Effects of two EBV-LMP1 variants on proliferation characteristics of CNE1 cell line

AIM: To investigate the possible effect of different Latent membrane protein 1 (LMP1) variants on proliferation characteristics of a human well-differentiated nasopharyngeal carcinoma (NPC) cell line CNE1. METHODS: The plasmids which carried EBV-LMP1 gene cloned from B95-8 lymphocyte (B95-8-LMP1) and NPC tissues (CAO-LMP1) were introduced into CNE1 by liposomal transfection. Expression of LMP1 in CNE1 was identified by RT-PCR and Western blot, respectively. Different Effects of the two EBV-LMP1 variants on proliferation characteristics of CNE1 including growth curve, cell cycle distribution and clony-forming efficiency were investigated by means of crystal violet staining proliferation assay, flow cytometry and plastic plate clony-forming assay, respectively. RESULTS: Two transfected cell lines stably expressing different LMP1 variants were established successfully. Either of the two LMP1 variants increased CNE1 growth rate, proliferation index (PI) and clony-forming rate significantly and CAO-LMP1 had more significant growth-promoting effect on CNE1 than B95-8-LMP1. CONCLUSION: It can be concluded from the study that CAO-LMP1 promotes CNE1 proliferation more effectively than B95-8-LMP1.     

Effect of Epstein-Barr virus latent membrane protein 1 on oncomiRs expression profile in nasopharyngeal carcinoma cell line CNE1

AIM: To investigate the differential expression profile between nasopharyngeal carcinoma cell line CNE1 and its steady EBV-LMP1-transfected cell line CNE1-LMP1, and to explore the regulatory effect of LMP1 on oncomiRs expression in CNE1 cell line. METHODS: A microRNA array that targets 132 of the most well studied oncomiRs was used to detect the expression profile of CNE1 and CNE1-LMP1. qRT-PCR assay were used to verify the expression data detected by microarray. RESULTS: Among the restricted 132 miRNAs, 30 were detectable. Among which, 30 were expressed in CNE1-LMP1, 19 in CNE1 and 11 were specifically expressed in CNE1-LMP1. Among the 19 shared miRNAs, the expression level of 6 miRNAs (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150 and hsa-miR-188) elevated over two folds in CNE1-LMP1. No decrease in miRNA expression more than two folds was observed. qRT-PCR confirmed the expression difference of these six miRNAs (P<0.01). Among the 11 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a showed the highest expression level surpassing the internal control sample. CONCLUSION: Our data suggest that LMP1 may play an important role in regulating the expression of miRNAs in tumor, which may be another important pathway employed by LMP1 in the development of nasopharyngeal carcinoma.     

Latent membrane protein 1 regulates expression of p27 and RPLP0 in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues

AIM: To observe the protein expression of p27 and ribosomal phosphoprotein large P0(RPLP0) regulated by latent membrane protein 1 (LMP1) in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues. METHODS: The protein levels of p27 and RPLP0 and the relationship with LMP1 were analyzed by Western blotting. The protein expression of LMP1, p27 and RPLP0 was also detected by the method of immunohistochemistry in 30 nasopharyngeal epithelial and 60 nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues. Meanwhile, the significance of clinical pathology was evaluated. RESULTS: The positive rate of LMP1 protein was 73.3% and 90.0% in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues, respectively. Compared with the LMP1-negative tissues, the protein levels of RPLP0 were low in the nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues with LMP1-positive expression, but the levels of RPLP0 protein were overexpressed. The protein expression of RPLP0 and RPLP0 was related to the age of nasopharyngeal carcinoma patients, the protein level of LMP1, the metastasis of lymph nodes and the TNM classification. The positive expression of p27 protein at high level was usually observed in the patients with young age, or had the characteristics of LMP1 (-), non-metastasis of lymph nodes, and in I or II stage of TNM classification. However, the protein expression of RPLP0 was low (P<0.05). CONCLUSION: LMP1 down-regulates p27 and up-regulates RPLP0 in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues.     

Establishment of nasopharyngeal carcinoma (NPC) cell lines stable expressing NPC-derived LMP1

AIM: To establish nasopharyngeal carcinoma(NPC) cell lines stable expressing NPC-derived latent membrane protein 1(LMP1) gene.METHODS:General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed by using recombinant techniques, then transfected these vectors into a poor differentiated NPC cell line named CNE-2 ,integration and expression of N-LMP1 in CNE-2 cells were detected by PCR,RT-PCR and Western blot. RESULTS:(1) General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed successfully.(2) It showed that N-LMP1 gene expressed in CNE-2 cells correctly.CONCLUSION: The first NPC cell lines which stable express NPC-LMP1 were established. The cell lines obtained will provide important basis for exploring the role of NPC-LMP1 in nasopharynx carcinogenesis.     

