SATB1高表达通过促进上皮-间充质转化而介导鼻咽癌细胞侵袭和转移

目的:探讨鼻咽癌(NPC)中特殊富含AT序列结合蛋白1(SATB1)的表达及其与肿瘤侵袭和转移的关系。方法:免疫组化检测76例临床NPC及61例对照鼻咽黏膜慢性炎症(NPI)组织样本中SATB1、上皮型钙黏蛋白(E-cadherin)和波形蛋白(vimentin)的表达,并分析NPC中SATB1与患者临床参数、E-cadherin和vimentin表达的相关性。体外培养不同分化状态的NPC细胞系CNE1、CNE2Z及C666-1,筛选出SATB1高表达细胞系,采用小干扰RNA(siRNA)沉默SATB1表达后,分析上皮-间充质转化(EMT)相关分子的表达及细胞侵袭能力的改变。结果:临床鼻咽组织中SATB1表达定位于胞质和胞核,NPC组中SATB1阳性表达率显著高于对照NPI组(P0.01);E-cadherin在NPI黏膜上皮中呈膜阳性表达,NPC中膜表达水平下降,但出现胞质阳性。NPI中E-cadherin膜阳性率为100%,显著高于NPC的32.89%(P0.01)。Vimentin呈胞质阳性,NPI上皮细胞中均阴性,但NPC中阳性率为51.32%(P0.01)。NPC中SATB1高表达与患者性别、年龄及N分期无关,但与T分期和M分期均呈正相关(P0.05);SATB1高表达与vimentin呈正相关(r=0.358,P=0.009)。NPC细胞系中SATB1表达与细胞分化水平呈负相关。采用siRNA敲减C666-1细胞中SATB1表达后,E-cadherin表达上升,vimentin表达下降,且细胞侵袭能力下降。结论:SATB1高表达通过促进EMT而介导NPC临床进展。     

Sp1 is over-expressed in nasopharyngeal cancer and is a poor prognostic indicator for patients receiving radiotherapy

Nasopharyngeal cancer (NPC) is a tumor of epithelial origin with complex etiology. Currently the standard treatment of NPC is radiotherapy, but therapy failure is quite common, making radioresistance an important issue. This study explores the association of specificity protein 1 (Sp1) protein expression with clinicopathological significance and disease prognosis in NPC patients receiving radiotherapy. A total of 82 NPC patients (55 males and 27 females, median age: 48 years old) were enrolled and received radiotherapy between September 2011 and March 2014. Tumor tissue and grossly adjacent normal mucosa were obtained in each patient. Sp1 expression was detected by western blot and immunohistochemical analysis, and the associations with clinicopathological status and radiotherapy response were analyzed. Our Results showed Sp1 protein expression was higher in CNE-1 and CNE-2 nasopharyngeal cancer cells than in normal nasopharyngeal mucosal NP69 cells. All 82 patients’ tissue sections were stained positive for the Sp1 protein, and 39 (47.6%) patients showed higher level than adjacent normal mucosa. Sp1-overexpression in the tumor tissue was correlated with a higher tumor stage, nodal status, clinical stage and distant metastasis (P < 0.01). Patients with higher Sp1 expression in pretreatment biopsies had a lower radiotherapy response compared to those with lower expression. In conclusion, Sp1 may play roles in radioresistance of nasopharyngeal cancer which attributes to tumor invasiveness, and serve as a novel prognostic marker of NPC radiotherapy. However, further studies are required to validate our findings in larger samples and explore more detailed mechanisms underlying radioresistance of Sp1.     

miR-141-3p promotes proliferation and metastasis of nasopharyngeal carcinoma by targeting NME1

