NGX6基因单核苷酸多态及与鼻咽癌的相关性

NGX6基因是本研究室在鼻咽癌 9p最小共同缺失区内新克隆的鼻咽癌候选抑瘤基因。通过采用病例 对照研究方法 ,利用动态等位基因杂交 (DASH)技术对 10 5例鼻咽癌患者和 183例正常人NGX6基因的 2个单核苷酸多态 (SNP)进行了分型 ,经相关分析发现 ,位于NGX6基因上游调控区的SNPrs8792 84与鼻咽癌发病存在显著相关性 ,基因型CT和TT的相对危险度分别为 3.93和 2 .2 7。实验结果进一步支持了NGX6基因与鼻咽癌的发生发展可能存在密切关系 ,SNPrs8792 84由于处在NGX6基因上游调控区域 ,其多态类型可能在某种程度上影响NGX6基因的表达调控 ,从而与鼻咽癌发病相关     

NGX6基因转染对鼻咽癌细胞基因表达谱的影响

鼻咽癌是我国南方的常见肿瘤 .NGX6是新近克隆的定位于 9p2 1 2 2 ,在鼻咽癌组织中表达下调的基因 .初步的研究结果显示 ,NGX6基因转染鼻咽癌细胞HNE1能够延缓其生长速度 ,使肿瘤细胞更多地停留在G0 G1期 .为探索NGX6基因在鼻咽癌发病机制中的作用 ,建立了高表达NGX6的鼻咽癌细胞系 .利用包含 14 0 0 0个基因的cDNA微阵列分析了NGX6基因转染对HNE1细胞基因表达谱的影响 ,发现NGX6基因的转染能够上调p2 9、APC7、NEU1、RNasek6、αE catenin和TFⅡEα等基因的表达 ;同时也下调properdinP因子、G0S2、BAZ2B、ZHX1,OS4和PBX3等基因的表达 .研究结果提示 ,NGX6基因对鼻咽癌细胞的生物学行为的影响可能与它对一些细胞周期调控因子和转录调控因子的影响相关 .作为一种高通量的分析技术 ,cDNA微阵列为新基因的功能研究提供了重要的线索     

NGX6 基因对高转移鼻咽癌细胞株 5-8F 细胞的影响

NGX6 是一个新克隆的鼻咽癌候选抑瘤基因 . 为进一步研究其功能,在构建 NGX6 的真核表达载体 NGX6/pcDNA3.1(+) 基础上,通过脂质体转染方法将 NGX6 基因导入鼻咽癌细胞株 SUNE-1 的亚株 5-8F 细胞 ( 具高成瘤高转移潜能 ) 中,并用 RT-PCR 和 RNA 印迹鉴定,建立了稳定表达 NGX6 基因的 5-8F 细胞系 . 借助细胞生长曲线、软琼脂集落形成实验对转染细胞的生物学行为进行了检测,同时采用包含 1 176 个与肿瘤学相关的基因 cDNA 微阵列,分析了 NGX6 基因转染对 5-8F 细胞基因表达谱的影响 . 结果显示：转染了 NGX6 基因的 5-8F 细胞的生长速度明显减慢,在软琼脂中集落形成率较对照组显著下降 (P < 0.05) ,发现 NGX6 基因的转染能够上调 5-8F 细胞中 p19 、 catenin α 2 、 desmoglein 1 等基因的表达,同时下调 EphB4 、 TIE2 、 vitronectin 等基因的表达 . 综上所述, NGX6 基因可以抑制 5-8F 细胞的恶性生物学行为,并影响一些与细胞周期、细胞黏附和血管生成有关的基因的表达 . 上述结果为鼻咽癌转移分子机制的阐明提供了重要的线索 .     

