[Correlation of Rab coupling protein expression with clinicopathological characteristics and prognosis in laryngeal squamous cell carcinoma].

OBJECTIVE: To explore the RCP protein expression and its clinicopathological significance in laryngeal squamous cell carcinoma (LSCC). METHODS: RCP protein expression in human head and neck squamous cell carcinoma cell lines (NP-69, Tu686, Tu212, M2 and M4) was analyzed by Western blotting. Besides, its expression in 87 cases of LSCC, 18 cases of adjacent epithelial mucosa and 16 cases of vocal cord leukoplakia was detected by immunohistochemistry, and their correlation with clinicopathological parameters and patients' outcome was analyzed. RESULTS: The NP-69, Tu212 Tu686, M2 and M4 cells showed a gradual increase in the expression of RCP protein. The average relative expression levels of RCP protein in the NP-69, Tu212, Tu686, M2 and M4 cells were 0.05±0.01, 0.38±0.05, 0.63±0.02, 0.84±0.06 and 0.96±0.04, respectively. The same situation occurred in the adjacent mucosa, vocal cord leukoplakia and LSCC. Specifically, only 3 of 18 adjacent mucosa showed a low RCP expression (scored 0-2). Although the 16 cases of vocal cord leukoplakia had a low RCP expression, all their scores ranged from 0 to 3. While in the LSCC specimens, 59 (67.8%) cases demonstrated a high RCP expression (scored 8-15), 18 cases showed a lower RCP expression (scored 4-7), and only 10 cases were scored 2-3. Among the 87 LSCC cases, there were 28 cases (32.2%) of low RCP expression and 59 cases of high RCP expression. All the 18 cases of cancer-adjacent tissues and 16 cases of vocal cord leukoplakia were of low RCP expression. RCP overexpression was significantly associated with T classification, clinical staging, lymph node metastasis and recurrence (P<0.05 for all). Survival analysis revealed that the 5-year survival rate was 40.0% in the patients with high RCP expression and 75.0% in the patients with low RCP expression, the tumor-free 5-year survival rate was 30.7% and 64.0%, respectively, both showing a significant difference between the two subgroups (P<0.05). Univariate analysis revealed that alcohol history; smoking, T classification, clinical staging, lymph node metastasis and RCP expression were significantly associated with a poor prognosis (P<0.05 for all). The multivariate analysis showed that only recurrence and RCP expression were independent prognostic factors affecting the prognosis for patients with LSCC (P<0.05 for both). CONCLUSIONS: Expression of RCP protein may contribute to the malignant progression of LSCC, and may become a novel marker predicting tumor recurrence, cervical lymph node metastasis and prognosis for patients with laryngeal squamous cell carcinoma.

Analysis of transcriptional factors and regulation networks in laryngeal squamous cell carcinoma patients with lymph node metastasis.

The present study was to identify and quantitate differentially expressed proteins in laryngeal squamous cell carcinoma (LSCC) tissues with or without lymph node metastasis and to explore transcriptional factors and regulation networks associated with the process. Tissue specimens were taken from 20 patients with LSCC, including 10 cases of LSCC without metastasis LSCC (N0) and 10 cases of LSCC with metastasis LSCC (Nx). Among the 643 unique proteins identified by using iTRAQ labeling and quantitative proteomic technology, 389 proteins showed an abundance change in LSCC (Nx) as compared to LSCC (N0). Cytoskeleton remodeling, cell adhesion, and immune response activation were found to be the main processes in LSCC metastasis. The construction of transcription regulation networks identified key transcription regulators for lymph node metastasis of LSCC, including Sp1, c-myc, and p53, which may affect LSCC metastasis through the epithelial-mesenchymal transition. Furthermore, our results suggest that ubiquitination may be a critical factor in the networks. The present study provides insights into transcriptional factors and regulation networks involved in LSCC metastasis, which may lead to new strategies for treatment of LSCC metastasis.

Generation of an induced pluripotent stem cell line (ZZUi034-A) from a 65 year old Chinese female donor with sendai virus reprogramming protocol.

Peripheral blood was extracted from a 65 year old Chinese female health donor. Induced pluripotent stem cells (iPSC) were reprogrammed with the 4 reprogramming factors: Klf-4, c-Myc, Oct-4, Sox-2, using sendai virus reprogramming protocol. The iPSC line showed pluripotency which was confirmed by immunofluorescent staining; the iPSC line was also able to form embryoid bodies in vitro and differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This healthy iPSC line can be served as healthy control for disease mechanism and disease modeling study.