TMT-tagged quantitative proteomics analysis of differentially expressed proteins and enrichment of signaling pathways in nasopharyngeal carcinoma cells with or without SATB1 over-expression

AIM: To analyze the effects of special AT-rich sequence binding protein 1 (SATB1) expression on the protein expression profiles in human nasopharyngeal carcinoma (NPC) cells, and to enrich the differential signaling pathways through bioinformatics analysis. METHODS: SATB1 over-expressing lentivirus and negative control lentivirus were used to infect the CNE1 cells, and then the cell lines were obtained by puromycin stressed method. The total proteins of the 2 cells were extracted, and the differentially expressed proteins were screened by TMT-labeled protein quantification technique and tandem mass spectrometry. The mRNA levels of the differential protein-coding genes were verified by RT-qPCR. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the signaling pathways of differential proteins. RESULTS: SATB1 over-expressing CNE1 cells were established through infected with associated lentivirus. Compared with the control group, 278 differentially expressed proteins were identified in SATB1 over-expressing CNE1 cells, in which 115 were up-regulated and 163 were down-regulated. 10 representative differential protein-coding genes were verified by RT-qPCR, which showed the consistence with the proteomic results. GO analysis indicated differentially expressed proteins were mainly involved in cellular processes, single-organism processes, biological regulation, metabolic processes, protein binding and catalysis. Cell components of differentially expressed proteins mainly existed in cell part, cells and organelles. KEGG analysis showed that differentially expressed proteins were involved in signaling pathways closely related to tumors, includeing MAPK, PI3K-Akt, AMPK, JAK-STAT, p53, PPAR, Hippo and HIF-1 signaling pathways. CONCLUSION: Over-expression of SATB1 significantly alters the protein expression profiles in the NPC cells and affects multiple signaling pathways closely related to tumors. Proteomics also provides a possible macro approach to the screening of molecular mechanisms, therapeutic and prognostic targets for NPC.     

Construction of SETD2 gene knockout nasopharyngeal carcinoma cell strains using CRISPR/Cas9 technique and analysis of their proliferation property

AIM:To establish SETD2 gene knockout nasopharyngeal carcinoma (NPC) cell strains based on CRIPSR/Cas9 technique and to analyze their proliferation characteristics. METHODS:Sub-quantitative RT-PCR and Western blot were used to detect the expression of SETD2 in immortalized nasopharyngeal epithelial cell line NP-69, well differentiated NPC cell line CNE1, poorly-differentiated NPC cell line CNE2Z and undifferentiated NPC cell line C666-1, and the SETD2 high expression cell line CNE1 was screened. The proliferation ability of CNE1 cells before and after the SETD2 gene knockout was analyzed by CCK-8 and colony formation assay. The cell cycle distribution was detected by flow cytometry, and the expression of cell cycle-related proteins was detected by Western blot. RESULTS:Compared with NP-69 cells, the expression of SETD2 was decreased gradually in CNE1, CNE2Z and C666-1 cells (P<0.01). Based on the CRISPR/Cas9 technique, 2 monoclonal cell strains with SETD2 gene stable knockout, named CNE1-SETD2-KO-#5 and #9, were successfully screened from total 15 monoclones. The results of CCK-8 and plate colony formation assay confirmed that the proliferation ability of CNE1-SETD2-KO-#5 and #9 cells was significantly enhanced compared with CNE1-WT cells (P<0.05). The results of flow cytometry analysis showed that the G₁ phase of CNE1-SETD2-KO-#5 and #9 cells was decreased, while the G₂/M and S phases were increased significantly (P<0.05). The results of Western blot confirmed the increases in the protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin B1, cyclin A2, cyclin E1, cyclin-dependent kinases 2 (CDK2) and CDK4, and the decrease in the protein level of p21 after SETD2 gene knockout (P<0.05). CONCLUSION:The NPC cell strains with SETD2 gene knockout were successfully constructed based on CRISPR/Cas9 technique. SETD2 expression correlates with cell differentiation status in the NPC cells. SETD2 gene knockout promotes NPC cell proliferation by up-regulating cyclin D1, cyclin B1, cyclin A2, cyclin E1, CDK2 and CDK4, and down-regulating p21 expression.     