结肠癌中NGX6抑制EGFR/K-ras/JNK/c-Jun/cyclin D1信号通路的研究

候选抑瘤基因NGX6具有抑制结肠癌增殖和转移的作用,研究表明其为表皮生长因子受体(epidermal growth factor receptor, EGFR)的负性调控因子,并可下调JNK通路中重要分子MADD (MAP-kinase activating death domain)的表达,其抑瘤机制是否与抑制EGFR介导的JNK信号通路的活性有关?在已建立的转染NGX6的细胞模型基础上,借助蛋白质印迹(Western blot)和免疫组化方法在细胞和组织水平检测NGX6转染前后EGFR/K-ras/JNK/c-Jun/cyclin D1信号通路中重要蛋白质的表达.结果表明,NGX6转染后结肠癌细胞HT-29在裸鼠体内成瘤明显受抑,差异有统计学意义.Western blot结果显示,在结肠癌细胞中NGX6可明显下调EGFR、K-ras、p-JNK、c-Jun和cyclin D1的表达;进一步采用Western blot和免疫组化法验证NGX6在体内对上述关键分子表达的影响,发现NGX6可抑制裸鼠移植瘤组织中EGFR、K-ras、p-JNK、c-Jun和cyclin D1的表达,与体外结果一致.上述研究表明,NGX6在结肠癌中主要通过抑制EGFR介导的JNK通路的活性而发挥其抑瘤作用,该研究为深入探讨NGX6的机制提供了重要的实验依据.     

A caffeic acid phenethyl ester analog inhibits the proliferation of nasopharyngeal carcinoma cells via targeting epidermal growth factor receptor

A previous study reported that compound 5A, a caffeic acid phenethyl ester (CAPE) analog, exhibited obvious neuroprotective activity, in particular, compound 5A possessed higher stability and membrane permeability than CAPE. CAPE displays antitumour function; therefore, evaluating the antitumour effect of its analog with higher stability and membrane permeability is worthwhile. We first investigated the antitumour activity of compound 5A. We found that compound 5A significantly inhibited the proliferation of tumor cells and showed low cytotoxicity in normal cells. Furthermore, compound 5A was found to induce the cell cycle arrest and apoptosis of CNE2 cells. Through the prediction of SwissTargetPrediction and subsequent confirmation, epidermal growth factor receptor (EGFR) was identified as a target of compound 5A. Compound 5A also influenced the expression of genes downstream of EGFR in nasopharyngeal carcinoma (NPC) cells. Based on these findings, compound 5A inhibits the proliferation of NPC cells by targeting EGFR and may become a new candidate compound for NPC treatment.     

microRNA‐124 inhibits stem‐like properties and enhances radiosensitivity in nasopharyngeal carcinoma cells via direct repression of expression of JAMA

Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA‐124 (miR‐124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR‐124 in NPC CSCs. qRT‐PCR was performed to measure miR‐124 expression in NPC tissues and cell lines and the effects of miR‐124 on stem‐like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR‐124 with the 3′UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR‐124 in an animal model and clinical samples. Down‐regulation of miR‐124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR‐124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up‐regulation of miR‐124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR‐124 can inhibit stem‐like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.     

Identification of a New Seven-span Transmembrane Protein: NGX6a Is Downregulated in Nasopharyngeal Carcinoma and Is Associated With Tumor Metastasis

Nasopharyngeal carcinoma (NPC)-associated gene 6 (NGX6) is a novel candidate metastasis suppressor gene that can significantly decrease the growth, motility, and invasion of NPC cells. In this study, we generated a highly specific NGX6 polyclonal antibody and analyzed its distribution in the human fetus by Western blot and immunohistochemistry. The result of the Western blot showed the protein of NGX6 had two types of isoforms, isoform a (NGX6a) and isoform b (NGX6b). Isoform a is composed of 472 amino acids with a calculated molecular mass of 52 kDa, whereas isoform b is composed of 338 amino acids with a calculated molecular mass of 37 kDa. It is predicated that there is an epidermal growth factor domain in the N terminal of both a and b isoforms, and seven transmembrane domains in NGX6a, but only two transmembrane domains in NGX6b. The expression level of NGX6a was higher than that of NGX6b in human fetal tissue. Obvious high expression of NGX6a protein presents in the nervous system and epithelial tissues of the human fetus, but the NGX6b protein (37 kDa) is mainly expressed in the nervous system. We further analyzed the tissue microarray, which contained 154 NPC biopsies and 70 non-NPC biopsies, and found that NGX6a was significantly downregulated in NPC and associated with tumor metastasis. (J Histochem Cytochem 58:41–51, 2010)     

EGFR plays a pivotal role in the regulation of polyamine-dependent apoptosis in intestinal epithelial cells