Generation of a heterozygous FUS-Q290X knock in human embryonic stem cell line (WAe009-A-83) using CRISPR/Cas9 system.

Fused in Sarcoma (FUS) gene encodes FUS RNA binding protein, a multifunctional protein component of the heterogeneous nuclear ribonucleoprotein complex, which is involved in pre-mRNA splicing and the export of fully processed mRNA to the cytoplasm, and it has been implicated in regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing. FUS gene mutations result in amyotrophic lateral sclerosis and Liposarcoma. This heterozygous FUS-Q290X knock in hESC line will be a valuable tool to investigate the disease mechanisms of amyotrophic lateral sclerosis and Liposarcoma.

[Correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in laryngeal squamous cell carcinoma].

OBJECTIVE: To evaluate the expression of EphA2 protein in tissue specimens and cell lines of laryngeal squamous cell carcinoma (LSCC), and to further study the correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in LSCC. METHODS: Western blot was applied to assess the EphA2 protein expression in LSCC cell line Hep-2 cells and the head and neck immortalized epithelial cell line NP-69 cells. Immunohistochemical staining was performed on paraffin sections of 88 cases of LSCC specimens and 16 cases of adjcent normal tissue samples to investigate the EphA2 protein expression, and to futher elucidate its correlation with clinicopathological characteristics. RESULTS: Compared with the NP-69 cells, EphA2 expression in LSCC cell line Hep-2 cells was upregulated. The positive rates of EphA2 expression in LSCC and adjcent normal tissues samples were 80.7% and 43.8%, respectively, with a significant difference between the two groups (P < 0.001). EphA2 overexpresion was closely correlated with clinical stage (I + II/III + IV, P = 0.005), metastasis (P = 0.025) and recurrence (P = 0.021) in LSCC. Furthermore, patients with EphA2 overexpression had poorer tumor-free survival and 5-year overall survival compared with that in patients with low EphA2 expression (33.3% vs. 63.2%, P = 0.003; 46.7% vs. 81.6%, P = 0.002). EphA2 expression combined with clinical stage provided a better predictive value in prognosis. Univariate and multivariate Cox regression analysis revealed that EphA2 expression is an independent prognostic factor for patients with LSCC (P = 0.019). CONCLUSIONS: The results of this study demonstrate that EphA2 protein expression is significantly increased in LSCC tissues and cell lines, and EphA2 protein overexpression is associated with tumor recurrence, metastasis and poorer prognosis in LSCC patients. These results suggest that EphA2 may play a critical role in the initiation and progression of LSCC, implicating EphA2 as a valuable marker for the prediction of recurrence, metastasis and prognosis in LSCC.

The Relevance of Regenerating Gene 1a Polymorphisms to Radiation Sensitivity and Survival of Nasopharyngeal Carcinoma Receiving Radiotherapy in a Southern Chinese Population.

OBJECTIVE: Gene polymorphism is closely related to tumor development, therapeutic response and prognosis. The relationship between regenerating gene 1A (Reg1A) polymorphism and nasopharyngeal carcinoma (NPC) is unclear. This retrospective study aimed to analyze the association between Reg1a polymorphisms and metastasis, radiation sensitivity and survivals in patients with NPC. METHODS: A total of 308 patients who had received radiotherapy at the Affiliated Xinhua Hospital, Hainan Medical College, between January 2010 and December 2018 with NPC, were enrolled for assessment of Reg1a polymorphisms through direct DNA sequencing. RESULTS: In the polymorphism of gene REG1A, patients with rs10165462 20CC genotype had later T stages (OR = 4.051, 95% CI: 1.775-9.244, P = 0.001), whereas carriers with rs12072 2922CC genotype had earlier T stages (OR = 1.891, 95% CI: 1.018-3.514, P = 0.044) after adjustments for age and gender, respectively. Among rs10165462 20 C/T polymorphism, 20TT wild-type was associated with better radiation response (P = 0.0019), and multivariate analysis showed that it was the only genotype of polymorphism that was significantly associated with better radiation response (OR = 0.265, 95% CI: 0.096-0.727, P = 0.01). Patients with the 20TT wild-type had a better five-year overall survival (60.9%) rate and five-year progression-free survival (60.8%) than those with the 20CC genotype (41.8% and 39.4%, P = 0.01 and P = 0.004, respectively). Patients with variant alleles (CC + CT) had significantly poorer OS (45.2%) and PFS (41.8%) compared with wild-type (TT) carriers (60.9% and 60.8%; P = 0.037 and P = 0.015, respectively). As for rs12072, patients with variant alleles (TT + TC) had significantly adverse OS and PFS compared with wild-type (CC) carriers (62.5% vs 44.8% and 62.5% vs 42.9%; P = 0.024 and P = 0.027, respectively). Cox regression showed that rs10165462 20CT was the only prognostic factor for OS (HR = 1.642, 95% CI 1.038-2.598, P = 0.034) and PFS (HR = 1.705, 95% CI 1.080-2.692, P = 0.022). CONCLUSION: Reg1a polymorphisms may be a predictor of radiation response, local invasion, OS and PFS in patients with NPC who undergo radiotherapy treatment.