Over-expression of SATB1 mediates invasion and metastasis of nasopharyngeal carcinoma cells through epithelial-mesenchymal transition

AIM:To analyze the high expression of special AT-rich sequence-binding protein 1 (SATB1) in nasopharyngeal carcinoma (NPC) and its role in tumor invasion and metastasis. METHODS:The method of immunohistochemistry was used to detect the expression of SATB1 and epithelial-mesenchymal transition (EMT)-related molecules E-cadherin and vimentin in 76 cases of NPC and 61 cases of nasopharyngeal chronic inflammation (NPI), and the correlations of over-expression of SATB1 with NPC patients' clinical parameters as well as the expression of E-cadherin and vi-mentin were analyzed. Variously differentiated NPC cell lines CNE1, CNE2Z and C666-1 were cultured in vitro, and then SATB1-overexpressing cell line was screened. After interfering with SATB1 expression by siRNA, the expression of EMT-related molecules and the change of cell invasiveness were analyzed. RESULTS:The expression of SATB1 in the nasopharyngeal tissue was dominantly localized in the nuclei. The positive rate of SATB1 in NPC group was significantly higher than that in NPI group (P<0.01). E-cadherin was membrane-positive in NPI epithelial cells, while membrane E-cadherin in NPC was decreased but cytoplasmic expression was increased. The positive expression rate of membrane E-cadherin in NPI was significantly higher than that of NPC (P<0.01). Vimentin was localized in cytoplasm and negative in NPI epithelial cells, but the positive rate in NPC parenchymal cells was significant higher than that in NPI (P<0.01). The high expression of SATB1 in NPC was not related to the patents' sex, age, clinical classification and N classification, but positively correlated with T and M classification (P<0.05). Besides, high expression of SATB1 was positively correlated with vi-mentin in NPC tissues (r=0.358, P=0.009). SATB1 expression in NPC cell lines was negatively correlated with the levels of cell differentiation. Knockdown of SATB1 expression in C666-1 cells with siRNA was accompanied by an increase in E-cadherin and a decrease in vimentin levels, as well as a decrease in cell invasiveness. CONCLUSION:High expression of SATB1 promotes the clinical progress of NPC through EMT mechanism.     

Effects of EBV infection on growth and apoptosis of nasopharyngeal carcinoma cell line

AIM: To explore the effect of EBV infection on growth and apoptosis of nasopharyngeal carcinoma(NPC) cell line.METHODS: NPC cell line CNE1 was directly infected by Epstein Barr virus (EBV). The expression of EBV-latent membrane protein 1 (EBV-LMP1) and bcl-2 were detected by immunohistochemistry method (LSAB). The growth of NPC cells was identified by MTT method. Apoptotic carcinoma cells were detected by flow cytometry analysis and the terminal deoxynucletidyl transferase-medicated dUTP-biotin nick end labeling (TUNEL) methods. RESULTS: EBV-LMP1 was positive in CNE1 infected by EBV(E-CNE1). Compared with CEN1, the growth of E-CNE1 apparently increased (P＜0.01). No apoptotic carcinoma cells were detected and bcl-2 postive cells were 2%~3% respectively in 2 kinds of NPC cells.CONCLUSION: Growth of NPC cells is enhanced by EBV infection and EBV-LMP1 expression, but no influence on expression of bcl-2 and apoptosis of NPC cells.     

Effects of EBV infection on growth and apoptosis of nasopharyngeal carcinoma cell line

AIM:To explore the effect of EBV infection on growth and apoptosis of nasopharyngeal carcinoma(NPC)cell line.METHODS:NPC cell line CNE1 was directly infected by Epstein Barr virus(EBV).The expression of EBV-latent membrane protein 1(EBV-LMP1)and bcl-2 were detected by immunohistochemistry method(LSAB).The growth of NPC cells was identified by MTT method.Apoptotic carcinoma cells were detected by flowcytometry analysis and the terminal deoxynucletidyl transferase-medicated dUTP-biotin nick end labeling(TUNEL)methods.RESULTS:EBV-LMP1 was positive in CNE1 infected by EBV(E-CNE1).Compared with CEN1, the growth of E-CNE1 apparently increased(P<0.01).No apoptotic carcinoma cel s were detected and bcl-2 postive cells were 2%~3%respectively in 2 kinds of NPC cells.CONCLUSION:Growth of NPC cells is enhanced by EBV infect ion and EBV-LMP1 expression, but no influence on expression of bcl-2 and apoptosis of NPC cells.     

