Integrated analysis of the differential cellular and EBV miRNA expression profiles in microdissected nasopharyngeal carcinoma and non-cancerous nasopharyngeal tissues.

Nasopharyngeal carcinoma (NPC) is commonly diagnosed in southern Asia. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. Increasing evidence suggests that the dysregulation of miRNAs promotes NPC tumorigenesis. Epstein-Barr virus (EBV) infection and EBV-encoded miRNAs are also associated with the development of NPC. However, it is unclear how cellular and EBV miRNAs jointly regulate target genes and signaling pathways in NPC. In the present study, we analyzed the differential cellular and EBV miRNA expression profiles in 20 pooled NPC tissues using microarrays. We found that 19 cellular miRNAs and 9 EBV miRNAs were upregulated and 31 cellular miRNAs were downregulated in NPC tissues. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the 19 upregulated miRNAs target mainly the p53 signaling pathway in cancer, whereas the downregulated miRNAs regulate pathways related to cancer, focal adhesion and Erb, and MAPK signaling. In contrast, the upregulated EBV miRNAs target primarily the TGF-ß and Wnt signaling pathways. Data also suggested that cellular miR-34b, miR-34c, miR-18a, miR­200a/b, miR-449a, miR-31 and let-7 may be dysregulated in NPCs, and that the aberrant activation of their target genes in the p53 pathway and cell cycle enhance NPC cell survival and proliferation. In addition, EBV-miRNAs such as BART3 and BART5 target genes in the p53, TGF-ß and Wnt signaling pathways to modulate NPC apoptosis and transformation. To better elucidate the interaction between miRNAs and target genes, we constructed an anti-correlated cellular and EBV miRNA/target gene regulatory network. The current findings may help dissect the roles played by cellular and EBV miRNAs during NPC tumorigenesis, and also provide useful biomarkers for the diagnosis and treatment of NPCs.

Integrated analysis of differential miRNA and mRNA expression profiles in human radioresistant and radiosensitive nasopharyngeal carcinoma cells.

BACKGROUND: The purpose of this study was to identify miRNAs and genes involved in nasopharyngeal carcinoma (NPC) radioresistance, and explore the underlying mechanisms in the development of radioresistance. METHODS: We used microarrays to compare the differences of both miRNA and mRNA expression profiles in the radioresistant NPC CNE2-IR and radiosensitive NPC CNE2 cells, applied qRT-PCR to confirm the reliability of microarray data, adopted databases prediction and anticorrelated analysis of miRNA and mRNA expression to identify the miRNA target genes, and employed bioinformatics tools to examine the functions and pathways in which miRNA target genes are involved, and construct a miRNA-target gene regulatory network. We further investigated the roles of miRNA-23a and its target gene IL-8 in the NPC radioresistance. RESULTS: THE MAIN FINDINGS WERE FOURFOLD: (1) fifteen differential miRNAs and 372 differential mRNAs were identified, and the reliability of microarray data was validated for randomly selected eight miRNAs and nine genes; (2) 174 miRNA target were identified, and most of their functions and regulating pathways were related to tumor therapeutic resistance; (3) a posttranscriptional regulatory network including 375 miRNA-target gene pairs was constructed, in which the ten genes were coregulated by the six miRNAs; (4) IL-8 was a direct target of miRNA-23a, the expression levels of IL-8 were elevated in the radioresistant NPC tissues and showed inverse correlation with miRNA-23a expression, and genetic upregulation of miRNA-23a and antibody neutralization of secretory IL-8 could reduce NPC cells radioresistance. CONCLUSIONS: We identified fifteen differential miRNAs and 372 differential mRNAs in the radioresistant NPC cells, constructed a posttranscriptional regulatory network including 375 miRNA-target gene pairs, discovered the ten target genes coregulated by the six miRNAs, and validated that downregulated miRNA-23a was involved in NPC radioresistance through directly targeting IL-8. Our data form a basis for further investigating the mechanisms of NPC radioresistance.