Downregulated long non-coding RNA SNHG7 restricts proliferation and boosts apoptosis of nasopharyngeal carcinoma cells by elevating microRNA-140-5p to suppress GLI3 expression.

Long non-coding RNAs (lncRNAs) have been proposed to correlate with various carcinomas, yet the role of lncRNA SNHG7 in nasopharyngeal carcinoma (NPC) is hardly studied. This study intends to examine the molecular mechanism of SNHG7 on NPC cells. The NPC tissues and nasopharyngeal tissues of mild inflammation of nasopharyngeal mucosa were obtained. SNHG7, miR-140-5p, and GLI3 mRNA and protein expression in tissues and in the CNE1, HONE1, C666-1, CNE2, and normal NP69 cell lines was detected. IC50 and the protein expression of related drug-resistant genes of CNE2 and CNE2/DDP cells were determined. Proliferative ability, cell colony formation rate, cell cycle, and apoptosis of CNE2 and CNE2/DDP cells were also detected. SNHG7, miR-140-5p, and GLI3 mRNA and protein expression in CNE2 and CNE2/DDP cells in each group was detected. SNHG7's cell localization, the binding sites of SNHG7 and miR-140-5p along with miR-140-5p and GLI3 were detected. Overexpressed SNHG7 and GLI3, and underexpressed miR-140-5p were found in NPC tissues and cells. SNHG7 silencing and miR-140-5p elevation declined the drug resistance of drug-resistant NPC cells and their parent cells, restrained NPC cell colony formation ability and proliferation, and boosted cell apoptosis. SNHG7 specially bound to miR-140-5p, and SNHG7 silencing elevated miR-140-5p expression. GLI3 was a direct target gene of miR-140-5p and miR-140-5p elevation diminished GLI3 expression. MiR-140-5p inhibition reversed the impacts of SNHG7 silencing on NPC cells. In summary, our study reveals that downregulated SNHG7 restricts GLI3 expression by upregulating miR-140-5p, which further suppresses cell proliferation, and promotes apoptosis of NPC.

Negative pressure catheter drainage and jejunal feeding for descending necrotizing mediastinitis by interventional techniques: a retrospective study.

Purpose: Descending necrotizing mediastinitis (DNM) is a lethal and acute suppurative disease. This report aimed to summarize our experience in the treatment of DNM with continuous negative pressure catheter drainage and transnasal jejunal feeding by interventional techniques. Materials and Methods: We retrospectively analyzed relevant clinical data of patients with DNM who underwent continuous negative pressure catheter drainage and transnasal jejunal feeding. All drainage catheters and jejunal feeding tubes were inserted by interventional techniques. Results: In total, 21 patients were diagnosed with DNM by esophagography and computed tomography (CT). Catheters for the drainage of mediastinal abscesses as well as transnasal jejunal feeding tubes were successfully placed in all patients, indicating a 100% success rate. Of all patients, 13 underwent insertion of abscess drainage catheters through percutaneous puncture under DynaCT guidance, while eight had drainage catheter insertion through fistula orifices in the posterior nasopharyngeal wall or esophagus under fluoroscopic guidance. In total, 26 drainage tubes were inserted. One patient with diabetes died of sepsis and diabetic ketoacidosis 5 days postoperatively, while the remaining 20 patients showed good recovery with successful removal of the drainage catheters. Durations of catheterization were 45.2±50.44 days. The overall clinical success rate was 95.2%. Conclusion: The above described methods are non-surgical, minimally invasive and efficacious, and may be alternative therapeutic tools for patients who are not eligible for surgical operation, have a high postoperative risk, or are more likely to choose minimally invasive techniques.

