Comparative Proteomics Analysis Of Nasopharyngeal Carcinoma And Normal Nasopharyngeal Epithelial Cells Purified By Laser Capture Microdissection

Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in southern China,with an incidence rate ranging from 20 to 50/100,000,and it poses one of the most serious public health problems in southern China.As an etiologically multi-factorial disease,carcinogenesis of the nasopharynx may result from combined effects of Epstein-Barr viral, genetic and environmental factors.Although numerous efforts have been made to reveal the molecular mechanism of NPC carcinogenesis,it remains poorly understood.Identification of NPC biomarkers will be helpful for diagnosis and treatment of NPC,and may provide new insights into its pathogenesis.High throughput omics technologies such as microarrays and proteomics offer the potential ability to find alterations previously unidentified in NPC. Analyses for gene expression profiles of NPC have been reported using a cDNA array,found the genes with aberrant expressions possibly contributed to pathogenesis of NPC.Because the functional molecules in cells are proteins, proteome analysis is believed to have an advantage over cDNA microarray for clinical use.Proteomics has introduced a new approach to cancer research which aims at identifying differential expression proteins associated with the development and progression of cancer,providing new opportunities to uncover biomarkers and therapeutic targets for cancer.Using tissue samples from patients may be the most direct and persuasive way to find biomarkers and therapeutic targets for cancers by a proteomic approach.A major obstacle,however,to the analysis of tumor specimens is tissue heterogeneity,which is particularly relevant to NPC as it often includes numerous infiltrating lymphocytes and stroma.Moreover,normal nasopharyngeal epithelial cells,from which the cancer is believed to arise, represent as little as 10%of nasopharyngeal mucosal tissue.Several approaches have been employed to obtain homogeneous cell populations from a heterogeneous tissue for proteomic analysis,such as short-term cell culture, and laser capture microdissection(LCM).Since 1996,LCM has emerged as a good choice for purifying cells from a tissue.There have been reports concerning proteomic research of laser capture microdissected tumor cells such as breast,hepatocellular and pancreatic cancers.To screen for NPC associated proteins,LCM was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues(NNET). Two-dimensional gel electrophoresis(2-DE)was performed to separate the total proteins of microdissected NPC and NNET,PDQuest software was applied to analyze 2-DE images,and the differential protein spots between the two types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS.The differential expression levels of the differential protein were validated by tissue microarray(TMA)immunohistochemistry and Western blotting.To explore the clincopathological significances of the NPC differential expressional proteins,immunohistochemistry was performed to detect the expression levels of the partial differential expressional proteins in paraffin-embedded archival tissue specimens,including 98 cases of primary NPC,30 cases of NNET,and 20 cases of cervical lymph node metastases (LMNPC),and the correlation of their expression level with clinicopathologic features and clinical outcomes were evaluated.Moreover,Western blotting was used to detect the expression levels of the partial differential expressional proteins in four NPC cell lines with different differentiated degrees and/or metastatic potentials.SiRNA was applied to inhibit the expression of cathepsin D,one of the differential proteins upregulated in 5-8F,and then in vitro cell invasion assay was performed to examine whether it associates with NPC metastasis。1.Screening for NPC differential expressional proteinsHighly reproducible and well-resolved 2-DE maps of pooled microdissected NPC and NNET were established.A total of 49 differential expressional protein spots between the tumor and normal tissues were found, and analyzed by MALDI-TOF MS and ESI-Q-TOF MS.A total of 36 differential proteins were successfully identified.Of the identified proteins, stathmin,nm23-H1,heat shock protein 27,vimentin,enolase, voltage-dependent anion channel 2,and guanine nucleotide binding protein etc.,were only expressed or up-regulated in NPC,whereas calcyphosine, annexin I,14-3-3σ,squamous cell carcinoma antigen 1(SCCA 1), cytokeratin8,and cathepsin D etc.,were down-regulated in NPC.To confirm the expression levels of the differential proteins identified by a proteomic approach,the expression of the five identified proteins(stathmin,14-3-3σ, annexin I,cytokeratin 8,and cathepsin D)in NPC and NNET were detected by tissue microarray immunohistochemistry and Western blotting.The results showed that stathmin was significantly up-regulated,whereas 14-3-3σ, annexin I,cytokeratin 8,and cathepsin D were significantly down-regulated in NPC versus.NNET(P＜0.01),which confirms the results of proteomic analysis.2.Clinicopathologic signicances of NPC differential expressional proteinsThe expression levels of the four differential proteins(stathmin,14-3-3σ, annexin I,and cathepsin D)were detected using immunohistochemistry in 98 cases of primary NPC,30 cases of NNET,and 20 cases of cervical LMNPC. Result showed:①14-3-3σand annexin I were significantly down-regulated in NPC versus NNET(P＜0.05).The expression levels of 14-3-3σand annexin I in primary NPC were significantly higher than those in LMNPC(P＜0.01);②Cathepsin D was significantly down-regulated in NPC versus NNET(P＜0.05).Whereas significant cathepsin D up-regulation was observed in LMNPC versus primary NPC(P＜0.01);③Stathmin was significantly up-regulated in NPC versus NNET(P＜0.05).But no significant difference in the expression level of stathmin was observed in the primary NPC and LMNPC.The statistics analysis showed:①Stathmin up-regulation,and 14-3-3σand annexin I down-regulation was correlated with poor histologic type/grade,advanced clinical stage,and recurrence(P＜0.01);②14-3-3σand annexin I down-regulation were correlated with regional lymphonode and distant metastasis(P＜0.01);③Cathepsin D down-regulation was significantly correlated with poor histological differentiation,whereas cathepsin D up-regulation was significantly correlated with an advanced clinical stage, recurrence,and lymph node and distant metastasis(P＜0.01).Western blotting was also done to detect the expression levels of stathmin, 14-3-3σ,annexin I,and cathepsin D in the four NPC cell lines with different metastatic potentials or differentiated degrees.The results showed:①Compared with well-differentiated CNE1,the expression level of stathmin was significantly increased in poorly-differentiated CNE2,but no obvious change of stathmin expression level in non-metastatic 6-10B and high metastatic 5-8F was observed;②14-3-3σand annexin I expression levels were significantly decreased in poorly-differentiated CNE2 compared with well-differentiated CNE1(P＜0.01),there were also significantly higher expression levels of 14-3-3σand annexin I in 6-10B without metastatic potential than those in 5-8F with high metastatic potential(P＜0.01);③Compared with well-differentiated CNE1,the expression level of cathepsin D was significantly decreased in poorly-differentiated CNE2.The expression level of cathepsin D was significantly higher in high metastatic 5-8F than that in non-metastatic 6-10B(P＜0.01).The results indicated that these four proteins were related to differentiation and/or metastatic potential of NPC cell lines. Furthermore,siRNA and in vitro cell invasion assay was performed to examine whether it associates with NPC metastasis.The results showed that downregulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells.In addition,the correlation of the expression levels of stathmin,14-3-3σ, annexin I,and cathepsin D in primary NPC with clinical outcomes were evaluated by the statistics and Kaplan-Meier survival curves.The result showed that patients with cathepsin D and stathmin up-regulation,14-3-3σand annexin I down-regulation had a poor prognosis.Multivariate analysis revealed that the expression status of stathmin,14-3-3σ,annexin I,and cathepsin D was an independent prognostic indicator.In summary,we identified 36 differential expression proteins between NPC and NNET by proteomic approach coupled with LCM.We further showed that four differential proteins(stathmin,14-3-3σ,annexin I,and cathepsin D)are potential biomarkers for differentiation,metastasis,and prognosis of NPC,and might contribute to NPC carcinogenesis.These findings reported here could have clinical value in distinguishing histological grades,predicting the prognosis of NPC and identifying NPC patients that are at high risk of progression and recurrence.