喉鳞癌Apaf-1基因表达及启动子区甲基化研究

喉鳞癌细胞存在多种癌基因和抑癌基因异常,Apaf-1(apoptotic protease activating factor-1)基因是诱导细胞凋亡的肿瘤抑制基因。为探讨Apaf-1基因在喉鳞癌发生中的作用,应用半定量PCR方法分析Apaf-1的表达,用比较基因组杂交(Comparative Genomic Hybrodization,CGH)和杂合性丢失(Loss of Heterozygosity,LOH)分析方法对喉鳞癌病人Apaf-1基因所在的12q22—23区域缺失情况进行研究．并用甲基化特异PCR对该基因启动子区甲基化情况进行了分析。结果表明：11例喉鳞癌组织出现Apaf-1 mRNA表达明显下调,占40．7％(11／27),而6例良性喉肿瘤未发现缺失或下调;CGH分析发现,18例喉鳞癌仅发现2例存在12q22-23区域缺失,未发现扩增;LOH分析发现,72例喉癌组织Apaf-1基因的5个多态位点LOH发生频率低,分别为18．2％(D12S346)、13．9％(D12S1706)、18．2％(D12S327)、22．2％(D12S1657)和16．6％(D12S393),11例出现Apaf-1 mRNA下调的喉癌组织均检测到启动子区甲基化,而16例Apaf-1 mRNA表达未下调者仅1例有甲基化,两者有显著差异(x^2检验,P=0．0001)。通过以上结果,首次证实Apaf-1基因与喉鳞癌相关,在喉鳞癌中Apaf-1基因缺失发生率低．提示启动子区甲基化是该基因失活的首要机制。     

Overexpression of lncRNA snaR is correlated with progression and predicts poor survival of laryngeal squamous cell carcinoma

Long noncoding RNAs (lncRNA) snaR is a characterized oncogenic lncRNA in triple negative breast cancer and ovarian cancer, while its role in other human diseases is unknown. In the present study, we found that plasma levels of snaR were upregulated in patients with laryngeal squamous cell carcinoma (LSCC) than in healthy controls. Plasma levels of snaR increased with increase in AJCC stages. Follow-up study showed that high plasma levels of snaR were correlated with poor overall survival. Plasma levels of snaR were positively correlated with transforming growth factor beta (TGF-β1) in patients with LSCC but not in healthy controls. Overexpression of snaR resulted in upregulation of TGF-β1 in cells of human LSCC cell lines, while exogenous TGF-β1 treatment showed no significant effect on snaR expression. snaR overexpression and exogenous TGF-β1 treatment promoted LSCC cell proliferation, migration, and invasion. In addition, TGF-β inhibitor partially reduced the enhancing effects of snaR overexpression on LSCC cell proliferation, migration, and invasion. Therefore, overexpression of lncRNA snaR is correlated with progression and predicts poor survival of LSCC and the mechanism of its actions is likely related to TGF-β1.     

Chromosome 3q26 Gain Is an Early Event Driving Coordinated Overexpression of the PRKCI,SOX2, and ECT2 Oncogenes in Lung Squamous Cell Carcinoma

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Genetic modification of the inner ear lateral semicircular canal phenotype of the Bmp4 haplo-insufficient mouse

In the mouse, development of the lateral semicircular canal of the inner ear is sensitive to Bmp4 heterozygosity. In the C57BL6 background 30% of the heterozygotes display circling behavior, 66% have a specific defect in the vestibular part of the inner ear, namely the constriction, interruption or absence of the lateral semicircular canal. Only mice having both ears affected display circling behavior. In the (C57BL6xCBA)N1 background, the penetrance of the canal phenotype is greatly reduced, and bilateral lateral canal defect is not sufficient to induce circling. We found association of the canal phenotype with the genotype of markers on chromosome 14 and 4, co-localizing with Ecs and Eclb identified in the Ecl mouse with similar lateral canal defects. Candidate genes to contain the causal mutation are Bmp4 on chromosome 14, and Rere on chromosome 4.     

Regulation of laryngeal squamous cell cancer progression by the lncRNA RP11‐159K7.2/miR‐206/DNMT3A axis

Long non‐coding RNAs (lncRNAs), which are longer than 200 nt, have been proved to play a role in promoting or inhibiting cancer progression. The following study investigated the role and underlying mechanisms of lncRNA RP11‐159K7.2 in laryngeal squamous cell carcinoma (LSCC) progression. Briefly, in situ hybridization (ISH) and real‐time quantitative PCR (RT‐qPCR) showed higher expression of RP11‐159K7.2 in LSCC tissues and cell lines. Patients with low expression level of RP11‐159K7.2 lived longer compared to those with high expression of RP11‐159K7.2 (χ² = 39.111, ***P < 0.001). Multivariate Cox regression analysis suggested that lncRNA RP11‐159K7.2 was an independent prognostic factor for LSCC patients (HR = 2.961, ***P < 0.001). Furthermore, to investigate the potential involvement of RP11‐159K7.2 in the development of LSCC, we knocked out the expression of endogenous RP11‐159K7.2 in TU‐212 cells and AMC‐HN‐8 cells via CRISPR/Cas9 double vector lentiviral system. RP11‐159K7.2 knockout decreased LSCC cell growth and invasion both in vitro and in vivo. Mechanically, we found that RP11‐159K7.2 could positively regulate the expression of DNMT3A by sponging miR‐206. In addition, a feedback loop was also discovered between DNMT3A and miR‐206. To sum up, these findings suggest that lncRNA RP11‐159K7.2 could be used as a potential biomarker for prognosis and treatment of LSCC.     