Development of one control and one tumor-specific induced pluripotent stem cell line from laryngeal carcinoma patient.

Skin fibroblasts and tumor fibroblasts were extracted from a 64-year old male patient clinically diagnosed with laryngeal carcinoma. Control and tumor specific induced pluripotent stem cells were reprogrammed with 5 reprogramming factors, Klf-4, c-Myc, Oct-4, Sox-2, and Lin-28, using the messenger RNA reprogramming system. The transgene-free iPSC lines showed pluripotency, confirmed by immunofluorescence staining. The iPSC lines also showed normal karyotype, and could form embryoid bodies in vitro and differentiate into the 3 germ layers in vivo. This in vitro cellular model can be used to study the oncogenesis and pathogenesis of laryngeal carcinoma.

Prevalence and Predisposing Factors for Depressive Status in Chinese Patients with Obstructive Sleep Apnoea: A Large-Sample Survey.

BACKGROUND AND OBJECTIVE: Recently, there are few studies reporting on depressive status and obstructive sleep apnoea (OSA) in China. A large-sample survey was to be performed to explore the prevalence of depressive status and related factors in Chinese patients with OSA. METHODS: From among a randomly-selected group of OSA patients, 1,327 met inclusion criteria. After screening with the Symptom Checklist 90 (SCL-90) and Self-Rating Depression Scale (SDS), patients were assigned to OSA without depressive status (control group, n = 698) and OSA with depressive status (n = 629) groups. Using chi-squared testing, the correlation analyses between the depressive status and OSA patient demographic and clinical variables were tested. Then depression-related risk factors in OSA patients were analysed using stepwise linear regression analysis. The effects of family and social factors on depressive status in OSA patients were investigated using Mann-Whitney U (one of nonparametric test). RESULTS: The prevalence of depressive status was 47.4% in OSA patients. Depressive status was significantly associated with female gender, single status, Family Burden Scale of Disease (FBS), Family APGAR Index (APGAR), apnoea-hypopnea index (AHI), and Perceived Social Support Scale (PSSS). Stepwise linear regression analysis further indicated that single status, hypoxemia, APGAR, AHI, PSSS, AHI, and FBS were all risk factors for depressive status in OSA patients. The total of the FBS score and three of its sub-factors scores (family daily activities, family relationships and mental health of family members) were higher, and the total of the APGAR score and two of its sub-factors scores (adaptability and affection) were lower in OSA with depressive status compared with the control group. Besides, the total score for the PSSS and scores for its two sub-factors (family support and social support) were all lower in OSA patients with depressive status than those of the control group. CONCLUSIONS: Depressive status has high comorbid rate in Chinese OSA patients and is significantly associated with single status, apnoea-hypopnea index, hypoxemia, family and social supports.

[Research on risk factors of depression symptoms in 1 327 patients with obstructive sleep apnea-hypopnea syndrome].

OBJECTIVE: To investigate the comorbidity rate of depression symptoms in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS) in Henan province and to ascertain the risk factors. METHODS: One thousand three hundred and twenty-seven patients with OSAHS determined by overnight polysomnogram (PSG) were enrolled in this study. After screening the Symptom Checklist 90 (SCL-90) and Self-rating Depression Scale (SDS), the patients were divided into two groups: OSAHS (control group, n = 698) and OSAHS+depression (n = 629). The correlation was explored between the depression symptoms in patients with OSAHS and the sociodemographic variables and health status including smoking, drinking, marital status, apnea hyponea index (AHI), anoxicity, Family Burden Scale of Disease (FBS), Family APGAR Index (APGAR), Perceived Social Support Scale (PSSS) and so on. Furthermore, In-depth analyses were carried out between the depression symptoms in patients with OSAHS and the social and family factor items (FBS, APGAR and PSSS). RESULTS: The comorbidity rate of depression symptoms in patients with OSAHS in Henan province was 47.4%, and was correlated with the gender, marital status, FBS, APGAR, AHI, PSSS and anoxicity. Logistic regression analysis indicated that single marital status, APGAR, AHI, PSSS, hypoxemia and heart disease were all independent risk factors for depression in OSAHS patients. The total of the FBS score and three of its subfactors scores (family daily activities, family relationships and mental health of family members) were higher, and the total of the APGAR score and two of its subfactors scores (adaptabilith and affection) were lower in OSAHS with depression compared with the control group (P < 0.05). Besides, the total score for the PSSS AND Scores for its two subfactors (family support and social support) were all lower in OSAHS patients with depression than those of the control group (P < 0.05). CONCLUSION: In patients with OSAHS, depression symptoms are common and are associated with marital status, AHI, anoxicity, concomitant diseases (hypertension, heart disease), concerns and supports from the family and society.

