Lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of nasopharyngeal carcinoma cell line C666-1 in vitro

Latent membrane protein 2A (LMP2A) is found to play a key role in the development of nasopharyngeal carcinoma (NPC). However, the role of LMP2A silencing in the inhibition of cell growth of NPC has not been clarified. In this study, we inhibited LMP2A gene expression by lentivirus-mediated RNAi, to explore the effects of LMP2A silencing on the growth of NPC cell line in vitro. A lentivirus-mediated RNAi technology was employed to specifically knock down the LMP2A gene in NPC cell line C666-1. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and colony formation assays were performed to evaluate the expression of LMP2A and biological behavior of cell line C666-1 in vitro. We successfully construct a highly efficient and stable lentivirus vector, which efficiently downregulate the expression of LMP2A gene in infected cell line C666-1. Down-regulation of the expression of LMP2A significantly inhibits the proliferation and colony formation of C666-1 cells. In addition, the specific down-regulation of LMP2A arrests cells in G0/G1 phase of cell cycle and increases apoptosis rate. Our findings suggest that lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of NPC cell line C666-1 in vitro, and LMP2A may be a potential target for gene therapy in treatment of NPC.     

Epstein-Barr Virus_Encoded LMP1 Upregulates MicroRNA-21 to Promote the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin-Induced Apoptosis by Suppressing PDCD4 and Fas-L

Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC₅₀ value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.     

Id1 interacts and stabilizes the Epstein-Barr virus latent membrane protein 1 (LMP1) in nasopharyngeal epithelial cells

The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells.     

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor.

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.     

Acetylated histone H3 and H4 mark the upregulated LMP2A promoter of Epstein-Barr virus in lymphoid cells

We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap.     

Loss of AKR1C1 is a good prognostic factor in advanced NPC cases and increases chemosensitivity to cisplatin in NPC cells

Cisplatin resistance is one of the main obstacles in the treatment of advanced nasopharyngeal carcinoma (NPC). AKR1C1 is a member of the Aldo-keto reductase superfamily (AKRs), which converts aldehydes and ketones to their corresponding alcohols and has been reported to be involved in chemotherapeutic resistance of multiple drugs. The expression and function of AKR1C1 in NPC have not been reported until now. The aim of this research was to investigate the expression of AKR1C1 and it is role in cisplatin resistance in NPC. AKR1C1 protein expression was detected by immunohistochemistry in human NPC tissues and by Western blot assays in NPC and immortalized nasopharyngeal epithelial cells. The effects of AKR1C1 knock-down by siRNA on proliferation, migration and invasion in NPC cells were evaluated by CCK8, wound healing and transwell assays. To evaluate the effects of AKR1C1 silencing on cisplatin sensitivity in NPC cells, CCK8 assays were used to detect cell proliferation, flow cytometry was used to detect cell cycle distribution, and flow cytometry and DAPI staining were used to detect cell apoptosis. AKR1C1 down-regulation was associated with advanced clinicopathological characters such as larger tumor size, more lymphatic nodes involvement, with metastasis and later clinical stages, while AKR1C1 down-regulation was a good prognostic factor for overall survival (OS) in NPC patients. In vitro study showed that AKR1C1 was not directly involved in the malignant biological behaviours such as proliferation, cell cycle progression and migration of NPC cells, whereas AKR1C1 knock-down could enhance cisplatin sensitivity of NPC cells. These results suggest that AKR1C1 is a potential marker for predicting cisplatin response and could serve as a molecular target to increase cisplatin sensitivity in NPC.     

应用cDNA微阵列技术研究EB病毒LMP1介导的鼻咽癌中转移相关基因的表达

探讨EB病毒基因组编码的癌蛋白LMP1对鼻咽癌细胞中转移相关基因表达的影响.采用蛋白质印迹法检测在强力霉素(Dox)诱导下,鼻咽癌细胞系pTet-on-LMP1 HNE2(L7细胞)中LMP1表达的时效和量效关系.应用cDNA微阵列技术建立诱导性LMP1介导鼻咽癌细胞中转移相关基因差异表达谱;运用RT-PCR验证cDNA微阵列筛选差异基因表达的可靠性.与LMP1不表达的L7细胞比较,LMP1高表达的L7细胞中7个基因的表达显著上调,12个基因的表达显著下调.随机选择其中4个基因进行RT-PCR,结果显示,这些基因表达阳性,且与微阵列中的变化趋势一致.LMP1可能通过激活和/或抑制一些转移相关基因的表达而参与鼻咽癌转移过程.     