PurposeThis study aimed to investigate the expression and biological function of miR-141-3p in nasopharyngeal carcinoma (NPC) via targeting neoplasm metastasis 1 (NME1).Materials and methodsThe expression of miR-141-3p and NME1 in 5-8F, C666-1, CNE-1, CNE-2, 6-10B and NP69 nasopharyngeal epithelial cells were detected using real-time Polymerase Chain Reaction (real-time PCR) and western blot, respectively. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8), and the metastasis was detected using Transwell. The binding of miR-141-3p to NME1 was detected by dual luciferase reporter gene detection system. The effects of miR-141-3p on tumor growth were also determined in vivo.ResultsThe results showed that the expression of miR-141-3p significantly increased in various tumor cell lines and the expression of NME1 was higher in NP69 cells and lower in 5-8F cells, which had significant negative correlation. Furthermore, the expression of NME1 was significantly reduced after transfection of miR-141-3p and miR-141-3p promoted cell proliferation and metastasis. The double luciferase reporter gene detection system confirmed that NME1 was the target gene of miR-141-3p. Knockout of NME1 promoted the proliferation and metastasis of NP69 or 6-10B cells and the activation of p-Akt, which were abrogated by miR-141-3p. In vivo, the tumor volumes and weights in the miR-141-3p group significantly increased followed by down-regulation of NME1 and activation of p-Akt.ConclusionsWe confirmed that miR-141-3p promotes the proliferation and metastasis of NPC by targeting NME1.     

Association between interferon gamma 13-CA-repeats polymorphism and metastasis of nasopharyngeal carcinoma in a population of Northern China

Interferon Gamma gamma (IFN-γ) 13-CA-repeats polymorphism is associated with a variety of diseases; here we report its association with nasopharyngeal carcinoma (NPC) metastasis in a retrospective analysis of a cohort of 220 NPC patients in the northern China. The results showed that the distributions of CA13-/CA13-genotypes were significantly higher in the patients with lymph node metastasis (P<0.05) and distant metastasis (P<0.001); there was a significant difference between NPC patients with stage I+II and those with stage III+IV regarding CA13+/CA13-(P<0.001) and CA13-/CA13- genotypes (P<0.001); further analysis showed a more pronounced difference between NPC patients with stage I+II+III and those with stage IV for CA13-/CA13-genotype (P<0.001), whereas no difference was found for CA13+/CA13- genotype (P=0.790). Thus, we identify that IFN-γ 13-CA-repeat polymorphism is significantly associated with the metastasis of NPC, which may provide insights into its prognosis and individualized treatment.     

miR-21 inhibitor suppresses proliferation and migration of nasopharyngeal carcinoma cells through down-regulation of BCL2 expression

This study is to investigate the expression of miR-21 in nasopharyngeal carcinoma (NPC) cells, and the effect of miR-21 in the biological behavior and expression of B-cell lymphoma 2 (BCL2) in NPC cells. Paired NPC and adjacent non-tumor tissues were obtained from 53 patients who underwent primary surgical resection of NPC tissues. Luciferase reporter assay was performed to test whether BCL2 is a direct target of miR-21. Methylthiazolyl blue tetrazolium assay and colony assay were used to evaluate the effect of miR-21 on NPC cell proliferation. Transwell and wound-healing assays were carried out to test the effect of low expression of miR-21 on cancer cell migration and invasion. QRT-PCR and Western blotting were used to measure the levels of mRNA and protein expression, respectively. Tumor tissues showed a positive correlation between the levels of miR-21 and BCL2 protein expression. Cells transfected with miR-21 inhibitor healed slower compared the control (P < 0.05). In addition, cell migration was notably inhibited by the down-regulation of miR-21 in vitro (P < 0.05). The reduction in miR-21 expression showed a remarkable effect on the biological behavior of NPC cell clone formation (P < 0.05). Low expression of miR-21 by transfection with miRNA expression plasmid led to a decrease in BCL2 expression, which was accompanied by reduced migration and proliferation of the cancer cells. Our results demonstrated that miR-21 inhibitor down-regulated BCL2 expression level, suggesting that BCL2 might be a target gene for the initiation and development of NPC cells.     

