Differential Proteomic Analysis Of NPC Cell Lines 5-8F And 6-10B

Nasopharyngeal carcinoma (NPC) is a high-incidence malignancy in southern China and Southeast Asia. Early metastasis is one of distinctive characteristics of NPC and the main reason of death in NPC patients. Furthermore, local recurrence after radiotherapy and distant metastasis are the bottlenecks which restrain therapeutic effect and prognosis for NPC. Therefore, elucidating NPC metastasis mechanism and screening the molecular biomarkers are crucial for treatment and prognosis, which have important theoretical and clinical value.A pair of NPC cell lines (5-8F and 6-10B) with different metastatic potential and the same genetic background was used as metastatic model of NPC in this study. Two-dimensional gel electrophoresis (2-DE) was used to separate the total and secretory proteins from 5-8F and 6-10B. PDQuest software was applied to analyze 2-DE maps of the total and secretory proteins in above two cell lines. Differentially expressed proteins between 5-8F and 6-10B were identified by MALDI-TOF-MS(matrix-assisted laser desorption/ionization time-of-flight mass Spectrometry) and ESI-Q-TOF MS/MS(electrospray ionization-quadrupole time-of-flight MS/MS). The biological functions and subcellular locations of identified proteins were further analyzed using bioinformatic resources. The expression levels of partial identified proteins were validated by Western blot analysis. The expression levels of differential protein nm23-H1 between NPC tissue and cervical lymph-node metastasis were detected by immunohistochemical staining. The results were as following: (1) The 2-DE maps of the total and secretory proteins from highly metastatic 5-8F and non-metastatic 6-10B were established respectively. One hundred and twenty-three differential proteins spots were found between 5-8F and 6-10B, and twenty-nine differential expression non-redundant proteins were identified by MS. Furthermore, the identified proteins were categorized into several protein groups according to their functions or subcellular locations; (2) The results of Western blot showed that Annexin A1, Stratifin, Keratin-8 and nm23-H1 were differential expression proteins in 5-8F and 6-10B, which was consistent with the results of our comparative proteomic analysis; (3) The expression level of nm23-H1 in the total proteins was significantly higher in 5-8F than that in 6-10B. And also the expression level of nm23-H1 in cervical lymph-node metastasis was significantly higher than that in primary NPC; (4) The differential protein—peptidyl-prolyl cis-trans isomerase A (PPIA) was found to have posttranslational modifications (PTM) including acetylation(ACET) and citrullination (CITR) or 3-phenyllactic acid(FLAC).In this study, twenty-nine differential expression proteins between 5-8F and 6-10B were identified, which may be associated with the metastasis of NPC, nm23-H1 may contribute to the metastasis of NPC, and PPIA was found to have PTM which may influence its function. These data will provide experimental evidences and new clues for elucidating the metastatic mechanism of NPC and screening the metastatic molecular biomarkers of NPC.