking behavior and the prevalence of hypertension. METHODS: Data was collected in a Kadoorie study of chronic disease in Wuzhong district, Suzhou city of Jiangsu province, China. Data from the baseline survey was used to describe the status of alcohol drinking and the prevalence of hypertension among local residents. Relationships between the frequency of alcohol drinking, consumption of alcohol, age when initiating weekly drinking behavior, drinking-related adverse conditions and the prevalence of hypertension, were studied by logistic regression. RESULTS: The rates on weekly alcohol drinking in the studied population were 40.7% in men and 0.6% in women. The amount of weekly average alcohol intake showed as 250.8 g in males and 47.2 g in females, with statistical significance seen between genders (P < 0.01). The prevalence rates of hypertension among male and female were 39.7% and 36.1% respectively, with significant difference (P < 0.01). Data from Multiple logistic regression analysis showed that when the frequency of alcohol drinking > or = 3 days per week or the weekly average alcohol intake > or = 100 grams, the risk would be higher to develop hypertension than in those non-drinkers (P < 0.01). The age of initiating behavior as weekly alcohol drinking younger than 20 years old or the dinking-related adverse condition appeared to be more than two kinds. The risks of developing hypertension were 1.50 times and 3.27 times than those non-drinkers in men but not in women. CONCLUSION: The frequency of drinking alcohol and the amount of alcohol intake per week was different between males and females. Along with the following factors as: increase of frequency on alcohol drinking per week, the amount of alcohol intake also increased. The advance of age related to the initiation of weekly drinking and the increase of alcohol-related adverse condition was also seen, the risk of hypertension showed an upward trend in males but not in females.

Experimental study on repair of the facial nerve with Schwann cells transfected with GDNF genes and PLGA conduits.

CONCLUSIONS: Schwann cells transfected by GDNF genes + PLGA were superior to Schwann cells + PLGA and direct anastomesis. This is a new and effective strategy for repair of facial nerve defects. OBJECTIVE: To evaluate the effect of bioactive artificial nerve conduits in the repair of facial nerve defects in Sprague-Dawley rats. MATERIALS AND METHODS: Schwann cells were harvested and transfected with PcDNA3.1 (+)/GDNF. After injection with Schwann cells, the conduits were cultured in the culture medium for 2 weeks. Thirty female Sprague-Dawley rats were selected and randomly divided into three groups (A, B, and C), which were treated as follows: A, direct anastomesis; B, Schwann cells + PLGA conduits; C, Schwann cells transfected by GDNF genes + PLGA conduits. General observation, electrophysiological study, histological study, and image analysis were performed 2 weeks, 1 month, 2 months, and 3 months postoperatively. RESULTS: The recovery of nerve regeneration and electrophysiological results in group C were superior to those in groups A and B; the difference was statistically significant (p < 0.01).

[Repair of facial nerve with bioactive artificial nerve conduit: experiment with rats].

OBJECTIVE: To evaluate the effect of bioactive artificial nerve conduits in the repair of facial nerve defects. METHODS: Schwann cells (SC) were collected from the sciatic nerve and brachial plexus of a newborn SD rat, cultured, transfected with the plasmid pcDNA3. 1 (+)/GDNF containing glial cell line-derived neurotrophic factor gene, digested by pancreatin to make mixture of cell suspension to be injected into poly (L-lactic-co-glycolic acid) conduits. After injected with SCs, the conduits were cultured in for 2 weeks. Thirty female SD rat underwent buccal branch of facial nerve and then were randomly divided into 3 groups: Group A, undergoing direct anastomosis of the amputated dextral nerve; Group B, in which the 2 sides of amputated nerve were put into the PLGA conduit without transfected GDNF; and Group C in which the 2 sides of amputated nerve were put into the PLGA conduit with transfected GDNF. The motion of the facial muscles and whisker was observed. Two weeks, and 1, 2, and 3 months after the operation electrophysiological study was conducted to measure the action potential of the facial nerve. Then the rata were killed with their bilateral facial nerve and pontes were taken out to undergo histological examination. RESULTS: Compound action potential could be detected in all groups at any time points except in Groups A and B 2 weeks after the operation. The values of compound action potential of Group C at different time points were all significantly higher than those of Groups A and B (all P < 0.01). However, there were not significant differences in the compound action potential values at any time point between Groups A and B (all P > 0.05). The mean number of myelinated nerve fibers, thickness of myelin sheath, and number of motor neuron of Group C were all significantly higher than those of Groups A and B (all P < 0.05). CONCLUSION: Conduit with SCs transfected with GDNF gene + PLGA is superior to that with SCs + PLGA and direct anastomosis in repair of facial nerve.