Effect of NP9 on gene expression profile of NPC cell line CNE1

AIM: To identify the gene expression profiles of CNE1 cells stably transfected with NP9 expressing plasmid, and to explore the potential molecular function of NP9 gene.METHODS: CNE1 cells stably expressing NP9 protein and CNE1 cells transfected with empty vector were used as test and control. Differentially expressed genes were screened with high- throughout human genome array. Differential expression of 6 genes was analyzed with quantitative RT-PCR. RESULTS: Of all the 14 500 human genes in array, 266 genes were revealed differential expression between test and control , of which 82 genes (RA>1)were up-regulated in test and 184 genes (RA<1) were down-regulated. 34 genes and 75 genes were found distinctively up-regulation (RA>1.5) and down-regulation (RA<1.5),respectively.CONCLUSION: NP9 expression in CNE1 cells leads to changes of some genes involved in regulation of cell cycle, cell proliferation and differentiation, cell signal transduction, cell adhesion. Some new clues may be provided for further studying the potential function and molecular mechanism of NP9 gene.     

Effect of transketolase-like protein 1 on proliferation of human nasopharyngeal carcinoma cells

AIM: To investigate the effect of transketolase-like protein 1 (TKTL1) on proliferation of human nasopharyngeal carcinoma cells in vitro. METHODS: The siRNA against TKTL1 mRNA was constructed and transfected into human nasopharyngeal carcinoma cells (CNE cell line). The activity of transketolase was detected before and after RNA interference.Real-time PCR was used to determine the mRNA expression of transketolase (TKT) gene family in the CNE cells.Flow cytometry and MTT test were used to detect the effect of anti-TKTL1 siRNA on cell proliferation and cell cycle in the CNE cells. RESULTS: The total transketolase activity was significantly decreased in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector or untransfected CNE cells. No significant difference in the expression level of TKT and TKTL2 gene between the CNE cells transfected with siRNA TKTL1 construct and the cells transfected with control vector or untransfected CNE cells was observed (P>0.05). However, the expression level of TKTL1 gene was significantly downregulated in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector.Cancer cells were arrested in G0/G1 phase, and cancer cell proliferation was significantly inhibited in the CNE cells transfected with siRNA TKTL1 construct. CONCLUSION: TKTL1 plays an important role in the total transketolase activity and cell proliferation of human nasopharyngeal carcinoma. TKTL1 may be considered as a potential target for novel anti-cancer therapy.     

Effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45

AIM: To investigate the effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45. METHODS: The protein levels of CADM1 in 3 human gastric carcinoma cell lines were detected by Western blotting. Eukaryotic expression vector pcDNA-CADM1 was constructed and transfected into MKN-45 cells. The MKN-45 cells stably expressing CADM1 were selected by G418 and identified by Western blotting. Furthermore, CCK-8 assay and Boyden chamber were used to analyze the effects of CADM1 overexpression on the prolife ration and invasion of gastric carcinoma cells. Western blotting was also utilized to detect the levels of cell proliferation- and invasion-related proteins. RESULTS: Relative level of CADM1 protein in MKN-45 cells was significantly lower than that in MKN-28 cells and SGC-7901 cells. Additionally, eukaryotic expression vector pcDNA-CADM1 was successfully constructed and MKN-45 cells stably expressing CADM1 were obtained. Compared with non-treatment and pcDNA3.1 groups, the proliferation of MKN-45 cells was obviously inhibited in pcDNA-CADM1 group. The result of Boyden chamber showed that the migrated cell numbers in pcDNA-CADM1 group (52.35±3.89) were significantly lower than that in untreated group (101.53±6.89) and pcDNA3.1 group (98.77±7.03). Compared with non-treatment and pcDNA3.1 groups, the protein level of p21 was significantly up-regulated and protein expression of MMP-2 and MMP-9 was obviously down-regulated. CONCLUSION: Overexpression of CADM1 may markedly inhibit cell proliferation and reduce invasion ability, and thus may be a novel target for treating gastric carcinoma.     

Expression of genes encoding gap junction protein in normal human nasopharyngeal epithelial tissue

AIM:To examine the expression of gap junction protein family genes, including thirteen independent genes, in normal human nasophryngeal epithelial tissue and to conjecture the possible roles of gap junction proteins in nasopharyngeal carcinoma.METHODS:With synthesized primers, the expression of thirteen genes encoding different gap junction proteins in human normal nasopharyngeal epithelial tissue were detected by RT-PCR.RESULTS:In 18 samples of normal human nasopharyngeal epithelial tissue, 16 of them were found the expression of Cx 30, 31.1, 17 of them were found the expression of Cx 37 and Cx 43, and Cx 40 expression were detected in 15 samples. Also the expression of Cx 26, 31, 32, 36, 45, 46, 46.6, 50 were detected respectively in 10, 11, 9, 1, 9, 0, 1,3 samples of the 18 cases.CONCLUSION:In normal human nasopharyngeal tissue, Cx 30, 31, 31.1,37, 40, 43 might be the key gap junction proteins.     

The effect of EBV latent membrane protein 1 on proliferation and cell cycle of nasopharyngeal carcinoma cells

AIM:To investigate the effect of Epstain-Barr virus latent membrane protein 1 (EBV-LMP1) on proliferation and cell cycle of nasopharyngeal carcinoma (NPC) cells. METHODS:The expression of EBV-LMP1 was detected by immunohistochemical method (LSAB). Proliferation of NPC cells was identified by MTT method. Cell cycle percentage was detected by flow cytometry analysis. RESULTS: OD value of EBV-LMP1 expressive NPC cells L-CEN1 was much higher than that of both EBV-LMP1 negative NPC cells V-CEN1 and CNE1 (P＜0.01). Compared with the cell cycle percentage in both V-CNE1 and CNE1, the percentage of G₁ was significantly decreased and the percentage of S was much increased in L-CNE1 (P＜0.01). But no obvious differences were observed in all cell cycle percentage between V-CNE1 and CNE1 (P＞0.05).CONCLUSION:The expression of EBV-LMP1 on NPC cell might cause some change of cell cycle and enhance cell proliferation.     

Calreticulin promotes migration and invasion of nasopharyngeal carcinoma cells by inducing cell EMT

AIM:To observe the expression of calreticulin (CRT) in nasopharyngeal carcinoma tissues, analyze the significance of clinical pathology and the influence on epithelial-mesencymal transition (EMT) of CNE2 cells. METHODS:The expression of calreticulin was detected by immunohistochemistry in 52 nasopharyngeal carcinoma and 57 nasopharyngeal benign tissues, and the significance of clinical pathology was evaluated. The calreticulin gene-specific small interfering RNA was constructed, and then was transfected into the NPC cell line CNE2 using the cationic liposome method. The effect of CRT on the morphological changes of the CNE2 cells was observed under light microscope. The effect of CRT on the cell migration and invasion abilities of the CNE2 cells was detected by Transwell migration and invasion assays. The expression of EMT-related proteins E-cadherin, vimentin, transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 in the CNE2 cells was determined by Western blot. RESULTS:The positive expression rate of CRT in the benign lesion tissues was 19.29% (11/57), which was significantly increased in the nasopharyngeal carcinoma tissues as 82.69% (43/52). The expression rate of CRT was positively correlated with the stage of nasopharyngeal carcinoma and lymph node metastasis (P<0.05). Knockdown of CRT expression made the CNE2 cells showing a spindle shape to a flat, cobblestone-like epithelial state change, arranged more compact, and the migration and invasion abilities were significantly decreased (P<0.05). Knockdown of CRT expression resulted in significant increase in the protein expression of E-cadhe-rin, and the decreases in the protein expression of vimentin, TGF-β and MMP-9 in the CNE2 cells (P<0.05). CONCLUSION:Calreticulin expression in nasopharyngeal carcinoma is significantly higher and positively correlated with nasopharyngeal carcinoma stage and lymph node metastasis. Calreticulin promotes cell migration and invasion of nasopharyngeal carcinoma CNE2 cells by inducing EMT.     

BPH-1 induces aromatase expression in prostatic stromal cells through paracrine of PGE2

AIM: To characterize the effect of prostatic epithelial cell paracrine on aromatase expression in prostatic stromal cells.METHODS: Conditioned medium (CM) of prostatic epithelial cell lines (BPH-1, LNCap, DU-145 and PC3) were collected and used to treat prostatic stromal cells. Expression of aromatase was determined by real-time RT-PCR and Western blotting. Expression of cyclooxygenase-2 mRNA in prostatic epithelial cell lines and prostaglandin (PGE2) in CMs were examined by real-time RT-PCR and ELISA, respectively. The CM of BPH-1 cells cultured with NS-398, specific inhibitor of cyclooxygenase-2, were collected, and the effect of NS-398 and PGE2 on aromatase expression was analyzed.RESULTS: CM of human benign prostate hyperplasia epithelial cell line (BPH-1) stimulated expression of aromatase mRNA and protein in stromal cells. But CM of prostate cancer epithelial cell lines (LNCap, DU145, PC3) had no effect on aromatase expression. COX-2 mRNA level in BPH-1 was much higher than that of other cell lines and PGE2 concentration in BPH-1 CM was much higher than that of other CMs. PGE2 concentration of the CM from BPH-1 cultured with NS-398 significantly decreased. CM from BPH-1 cultured with NS-398 failed to stimulate aromatase expression, while PGE2 induced aromatase expression in prostatic stromal cells.CONCLUSION: BPH-1 could induce aromatase expression in prostatic stromal cells through paracrine of PGE2.