miR-21 modulates chemosensitivity of tongue squamous cell carcinoma cells to cisplatin by targeting PDCD4

Chemoresistance is a challenge for clinician in management of tongue cancer. Therefore, it is necessary to explore alternative therapeutic methods to overcome drug resistance. miRNAs are endogenous ?22nt RNAs that play important regulatory roles by targeting mRNAs. miR-21, an essential oncogenic molecule, is associated with chemosensitivity of several human cancer cells to anticancer agents. In this study, we investigated the effects and molecular mechanisms of miR-21 in chemosensitivity of tongue squamous cell carcinoma cells (TSCC) to cisplatin. miR-21 expression was detected in tongue cancer tissue using RT-PCR and PDCD4 protein expression was measured using immunohistochemistry. miR-21 and(or) PDCD4 depleted cell lines were generated using miR-21 inhibitor and(or) siRNA. The viabilities of treated cells were analyzed using MTT assay. RT-PCR was used to detect miR-21 expression and immunoblotting was used to detect protein levels. Cell cycle and apoptosis were analyzed using propidium iodide (PI) staining and Annexin V/PI staining, respectively. The expression of miR-21 in tumorous tissue was significantly higher compared with adjacent normal tissue and loss of PDCD4 expression was observed in TSCCs. Transfection of miR-21 inhibitor induced sensitivity of TSCC cells (Tca8113 and CAL-27) to cisplatin. TSCC cells transfected with PDCD4 siRNA became more resistant to cisplatin therapy. We found an increase PDCD4 protein level following the transfection of miR-21 inhibitor using Western blot analysis. In addition, the enhanced growth-inhibitory effect by miR-21 inhibitor was weakened after the addition of PDCD4 siRNA. Suppression of miR-21 or PDCD4 could significantly promote or reduce cisplatin-induced apoptosis, respectively. Our data suggest that miR-21 could modulate chemosensitivity of TSCC cells to cisplatin by targeting PDCD4, and miR-21 may serve as a potential target for TSCC therapy.     

Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.     

Upregulated expression of kappa light chain by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-kappaB and AP-1 pathways

B lymphocytes are generally considered to be the only source of immunoglobulins. However, increasing evidence revealed that some human epithelial cancer cell lines, including nasopharyngeal carcinoma (NPC) cell lines, expressed immunoglobulins. Moreover, we previously found that expression of kappa light chain in NPC cells could be upregulated by EBV-encoded latent membrane protein 1 (LMP1). Here, Western blot and flow cytometric analysis of intracellular kappa staining indicated that upregulation of the expression of kappa was inhibited by using LMP1-targeted DNAzyme and that Bay11-7082 and SP600125, inhibitors of JNK and NF-kappaB, respectively, inhibited LMP1-augmented kappa light chain expression in NPC cells. LMP1-positive NPC cells expressing the dominant-negative mutant of IkappaBalpha (DNMIkappaBalpha) or of c-Jun (TAM67) exhibited significantly decreasing kappa production compared with their parental cells. These results suggest that LMP1 elevated kappa light chain through activation of the NF-kappaB and AP-1 signaling pathways. The present study provided some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins.     

Epstein–Barr virus latent membrane protein 1 induces the chemotherapeutic target,thymidine phosphorylase,via NF-κB and p38 MAPK pathways