Intracellular polyamine synthesis is regulated by the enzyme ornithine decarboxylase (ODC), and its inhibition by -difluromethylornithine (DFMO), confers resistance to apoptosis. We have previously shown that DFMO leads to the inhibition of de novo polyamine synthesis, which in turn rapidly activates Src, STAT3 and NF-κB via integrin β3 in intestinal epithelial cells. One mechanism to explain these effects involves the activation of upstream growth factor receptors, such as the epidermal growth factor receptor (EGFR). We therefore hypothesized that EGFR phosphorylation regulates the early response to polyamine depletion. DFMO increased EGFR phosphorylation on tyrosine residues 1173 (pY1173) and 845 (pY845) within 5 min. Phosphorylation declined after 10 min and was prevented by the addition of exogenous putrescine to DFMO containing medium. Phosphorylation of EGFR was concomitant with the activation of ERK1/2. Pretreatment with either DFMO or EGF for 1 h protected cells from TNF-/CHX-induced apoptosis. Exogenous addition of polyamines prevented the protective effect of DFMO. In addition, inhibition of integrin β3 activity (with RGDS), Src activity (with PP2), or EGFR kinase activity (with AG1478), increased basal apoptosis and prevented protection conferred by either DFMO or EGF. Polyamine depletion failed to protect B82L fibroblasts lacking the EGFR (PRN) and PRN cells expressing either a kinase dead EGFR (K721A) or an EGFR (Y845F) mutant lacking the Src phosphorylation site. Conversely, expression of WT-EGFR (WT) restored the protective effect of polyamine depletion. Fibronectin activated the EGFR, Src, ERKs and protected cells from apoptosis. Taken together, our data indicate an essential role of EGFR kinase activity in MEK/ERK-mediated protection, which synergizes with integrin β3 leading to Src-mediated protective responses in polyamine depleted cells.     

Hippo pathway contributes to cisplatin resistant-induced EMT in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is a kind of head-neck malignant tumor derived from the nasopharyngeal epithelium and is mainly prevalent in Southern China and Southeast Asia countries. Cisplatin (DDP) provides the first-line therapeutic administration in NPC patients. However, chemoresistance has been a main barrier and caused bad treatment outcome in NPC therapy. To understand the molecular mechanism of acquired resistance to DDP, multiple methods were performed to examine the morphocytology and molecular changes in DDP-resistant NPC cells. We found that drug resistance cells displayed epithelial-mesenchymal transition (EMT) characteristics. DDP-resistant NPC cells exhibited enhanced migration and invasion potential. Moreover, overexpression of TAZ, one key gene in Hippo pathway, is closely associated with the DDP resistance of NPC cells and its EMT properties. Depletion of TAZ in DDP-resistant cells reversed EMT phenotypes to MET characteristics and restored chemosensitivity of DDP-resistant cells to DDP treatment. These results suggest that inactivation of TAZ could be a promising approach for the treatment of NPC patients.     

miR-143 inhibits proliferation and metastasis of nasopharyngeal carcinoma cells via targeting FMNL1 based on clinical and radiologic findings

Mounting evidence has reported that microRNA-143 (miR-143) is involved in the development of multiple cancers. To investigate the underlying mechanisms of miR-143 regulating proliferation and metastasis in nasopharyngeal carcinoma (NPC) cells, we evaluated the levels of miR-143 and formin-like protein 1 (FMNL1) in NPC tissues. The results of qRT-PCR and Western blot analysis showed that the expression of miR-143 was decreased, while FMNL1 was increased in NPC tissues. The expression of miR-143 was significantly elevated in NPC cells compared with that of human nasopharyngeal epithelial cells. The results of MiRcode prediction, dual-luciferase reporter, and Western blot analysis assays indicated that miR-143 negatively regulated the expression of FMNL1 (r² = 0.4365P = 0.0001). Overexperssion of miR-143 or FMNL1 knockdown inhibited cell proliferation, migration, and invasion in NPC cells (P < 0.05). Ectopic expression of FMNL1 undermined the inhibition effect of miR-143 on proliferation, migration, and invasion in NPC cells. The findings of this study revealed that miR-143 functioned as a tumor suppressor and inhibited the NPC progression by targeting FMNL1.     