H19/miR-107/HMGB1 axis sensitizes laryngeal squamous cell carcinoma to cisplatin by suppressing autophagy in vitro and in vivo

Laryngeal squamous cell carcinoma (LSCC) is the most common malignant tumor, which occurs in the head and neck. Current treatments for LSCC are all largely weakened by increasing drug resistance. Our study aimed to investigate the effects of long noncoding RNA (lncRNA) H19 on drug resistance in LSCC. In our study, we found that the level of H19 was sharply upregulated in LSCC tissues and drug-resistant cells compared with the control. Besides, the expression of high-mobility group B1 (HMGB1) was elevated, and microRNA107 (miR-107) was suppressed in drug-resistant cells compared with the control. Further study revealed that the interference of H19 by short hairpin RNA (shRNA) effectively suppressed high autophagy level and obvious drug resistance in drug-resistant cells. Besides that, miR-107 was predicted as a target of H19 and inhibiting effects of H19 shRNA on autophagy and drug resistance were both reversed by miR-107 inhibitor. Moreover, HMGB1 was predicted as a target of miR-107 in LSCC cells and knockdown of HMGB1 was able to suppress autophagy and drug resistance in LSCC cells. In addition, our investigation demonstrated that H19 shRNA exerted an inhibiting effect on autophagy and drug resistance by downregulating HMGB1 by targeting miR-107. Finally, the in vivo experiment revealed that LV-H19 shRNA strongly suppressed drug resistance compared with the usage of cisplatin individually. Taken together, our research indicated an H19–miR-107–HMGB1 axis in regulating the autophagy-induced drug resistance in LSCC in vitro and in vivo, providing novel targets for molecular-targeted therapy and broadening the research for LSCC.     

MiR-143-3p suppresses cell proliferation,migration, and invasion by targeting Melanoma-Associated Antigen A9 in laryngeal squamous cell carcinoma

Previously we found that melanoma-associated antigen-A9 (MAGE-A9) was a significantly upregulated biomarker in laryngeal squamous cell carcinoma (LSCC). A high expression of MAGE-A9 indicates an unfavorable survival outcome, and the MAGE-A9 expression level is an independent prognostic factor of LSCC. To explore the mechanism of MAGE-A9 upregulation, several predicted regulatory microRNAs were screened and validated in LSCC cells. In the current study, we found that miR-143-3p (MAGE-A9 related miRNAs) expression levels correlated negatively with the MAGE-A9 protein expression in LSCC tissues. Dual-luciferase reporter assays and Western blot analysis revealed MAGE-A9 to be a direct target of miR-143-3p. Furthermore, a series of in vitro gain- and loss-of-function assays revealed that miR-143-3p inhibited LSCC cell proliferation, migration, and invasion. Also, miR-143-3p suppressed LSCC tumorigenesis in vivo. These effects were clinically relevant, as a lower expression of miR-143-3p occurred in severer clinical stages and represented poor overall survival in patients with LSCC. Taken together, these results suggest that downregulation of miR-143-3p contributes to tumor progression through upregulation of MAGE-A9. The expression level of these two key molecules maintained LSCC progression, thus, highlighting the potential of miR-143-3p as a therapeutic target for human LSCC.     

Identification of a differential expression signature associated with tumorigenesis and metastasis of laryngeal carcinoma

Objectives

Metastasis is the most significant prognostic factor for laryngeal carcinoma which necessitates the identification of molecular alterations associated with metastasis. The identification of such molecular alterations will not only prove useful in treatment but also provide insight into mechanisms of cancer metastasis. The studies conducted so far have not specifically focused on metastasis or invasion pathways. Therefore we investigated the expression profiles with a pathway focused approach.

Materials and methods

Total RNA was extracted from 36 laryngeal tumors and paired cancer free tissue. Expression levels of 88 genes were determined using a PCR array system following cDNA synthesis. Obtained data was used for the calculation of altered expression levels, facilitating relevant algorithms. Significant alterations were determined according to their p-value obtained by Student's t-test.

Results

Sixteen genes have shown altered expression when compared with adjacent cancer-free tissue. 2 of these 16 genes have shown differential expression in tumors with neck metastasis in respect to non-metastatic tumors.

Conclusion

We found that TGFB1, TIMP1, c-Myc, SPARC, COL4A2 and SOX4 show altered expression in laryngeal tumors. c-Myc and SOX4 expression is decreased as laryngeal tumors switch to metastatic phenotype.