p62/SQSTM1在鼻咽癌中的表达

目的:研究p62在鼻咽癌(nasopharyngeal carcinoma,NPC)细胞、组织中的表达情况及其自身抗体在外周血中的水平,探究p62与NPC发生、转移的关系及其在临床中的应用价值.方法:采用Western印迹法和免疫组织化学染色法分别检测NPC细胞株NP69,6-10B和5-8F和NPC患者组织中的p62表达;ELISA检测患者血清中抗p62抗体浓度,并分析其与临床病理特征的关系.结果:p62在NP69,6-10B和5-8F细胞中的表达水平依次升高(P＜0.05);NPC组织中p62高表达,其水平与患者有无转移(x2=9.332,P=0.002)有关.NPC患者血清中抗p62水平较慢性鼻咽炎组明显升高(t=-4.653,P＜0.001),且抗p62抗体水平与患者是否转移有关(t=14.255,P=0.016);血清抗p62抗体筛查NPC的最佳临界值为51.82 μg/L,判别是否发生转移的临界值为62.03 μg/L.结论:p62高表达与NPC发生及转移密切相关,组织中p62蛋白和血清中抗p62抗体可能作为NPC病情监测和判断预后的新指标.     

细胞周期蛋白E2在人鼻咽癌组织中的表达及临床意义

目的:研究细胞周期蛋白E2(Cyclin E2)在人鼻咽癌(NPC)组织中的表达及临床意义。方法:应用免疫组织化学法和RT-PCR法检测NPC组织及鼻咽非肿瘤组织Cyclin E2蛋白和mRNA的表达。结果:Cyclin E2蛋白在NPC和鼻咽非肿瘤组织中的表达率分别为75%(45/60)和9.5%(2/21),两者相比有统计学意义(P<0.01);鼻咽癌组织中Cyclin E2蛋白的过表达对同年龄、性别的患者无统计学差异(P>0.05),但与淋巴结转移、分期和5年生存率有相关性(P<0.01)。鼻咽癌组与对照组相比,Cyclin E2 mRNA表达亦有统计学差异(P<0.01)。结论:Cyclin E2表达对判断鼻咽癌病变进展、生存率及转移有重要参考价值。     

Kank1 reexpression induced by 5-Aza-2’-deoxycytidine suppresses nasopharyngeal carcinoma cell proliferation and promotes apoptosis

Kank1, which was first described as a potential tumor suppressor for renal cell carcinoma (RCC), mapped to 9p24.3 and encoded an ankyrin-repeat domain-containing protein. Its frequent deletion was found to be associated with several human malignant tumors, cerebral palsy, and neuronal and developmental diseases. However, its functional role in nasopharyngeal cancer (NPC) was still unknown. In the present study, we found that Kank1 expression was down-regulated in NPC cells than in human nasopharyngeal epithelial cell line NP69 and demethylating agent 5-aza-2’-deoxycytidine (5-aza-CdR) could improve its mRNA and protein expression level. Further studies demonstrated that DNA methylation might be the mainly cause for Kank1 decreased expression and restored Kank1 expression mediated by 5-aza-CdR played a key role in suppressing NPC cells growth and inducing its apoptosis. Our primary results revealed new function of Kank1 for NPC and implied that epigenetic regulation especially demethylation may have a potential value for NPC treatment.     

Periostin, a stroma-associated protein, correlates with tumor invasiveness and progression in nasopharyngeal carcinoma

Recently, the tumor microenvironment is increasingly recognized as playing an important role in cancer proliferation, invasion, and metastasis. To screen stroma-associated proteins involved in nasopharyngeal carcinoma (NPC) carcinogenesis, laser capture microdissection (LCM) and quantitative proteomic analysis were employed to assess different protein expression of the stroma between NPC and normal nasopharyngeal mucosa (NNM). In this study, periostin was identified to be significantly up-regulated in NPC stroma compared with NNM stroma and the result was further confirmed by Western blotting. Immunohistochemistry showed that over-expression of periostin was frequently observed in the stroma of NPC and matched lymph node metastases (LNM) compared with the stroma of NNM. Statistical analysis showed over-expression of periostin was significantly associated with advanced clinical stage (P?<?0.001) and lymph node metastasis (P?<?0.001) and decreased overall survival (P?<?0.001) in NPC. Cox regression analysis indicated over-expression of periostin was an independent prognostic factor. Furthermore, ectopic expression of periostin was used to examine its effect on invasiveness of NPC cell in vitro and the result showed that periostin was able to promote invasiveness of NPC cell. In conclusion, periostin expression is correlated with tumor stage, lymph node metastasis, and patient survival. Periostin is a potential biomarker for the differentiation and prognosis of NPC, and it might play an important role in the progression of NPC.     