High thymidine phosphorylase (TP) expression is significantly correlated with poor prognosis in patients with nasopharyngeal carcinoma (NPC). NPC is an Epstein-Barr Virus (EBV)-associated cancer in which the EBV-encoded oncogene product, latent membrane protein 1 (LMP1), is expressed in approximately 60% of tumor tissues. However, no previous study has examined whether LMP1 is involved in up-regulating TP expression in NPC tissues. We herein show that LMP1 expression is correlated with TP expression in tumor cells, as examined by quantitative RT-PCR and immunohistochemical staining. We further show that the CTAR1 and CTAR2 domains of LMP1 mediate TP induction, as demonstrated by quantitative RT-PCR and Western blot analyses using LMP1 deletion and site-specific mutants. Mechanistically, LMP1-mediated TP induction is abolished by inhibitors of NF-κB and p38 MAPK, dominant-negative IκB and p38, and siRNA-mediated knockdown of p38 MAPK. Clinically, there were significant correlations among the expression levels of TP, activated p65, and phospho-p38 MAPK in NPC biopsy samples. Functionally, LMP1-mediated induction of TP expression enhanced the sensitivity of NPC cells to the chemotherapeutic prodrug, 5'-DFUR. Our results provide new insights into the roles of LMP1-mediated NF-κB and p38 MAPK signaling pathways in TP induction, potentially suggesting new therapeutic strategies for the treatment of NPC.     

Cyclophilin A binds to AKT1 and facilitates the tumorigenicity of Epstein-Barr virus by mediating the activation of AKT/mTOR/NF-κB positive feedback loop

The AKT/mTOR and NF-κB signalings are crucial pathways activated in cancers including nasopharyngeal carcinoma (NPC), which is prevalent in southern China and closely related to Epstein-Barr virus (EBV) infection. How these master pathways are persistently activated in EBV-associated NPC remains to be investigated. Here we demonstrated that EBV-encoded latent membrane protein 1 (LMP1) promoted cyclophilin A (CYPA) expression through the activation of NF-κB. The depletion of CYPA suppressed cell proliferation and facilitated apoptosis. CYPA was able to bind to AKT1, thus activating AKT/mTOR/NF-κB signaling cascade. Moreover, the use of mTOR inhibitor, rapamycin, subverted the activation of the positive feedback loop, NF-κB/CYPA/AKT/mTOR. It is reasonable that LMP1 expression derived from initial viral infection is enough to assure the constant potentiation of AKT/mTOR and NF-κB signalings. This may partly explain the fact that EBV serves as a tumor-promoting factor with minimal expression of the viral oncoprotein LMP1 in malignancies. Our findings provide new insight into the understanding of causative role of EBV in tumorigenicity during latent infection.     

Downregulation of BMI-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for nasopharyngeal carcinoma (NPC). However, drug resistance may occur after several cycles of 5-FU-based chemotherapy. The oncogene B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI-1) has been shown to be involved in the protection of cancer cells from apoptosis. In this study, 5-FU treatment could increase the percentage of apoptotic NPC cells among BMI-1/RNAi-transfected cells than that among cells transfected with the empty vector. The 50% inhibitory concentration (IC₅₀) values of 5-FU were significantly decreased to a greater extent in the cells transfected with BMI-1/RNAi. Most importantly, the expression of phospho-AKT and the anti-apoptotic protein BCL-2 were downregulated in the cells in which BMI-1 expression was inhibited, whereas the apoptosis-inducer BAX was observed to be upregulated. Abrogation of AKT pathway by a PI3K inhibitor could not further increase the sensitivity to 5-FU in the cells with reduced BMI-1 expression. Taken together, BMI-1 depletion enhanced the chemosensitivity of NPC cells by inducing apoptosis; which is associated with inhibition of the PI3K/AKT pathway.     

一株受四环素及其衍生物诱导表达的Tet-on鼻咽癌细胞系

Ｔｅｔ－ｏｎ基因表达系统是新近发展的一种真核生物体外表达系统．该系统利用四环素及其衍生物对所感兴趣基因进行诱导表达．这种诱导表达具有严密、高效、可控性强、表达泄露小等优点．对于研究一些致死基因及毒性强的基因在细胞或体内的表达提供了极好的工具．利用Ｔｅｔ－ｏｎ基因表达系统,构建了一株鼻咽癌细胞系,该细胞系的建立为进一步研究一些鼻咽癌发病相关基因,如ＥＢ病毒潜伏膜蛋白基因、瘤基因、抑瘤基因等与鼻咽癌的相关性,提供了理想的细胞模型．     

Down-regulation of EBV-LMP1 radio-sensitizes nasal pharyngeal carcinoma cells via NF-κB regulated ATM expression

Background

The latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice.

Results

In this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells.

Conclusions

Together, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs.