Quantitative Proteomics Analysis Of The Stroma In Nasopharyngeal Carcinoma

The role which stroma plays in tumor carcinogenesis is more and more important and now is becoming a hotspot.Nasopharyngeal carcinoma(NPC) is one of the most common malignant tumors in southern China,its incidence and mortality occupies the first place of the world,and is a great threat to people's health and lives.To delineate the stromal proteins involved in NPC carcinogenesis,we assessed differences in protein expression of the stroma from NPC and normal nasopharyngeal mucosa(NNM) using a quantitative proteomic approach combined with laser capture microdissection(LCM).LCM was performed to purify stromal tissue from the NPC and NNM,respectively.The protein expression profiles of the stroma tissue from NPC and NNM were compared by fluorescent two-dimensional difference gel electrophoresis(2D-DIGE),and 34 differential protein spots between tumor stroma(TS) and normal stroma (NS) were chosen to be identified by mass spectrometry(MS).2D-DIGE patterns of the purified stroma of NPC and NNM were established.A total of 20 differential proteins were identified,and part of the differential proteins (periostin,S100A9,CapG and L-plastin) were selectively further analyzed by Western blotting to validate the results of 2D-DIGE.Of all the identified proteins,periostin,S100A9,CapG,PYCARD et al.were up-regulated in NPC stroma,whereas L-plastin,Rho-GDI-β,B23,hnRNP K et al.were down-regulated in NPC stroma.To explore the function and clincopathological significances of the differential expression proteins, immunohistochemical(IHC) analysis was performed to detect the expression levels of the differential proteins in paraffin-embedded archival tissue specimens,including 66 cases of primary NPC,30 cases of NNM,and 20 cases of cervical metastatic lymph node from NPC(LMNPC),and the correlation of their expression levels with clinicopathologic features were evaluated.The expression levels of periostin,S100A and CapG in primary NPC were significantly higher than those in NNM(P＜0.01),and L-plastin was significantly down-regulated in NPC versus NNET(P＜0.01);the expression levels of periostin and S100A9 in LMNPC were significantly higher than those in primary NPC(P＜0.05),but no significant difference in the expression level of CapG and L-plastin were observed in the primary NPC and LMNPC.The statistics analysis showed:periostin,S100A9,CapG up-regulated and L-plastin down-regulated were correlated with poor histologic type/grade,advanced clinical stage,periostin and S100A9 up-regulated were correlated with lymph node metastases(P＜0.01).On the other hand,periostin was selected to explore the function in NPC cells.First,Western blotting was employed to detect the expression levels of periostin in four NPC cell lines(CNE1,CNE2,5-8F and 6-10B) with different differentiated degrees and/or metastatic potentials and NIH 3T3 fibroblasts, and found that periostin did not expressed in CNE1,CNE2,6-10B and NIH 3T3 cells,and 5-8F cells show weak expression.Sequently,the recombinant plasmids[pCMV-neo(+)-Periostin]and control plasmids[pCMV-neo(+)] were transfected into 6-10B cells(low metastatic potentials) using lipofectamine 2000^TM reagent to build stable transfection clone cells,at the same time,the recombinant plasmids[pCMV-neo(+)-Periostin]and control plasmids[pCMV-neo(+)]were also transfected into NIH 3T3 fibroblasts to obtain transient transfection cells.With stable transfection 6-10B cells (6-10B^periostin) and transient transfection NIH 3T3 cells,their biological characteristic analysis was performed as follows:(1) Flow cytometry(FCM) analysis of 6-10B^periostin cell revealed a significant decrease of G1 phase with a corresponding increase of S phase and G2 phase populations when compared with the control cell lines;(2) The MTT assay showed that up-regulated periostin in 6-10B cells was associated with a marked increase of cell growth; (3) The monolayer growth experiment showed that the clonoogenicity of 6-10B^periostin was significantly higher than those of the controls;(4) Soft agar growth experiments revealed that the colony formation abilities of 6-10B^periostin was higher than those of the controls;(5) Transwell chamber invasion assay showed that up-regulation of periostin expression in 6-10B cells was associated with increased in vitro cell invasion and migration;(6) A gelatin zymogram for MMPs activation demonstrated an increase in MMP-2 and MMP-9 activity in cultivated supernatant of 6-10B(periostin) and mixed cultured 6-10B and 3T3(+) cells.Furthermore,the expression of integrin-α_vβ₅ was also detected by IHC in NPC and NNM,6-10B(periostin) cells,6-10B(vector) cells and 6-10B cells,the expression levels of integrin-α_vβ₅ in primary NPC and 6-10B(periostin) cells were significantly higher than those in NNM and 6-10B(vector), 6-10B cells.The expression in NPC of integrin-α_vβ₅ showed positively correlated with the expression of periostin(r=0.682,p=0.000).Our results from above experiments demonstrated that periostin plays an important role in regulation of cell proliferation,the activity of MMPs,cell migration and invasion probably by combining with integrin-α_vβ₅。Taken together,we identified 20 differential proteins between the stroma from NPC and NNM by quantitative proteomic approach coupled with LCM. Sequently,the differential expression protein periostin was further analyzed by IHC,molecular biology technology and cell biology technology.The results indicate that Periostin plays a positive role in regulation of the evolution of NPC:periostin is related to the differentiation,metastasis and prognosis of NPC and exert its functions probably by combining with integrin-α_vβ₅. Periostin promotes the invasion of NPC through regulating the activity of MMPs.Our results will be helpful to study the role of stroma in the NPC carcinogenesis,as well as discover the interaction between NPC cells and their surrounding microenvironment,and provide a new idea for finding stromal targets of tumor therapy.