Increased expression of Rab coupling protein in squamous cell carcinoma of the head and neck and its clinical significance.

The role of Rab coupling protein (RCP) has not been previously investigated in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to explore RCP protein expression and its clinicopathological significance in SCCHN. RCP protein expression in 95 SCCHN samples, 18 vocal nodule epithelia and 16 leukoplakia epithelia samples was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Our data indicated that vocal nodule epithelia, leukoplakia epithelia and SCCHN showed a gradual increase in the expression of RCP protein. RCP overexpression was significantly associated with T classification, clinical staging, lymph node metastasis and recurrence. Survival analysis revealed that a high RCP expression was significantly correlated with shorter overall survival and disease-free survival. In conclusion, RCP protein may contribute to the malignant progression of SCCHN, and serves as a novel prognostic marker in patients with SCCHN.

Elevated expression of iASPP in head and neck squamous cell carcinoma and its clinical significance.

iASPP is shown to be elevated in several cancers. However, the role of iASPP in head and neck squamous cell carcinoma (HNSCC) remains unknown. We have investigated iASPP expression in HNSCC tissue and cell lines and evaluated its prognostic significance in HNSCC. The expression of iASPP in 109 primary HNSCC tissue specimens was examined by immunohistochemistry and its association with clinicopathological parameters and prognosis was analyzed. Additionally, expression status of iASPP in 16 paired HNSCC tissues and 7 HNSCC cell lines was evaluated by quantitative real-time PCR (qPCR) and immunoblotting. The protein and mRNA expression of iASPP were increased in HNSCC tissues and cell lines. Immunohistochemical staining indicated iASPP was detected in both cytoplasm and nucleus. Importantly, overexpression of cytoplasmic and nuclear iASPP was significantly associated with T classification (p = 0.002 and p = 0.033, respectively), clinical stage (p < 0.001 and p = 0.004), lymph node metastasis (p = 0.001 and p < 0.001), and recurrence (both p < 0.001). Survival analysis demonstrated high iASPP expression significantly correlated with shorter disease-free survival (DFS) (both p < 0.001 for cytoplasmic and nuclear expression) and overall survival (OS) (both p < 0.001 for cytoplasmic and nuclear expression). Multivariate analysis revealed that cytoplasmic iASPP was the only independent prognostic factor for HNSCC patients. iASPP expression is elevated in HNSCC tissues and cell lines, which suggests iASPP may contribute to the malignant progression of HNSCC, and serves as a novel prognostic marker and a potential therapeutic target in HNSCC.

TGF-ß1 mediates epithelial to mesenchymal transition via the TGF-ß/Smad pathway in squamous cell carcinoma of the head and neck.

Development of metastasis is a major cause of death for squamous cell carcinoma of the head and neck (SCCHN) patients. Epithelial to mesenchymal transition (EMT) is now regarded as a correlate of tumor metastasis. Given that transforming growth factor-ß1 (TGF-ß1) is an important inducer of EMT, we examined the effects of TGF-ß1 on the human SCCHN cell line Tu686. We found that TGF-ß1 mediated cell morphological changes. Phase-contrast microscopy revealed a loss of the adherent phenotype with cellular elongation, decrease in cell-to-cell contact, and the induction of a fibroblast-like state. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that TGF-ß1 could induce down-regulation of the epithelial marker E-cadherin and up-regulation of the mesenchymal marker vimentin in Tu686 cells in a concentration- and time-dependent manner. Wound- healing and transwell invasion assay indicated that TGF-ß1 promoted Tu686 cell migration and invasion dramatically. In addition, these changes were mediated via canonical TGF-ß/Smad signaling with concomitant up-regulation of phosphorylated Smad2. Smad2 RNAi abrogated both expression and functional effects of TGF-ß1 on Tu686 cells. In conclusion, the present study demonstrates that TGF-ß1 could induce EMT in the SCCHN cell line via the TGF-ß/Smad signaling pathway. More importantly, a cell model for EMT was established, which is valuable for future studies on the metastasis of SCCHN.