Increased expression of IRS-1 is associated with lymph node metastasis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a head and neck malignant tumor rare throughout most of the world but common in Southeast Asia, especially in Southern China, which is with characteristics of early cervical lymph node metastasis and high incidence rate of distant metastasis. Insulin receptor substrate 1 (IRS-1) is a signaling adapter protein that is encoded by the IRS-1 gene in humans, plays an important role in the development, progression, invasion and metastasis of tumors. The aim of the present study was to investigate the association between the expression of IRS-1 protein and clinicopathological characteristics in NPC by immunohistochemistry. The results showed that the expression level of IRS-1 was significant higher in NPC than that in the control nasopharyngeal epithelia (P = 0.042). The positive percentage of IRS-1 expression in NPC with lymph node metastasis was also significantly higher than those without lymph node metastasis (P = 0.008). Positive expression of IRS-1 was proved to be the independent predicted factor for lymph node metastasis of NPC (P = 0.025) regardless of age, gender, histological type and clinical stages by multivariate logistic regression analysis. In addition, results showed higher sensitivity and agreement rate of IRS-1 for predicting lymph node metastasis of NPC patients. Taken together, high expression of IRS-1 might be closely correlated with lymph node metastasis in NPC and positive expression of IRS-1 could be used as an independent biomarker for predicting lymph node metastasis of NPC.     

Silencing SATB1 influences cell invasion,migration, proliferation,and drug resistance in nasopharyngeal carcinoma

Special AT rich sequence binding protein 1 (SATB1) play an important role in many cancers, but the role of SATB1 in nasopharyngeal carcinoma (NPC) is still not full understand. Immunofluorescence staining showed that SATB1 was mainly localized in the nuclei in CNE-2 cell. After successful down-regulation of SABT1 in NPC cell line CNE-2 by shRNA, compared to parental CNE-2 and control shRNA group, the capacity of the proliferation, migration, invasion and drug resistance of CNE-2 cell was reduced, which indicated that SATB1 may be involved in NPC development and progression. SATB1 may be a promising therapeutic target for nasopharyngeal carcinoma.     

Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein-Barr virus-encoded latent membrane protein 1

Undifferentiated nasopharyngeal carcinoma (NPC) is closely associated with EBV infection, and the EBV-encoded latent membrane protein 1 (LMP1) is frequently detected in NPC. However, little is known about the pathologic roles of LMP1 in this disease. Recently, we reported the morphologic transformation and increased expression of the LAMC2 and ITGalpha6 genes in LMP1-expressing NPC cell lines. In this study, we further examine the effects of LMP1 in an immortalized nasopharyngeal epithelial cell line called NP69. This cell line was established from primary nonmalignant nasopharyngeal epithelial cells and may represent a model of premalignant nasopharyngeal epithelial cells. LMP1 induced many phenotypic changes in NP69 cells. These include morphologic transformation, increased cell proliferation, anchorage-independent growth, resistance to serum free-induced cell death, and enhanced cell migration and invasion. In addition, expression array analysis identified 28 genes that demonstrated a more than 2-fold difference in expression of NP69 cells expressing LMP1 when compared with a vector control. Two of the up-regulated genes (VEGF and vimentin) identified have been previously reported as LMP1 targets. The majority of the identified genes are associated with cell growth, differentiation, cell shape, and invasion. The present findings support the proposed roles of LMP1 in promoting cell transformation, migration, and invasion in premalignant nasopharyngeal epithelial cells. The present study also indicates the activation of the Ras/MAPK pathway in LMP1-expressing cells, which may be involved in mediating some of the transforming effects of LMP1 observed in nasopharyngeal epithelial cells.     