Identification of integrin ß1 as a prognostic biomarker for human lung adenocarcinoma using 2D-LC-MS/MS combined with iTRAQ technology.

To discover novel lung adenocarcinoma (AdC) biomarkers, isobaric tags for relative and absolute quantitation (iTRAQ)-tagging combined with 2D-LC-MS/MS analysis was used to identify differentially expressed plasma membrane proteins in lung AdC and paired paraneoplastic normal lung tissues (PNLTs) adjacent to tumors. In this study, significant caveolin-1 downregulation and integrin ß1 upregulation was observed in primary lung AdC vs. PNLT. As there has been no report on the association of integrin ß1 with lung AdC, immunohistochemical staining was performed to detect the expression of integrin ß1 in an independent set of archival tissue specimens including 42 cases of PLNT, 46 cases of without lymph node metastasis primary AdC (non-LNM AdC) and 62 cases of LNM AdC; the correlation of their expression levels with clinicopathological characteristics and clinical outcomes were evaluated. Based on the data, upregulation of integrin ß1 was significantly correlated with advanced clinical stage and lymph node metastasis. Integrin ß1 overexpression was significantly associated with advanced clinical stage (P<0.05), lymph node metastasis (P<0.05), increased relapse rate (P<0.05) and decreased overall survival (P<0.05) in AdCs. Cox regression analysis indicated that integrin ß1 overexpression is an independent prognostic factor. The data suggest that integrin ß1 is a potential biomarker for LNM and prognosis of AdC and integrin ß1 upregulation may play an important role in the pathogenesis of AdC.

Identification of flotillin-1 as a novel biomarker for lymph node metastasis and prognosis of lung adenocarcinoma by quantitative plasma membrane proteome analysis.

To identify a novel lung adenocarcinoma (AdC) biomarker, iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed plasma membrane (PM) proteins in primary lung AdCs and paraneoplastic normal lung tissues (PNLTs). As a result, 36 differentially expressed membrane proteins were identified. Two differential PM proteins flotillin-1 and caveolin-1 were selectively validated by Western blotting. As there has been no report on the association of flotillin-1 with lung AdC, immunohistochemistry was further performed to detect the expression of flotillin-1 in the archival tissue specimens including 42 cases of PNLTs, 62 cases of primary lung AdCs with lymph node metastasis (LNM AdCs), and 46 cases of primary lung AdCs without lymph node metastasis (non-LNM AdCs), and the correlation of flotillin-1 expression levels in lung AdCs with clinicopathological features and clinical outcomes were evaluated. The results showed that up-regulation of flotillin-1 expression in lung AdCs was significantly correlated with advanced clinical stage, lymph node metastasis, increased postoperative relapse and decreased overall survival. Cox regression analysis revealed that the expressional level of flotillin-1 was an independent prognostic factor. The data suggest that flotillin-1 is a potential novel biomarker for lymph node metastasis and prognosis of lung AdC, and flotillin-1 up-regulation might play an important role in the pathogenesis of lung AdC.

Identification of heat shock protein 27 as a radioresistance-related protein in nasopharyngeal carcinoma cells.

PURPOSE: To identify the proteins involved in radioresistance in nasopharyngeal cancer (NPC) cells. METHODS: Sublethal ionizing radiation was applied to establish a radioresistant NPC cell line from its parental NPC cell line CNE1. Clonogenic survival assay, cell growth assay and flow cytometry analysis were used to examine the difference of radiosensitivity in the radioresistant CNE1 cells (CNE1-IR) and control CNE1 cells. Comparative proteomics was performed to identify the differential proteins in the two cell lines. Association of HSP27, one of upregulated proteins in CNE1-IR cells, with NPC cell radioresistance was selected for further investigation using antisense oligonucleotides (ASOs), clonogenic survival assay, Hoechst 33258 staining of apoptotic cells and MTT assay of cell viability. RESULTS: Radioresistant NPC cell line CNE1-IR derived from its parental cell line CNE1 was established. Thirteen differential proteins in the CNE1-IR and CNE1 cells were identified by proteomics, and differential expression of HSP27, one of identified proteins, was selectively confirmed by western blot. Inhibition of HSP27 expression by HSP27 ASOs decreased clonogenic survival and cell viability and increased cell apoptosis of CNE1-IR cells after irradiation, that is, enhanced radiosensitivity of CNE1-IR cells. CONCLUSION: The data suggest that HSP27 is a radioresistant protein in NPC cells, and its upregulation may be involved in the NPC radioresistance.

Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics.

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.

Identification of GLIPR1 tumor suppressor as methylation-silenced gene in acute myeloid leukemia by microarray analysis.

PURPOSE: To identify methylation-silenced genes in acute myeloid leukemia (AML). METHODS: Microarray analyses were performed in AML cell line HL-60 cells exposed to the demethylating agent 5-aza-2dC. The methylation status and expression of glioma pathogenesis-related protein 1 (GLIPR1), one of highly induced genes by demethylation, were further detected in six hematopoietic malignancy cell lines and 260 bone marrow samples from leukemia patients and nonmalignant diseases as control, as well as pre-treated and post-treated bone marrow samples from 24 complete remission AML patients received chemotherapy using MS-PCR, bisulfite DNA sequencing, RT-PCR, and Western blotting. RESULTS: One hundred and nine genes were significantly induced by demethylation in HL-60 cells, 12 genes of which were confirmed by RT-PCR. GLIPR1, a tumor suppressor gene, was frequently methylation-silenced in AML cell lines and AML patients, but not in the other hematopoietic malignancy cell lines and patients. The frequencies of methylation-silenced GLIPR1 in the pre-treatment were significantly higher than those in the post-treatment in complete remission AML patients. CONCLUSION: We identify 109 genes induced by demethylation in HL-60 cells, and demonstrate that GLIPR1 is a methylation-silenced gene in the AML patients, and may serve as a marker for monitoring disease activity during therapy in the AML patients. The data provide the important information for studying the pathogenesis of AML and discovering the target genes of methylating agents.

Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

BACKGROUND: The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells. RESULTS: We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins. CONCLUSION: The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin.

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

Identification of the proteins related to p53-mediated radioresponse in nasopharyngeal carcinoma by proteomic analysis.

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), and p53 is closely associated with the radiosensitivity of cancer, but the molecular mechanisms of p53-mediated radioresponse in NPC remains unclear. We previously established NPC CNE2sip53 cell line with p53 knockdown and paired control cell line CNE2/pSUPER, which provides a cell model system to investigate mechanisms of p53-mediated radioresponse in NPC. In this study, we first compared the radiosensitivity of CNE2sip53 and CNE2/pSUPER by a clonogenic survival assay, cell growth assay, and Hoechst 33258 staining and flow cytometry analysis of apoptotic cells. The results showed that the radiosensitivity of CNE2sip53 was significantly lower than that of CNE2/pSUPER, indicating that p53 plays a role in mediating NPC radiosensitivity. To search for the proteins associated with the p53-mediated radioresponse in NPC, a proteomic approach was performed to identify the radioresponsive proteins in CNE2sip53 and CNE2p/SUPER, respectively, and then the difference of radioresponsive proteins in CNE2sip53 and CNE2p/SUPER was compared. As a result, 14 differential radioresponsive proteins were identified in the two cell lines, 4 proteins of which were conformed by Western blot. Among them, 9 and 5 proteins were identified solely from CNE2p/SUPER and CNE2sip53, respectively. Furthermore, protein-protein interaction analysis showed that 7 differential radioresponsive proteins identified only in CNE2p/SUPER were related to p53 protein. Our results suggest that the differential radioresponsive proteins unique to CNE2p/SUPER may be involved in p53-mediated radioresponse in NPC, which will be helpful for elucidating the mechanisms of p53-mediated NPC cellular response to radiotherapy.