TIPE1‐mediated autophagy suppression promotes nasopharyngeal carcinoma cell proliferation via the AMPK/mTOR signalling pathway

Recent studies have shown that tumour necrosis factor‐α–induced protein 8 like‐1(TIPE1) plays distinct roles in different cancers. TIPE1 inhibits tumour proliferation and metastasis in a variety of tumours but acts as an oncogene in cervical cancer. The role of TIPE1 in nasopharyngeal carcinoma (NPC) remains unknown. Interestingly, TIPE1 expression was remarkably increased in NPC tissue samples compared to adjacent normal nasopharyngeal epithelial tissue samples in our study. TIPE1 expression was positively correlated with that of the proliferation marker Ki67 and negatively correlated with patient lifespan. In vitro, TIPE1 inhibited autophagy and induced cell proliferation in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells. In addition, knocking down TIPE1 expression promoted autophagy and decreased proliferation, whereas overexpressing TIPE1 increased the levels of pmTOR, pS6 and P62 and decreased the level of pAMPK and the LC3B. Furthermore, the decrease in autophagy was remarkably rescued in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells treated with the AMPK activator AICAR. In addition, TIPE1 promoted tumour growth in BALB/c nude mice. Taken together, results indicate that TIPE1 promotes NPC progression by inhibiting autophagy and inducing cell proliferation via the AMPK/mTOR signalling pathway. Thus, TIPE1 could potentially be used as a valuable diagnostic and prognostic biomarker for NPC.     

MicroRNA-320a inhibits cell proliferation,migration and invasion by targeting BMI-1 in nasopharyngeal carcinoma

In the present study, we investigated the roles and molecular mechanisms of miR-320a in human nasopharyngeal carcinoma (NPC). miR-320a expression was strongly reduced in NPC tissues and cell lines. Overexpression of miR-320a significantly suppressed NPC cell growth, migration, invasion and tumor growth in a xenograft mouse model. A luciferase reporter assay revealed that miR-320a could directly bind to the 3′ UTR of BMI-1. Overexpression of BMI-1 rescued miR-320a-mediated biological function. BMI-1 expression was found to be up-regulated and inversely correlated with miR-320a expression in NPC. Collectively, our data indicate that miR-320a plays a tumor suppressor role in the development and progression of NPC and may be a novel therapeutic target against NPC.     

Let‐7b‐5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377

KIAA1377 has been found to be linked with lymph node metastasis in esophageal squamous cell carcinoma (SCC) in our previous study; however, the regulation of KIAA1377 remains far from understood. Herein, to understand the regulation of KIAA1377 from the angle of microRNA (miRNA)–messenger RNA (mRNA) modulation in the setting of SCC cells, the basal level of KIAA1377 was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis in KYSE‐150 and HeLa cells; biological roles of KIAA1377 contributing in the proliferation, migration, and invasion were evaluated using 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), wound‐healing and Transwell assays, respectively, after KIAA1377 was knocked out mediated by the CRISPR‐Cas9 system. Bioinformatic prediction revealed that let‐7b‐5p was a putative miRNA regulating KIAA1377, which was ensuingly validated by the luciferase reporter assay; after which, variation of KIAA1377 expression was further verified by qRT‐PCR and western blot analysis. Moreover, the biological roles of let‐7b‐5p in proliferation, migration, and invasion of KYSE‐150 and HeLa cells were also evaluated. It was exhibited that KIAA1377 was able to promote the proliferation and motility of both KYSE‐150 and HeLa cells, which can be reverted by re‐expression of let‐7b‐5p. The luciferase reporter assay verified that let‐7b‐5p can diametrically target KIAA1377. Collectively, our data demonstrated that let‐7b‐5p can directly but negatively regulate KIAA1377 in SCC cell lines, Ecal109, and HeLa cells.     

Plac8‐mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway

To explore the relationship between autophagy and cell function, we investigated how PLAC8‐mediated autophagy influences proliferation, apoptosis and epithelial‐mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP‐GFP‐tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3‐methyladenine or by knocking down Beclin‐1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co‐immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.     

Suppression of bcl-2 gene by RNA interference increases chemosensitivity to cisplatin in nasopharyngeal carcinoma cell line CNE1

To explore the effect of suppressing BCL-2 expression using RNA interference (RNAi) technique in nasopharyngeal carcinoma cell line CNE1. CNE1 cell lines stably expressing shRNAs targeted bcl-2 and GL3 gene were established and gene expression inhibition was assessed by Western blotting analysis. The effect of suppressing bcl-2 by RNAi on cell growth was studied, the apoptosis induction and the sensitization of CNE1 cells to cisplatin were quantified by MTT assay and flow cytometry. The results showed that: stable transfection of CNE1