Increase in tumour‐infiltrating lymphocytes with regulatory T cell immunophenotypes and reduced ζ‐chain expression in nasopharyngeal carcinoma patients

The pathological significance of the mechanisms of tumour immune-evasion and/or immunosuppression, such as loss of T cell signalling and increase in regulatory T cells (T_regs), has not been well established in the nasopharyngeal carcinoma (NPC) microenvironment. To evaluate the T_reg immunophenotypes in tumour-infiltrating lymphocytes (TILs), we performed a double-enzymatic immunostaining for detection of forkhead box P3 (FoxP3) and other markers including CD4, CD8, and CD25 on 64 NPC and 36 non-malignant nasopharyngeal (NP) paraffin-embedded tissues. Expression of CD3ζ and CD3ε was also determined. The prevalence of CD4⁺FoxP3⁺ cells in CD4⁺ T cells and the ratio of FoxP3⁺/CD8⁺ were increased significantly in NPC compared with those in NP tissues (P < 0·001 and P = 0·025 respectively). Moreover, the ratio of FoxP3⁺/CD25⁺FoxP3⁻ in NPC was significantly lower than that in NP tissues (P = 0·005), suggesting an imbalance favouring activated phenotype of T cells in NPC. A significant negative correlation between the abundance of FoxP3⁺ and CD25⁺FoxP3⁻ cells (P < 0·001) was also identified. When histological types of NPC were considered, a lower ratio of FoxP3⁺/CD25⁺FoxP3⁻ was found in non-keratinizing and undifferentiated carcinomas. Increased CD4⁺FoxP3⁺/CD4⁺ proportion and FoxP3⁺/CD8⁺ ratio were associated with keratinizing squamous cell carcinoma. A reduced expression of CD3ζ in TILs was found in 20·6% of the NPC tissues but none of the NP tissues. These data provide evidence for the imbalances of T_reg and effector T cell phenotypes and down-regulation of signal-transducing molecules in TILs, supporting their role in suppression of immune response and immune evasion of NPC.     

EB病毒LMP1促鼻咽上皮细胞增殖的蛋白分子鉴定

目的： 探讨EB病毒潜伏性膜蛋白 1(LMP1)促鼻咽上皮细胞增殖的分子机制。方法: 采用逆病毒感染的方法,将浓缩的逆病毒RV-pLNSX(空载体)和RV-LMP1分别感染鼻咽上皮细胞NP69,建立NP69-pLNSX与NP69-LMP1稳定表达细胞系,通过绘制细胞生长曲线、平皿克隆形成实验和软琼脂集落形成实验检测LMP1对NP69细胞增殖的影响;运用比较蛋白质组学方法鉴定LMP1在NP69细胞中参与调节的蛋白,并对部分蛋白表达进行验证。结果: (1) LMP1具有促鼻咽上皮细胞NP69增殖的作用(n=3,P<0.05)。(2) 鉴定了22个LMP1参与调节NP69细胞的蛋白(表达上调的蛋白9个,下调的13个),实时荧光定量RT-PCR和Western blotting证实了部分上述蛋白的差异表达。结论: LMP1可能通过参与调节keratin 19和vimentin等蛋白的表达促鼻咽上皮细胞NP69增殖。     

共刺激分子PD-L1在鼻咽癌组织中的表达和意义

目的探讨共刺激分子PD-L1在鼻咽癌组织的表达和临床意义,以及PD-L1的表达和鼻咽癌Foxp3＋淋巴细胞增多的相关性。方法采用免疫组化方法检测共刺激分子PD-L1在64例鼻咽癌的肿瘤组织和20例慢性鼻咽炎组织的表达情况及Foxp3＋淋巴细胞的分布。结果 PD-L1在鼻咽癌组织中阳性表达率为67.2%,明显高于其在慢性鼻咽炎组织中的表达,差异具有统计学意义（P〈0.05）。PD-L1的阳性表达与鼻咽癌病人性别、年龄、临床分期及是否转移等指标均无显著相关性（P〉0.05）,但与鼻咽癌组织中Foxp3＋淋巴细胞增加有关,差异有统计学意义（P〈0.05）,并且鼻咽癌PD-L1的表达也与Foxp3＋/CD8＋淋巴细胞比率增加正相关（P〈0.05）。结论鼻咽癌组织PD-L1在鼻咽癌组织中高表达,并与瘤内的调节性T细胞升高及CD8＋细胞下降有关,其可能在鼻咽癌的发展中起到一定作用,有望成为免疫治疗的一个新靶点。     

Overexpression of AIB1 in nasopharyngeal carcinomas correlates closely with advanced tumor stage

AIB1, a candidate oncogene in breast cancer, is commonly amplified and overexpressed in several types of human cancers. In this study, expression and amplification of AIB1 in nasopharyngeal carcinoma (NPC) were studied by immunohistochemical analysis and fluorescence in situ hybridization using tissue microarrays, including 80 specimens of NPC and 20 specimens of nonneoplastic nasopharyngeal mucosa. In this NPC cohort, overexpression and amplification of AIB1 was detected in 36 (51%) of 71 and 3 (7%) of 46 NPCs, respectively. Overexpression of AIB1 was observed more frequently in NPCs in late T stages (T3/T4, 24/35 [69%]) than in earlier stages (T1/T2, 12/36 [33%]; P < .05). In addition, 18 (72%) of 25 NPCs with lymph node metastasis (N1-3) showed overexpression of AIB1; the frequency was significantly higher than that in NPCs without node metastasis (N0, 18/49 [39%]; P < .05). These findings suggest that overexpression of AIB1 in NPCs may be important in the acquisition of an invasive and/or metastatic phenotype.     

Expression and clinical significances of Beclin1, LC3 and mTOR in colorectal cancer

Autophagy is related to cancer and other diseases, and compromised autophagy could promote chromosome instability associated with carcinogenesis and tumor progression. The role of autophagy in the growth and metastasis of colorectal cancer (CRC) remains poorly understood. Beclin1 mediates autophagic initiation, and LC3 is a specific marker for autophagy. Inactivation of mTOR caused by cellular hypoxia or energy deficiency induces autophagic activity. This study aims to examine the expression and clinical significance of these proteins in CRC. Immunohistochemistry results showed that the positive expression rates of Beclin1, LC3, and mTOR in cancer tissues were 90.50%, 87.19%, and 46.28%, respectively, which were higher than those in adjacent tissues (P < 0.05). Differentiation degree and lymph node metastasis were associated with LC3 overexpression (P < 0.05) but not with Beclin1 (P > 0.05). Lymph node metastasis was also related to mTOR. Spearman analysis results showed that LC3 expression was positively correlated with Beclin1 but negatively correlated with mTOR (r = 0.593 and -0.165, respectively; P < 0.01). Beclin1 expression was also not associated with mTOR (P > 0.05). Survival analysis further indicated that LC3, mTOR, and lymph node metastasis were independent prognostic factors in CRC. Real-time PCR results and Western blot indicated that Beclin1, LC3, and mTOR expression in CRC was significantly higher than that in adjacent tissues (P < 0.01). The aberrant protein expression may be associated with the development and progression of CRC. The LC3 and mTOR genes must be simultaneously detected to evaluate progression and prognosis of CRC.     

受体酪氨酸激酶Axl高表达促进鼻咽癌临床进展

目的:探讨受体酪氨酸激酶anexelekto(Axl)在鼻咽癌(nasopharyngeal carcinoma,NPC)中的表达及意义。方法:采用免疫组化法检测78例NPC和32例鼻咽黏膜慢性炎中Axl的表达,分析Axl蛋白表达与NPC患者临床参数的相关性。常规培养NPC细胞,免疫荧光法检测不同分化NPC细胞系CNE1、CNE2Z及C666-1中Axl的蛋白表达情况。应用Axl特异性抑制剂TP-0903处理CNE1和C666-1细胞,CCK-8实验检测细胞的活力,流式细胞术检测细胞周期的分布,q PCR检测Axl和增殖细胞核抗原(PCNA)的mRNA表达,Western blot检测Axl及p-Axl蛋白的表达。结果:Axl蛋白定位于胞膜和胞质。NPC中Axl高表达阳性率显著高于鼻咽黏膜慢性炎(P0.01)。Axl高表达与患者年龄、性别及M分期无关,与临床分期、T分期和N分期呈正相关(P0.05)。Axl在高分化CNE1细胞中低表达,在低分化CNE2Z细胞和未分化C666-1细胞中表达水平明显增高。TP-0903呈浓度和时间依赖性抑制NPC细胞的活性,2 nmol/L TP-0903即具有显著抑制效应,能阻滞细胞周期于G0期,在降低Axl活性的同时也显著抑制PCNA的表达。结论:Axl高表达可促进NPC的临床进展;TP-0903显著抑制NPC细胞的增殖,提示Axl可能在NPC靶向治疗中具有一定的价值。