Role Of Nestin In Maintaining Stemness Of Nasopharyngeal Carcinoma Cell

Background and ObjectivesNasopharyngeal carcinoma (NPC) is one of the commonest carcinomas with a high degree of malignant phenotype in Southern China and Southeast Asia.Due to its originating form a hidden anatomical site,it is usually in the advanced clinical stage at the time of diagnosis.In addition,NPC tends to be early invasive and metastatic.Hence,the prognosis for NPC is poor with a 5-year survival rate of less than 60%.For patients with advanced tumors, although surgical treatment by combined radiotherapy and chemotherapy to shrink tumor size, but can not completely remove the tumor, the patient eventually died of tumor metastasis or recurrence. Therefore, tumor metastasis and recurrence has become an urgent problem in clinical treatment. In recent years, "tumor stem cells (cancer/tumor stem cells) " theory understands the tumor from a new perspective.There is a new interpretation of tumors from the tumorigenicity, tumor metastasis as well as oncotherapy aspects.They put forward that the root causes of cancer metastasis and recurrence is tumor stem cells, so that people could try to explore the complete way of curing tumor.Nestin is a class VI intermediate filament protein mainly expressed in neural tissue during embryonic development.In adults, nestin is present only in a small subset of cells and tissues. For a long time, nestin is a marker of neural stem cell and progenitor cell, The name "nestin" is derived from its location:neural stem cell protein.In recent years,nestin expression has also been detected in various types of human solid tumors and their corresponding cell lines. Furthermore,there is a great relationship between nestin expression and the malignant phenotype of tumor. In the central nervous system, the close relationship between nestin expression and cell proliferation has been confirmed many times. Has been reported in brain tumors, CD133/nestin positive cells is considered to be potential cancer stem cell population, but the specific mechanism is still unclear.In summary, whether in normal or abnormal tissue, nestin expression indicates an undifferentiated state, primitiveness,plasticity, and an increased proliferative potential. As the evidence of nestin as a marker of central nervous system cancer stem cells increased, and in a growing number of solid tumors found in the expression of nestin, nestin in the role of cancer stem cells, has begun to attract more attention of scholars. Up to now,there is little article which is involved in the investigation of nasopharyngeal normal and cancer stem cells.Zhang found long-term BrdU-labeled LRCs in nasopharyngeal epithelia of adult mice and subcutaneous xenografts of human NPC cell lines in nude mice.Wang isolated SP cells in nasopharyngeal carcinoma cell line CNE2. However, how the relationship between nestin and nasopharyngeal carcinoma, and whether it is an important marker of nasopharyngeal carcinoma cancer stem cells, so far no related reports.Based on the research review of nestin and its potential significance in the molecular mechnisms of tumor, the identification of its expression characteristics in NPC tissues and cell lines will help understand the mechanisms by which nestin contributes to the origin and progression of NPC, in particular,stemness that nestin contributes to NPC cells.Furthermore,the implication of the role of nestin in NPC might provide new insight into understanding the molecular mechanism involved in NPC carcinogenesis and progression,and may lead to the development of new approaches for effective diagnosis and therapy.In this study,we performed the following researches to elucidate the characteristics and regulatory mechanisms of nestin.Contents and methods1.Identification of expression characteristics of nestin in NPCThe expression of nestin mRNA in 4 NPC cell lines (5-8F,6-10B,CNE1,CNE2) was detected by real-time PCR.The expression of nestin protein in 4 NPC cell lines and NPC tissues was detected by western blot,immunofluorescence and immunohisto chemistry(IHC) respectively.2.Construction of shRNA lentivirus interference vector targeting nestin and establishing NPC cell lines with nestin silenced by stable RNA interference.The recombinant lentiviral shRNA expression plasmid targeting nestin was packaged into mature lentivirus by 293T cells, and the supernatant containing the virus was harvested, concentrated and titrated. The best target for RNA interference was selected by real-time PCR.5-8F cells was then infected by the recombinant lentiviral vector, and colonies with GFP expression were selected by limiting dilution assay of 96-well plate to expand culture. Finally,5-8F NPC cells with nestin silenced and negative empty vector 5-8F cells were respectively established for further investigation.3.Identification of effects of suppressing the expression of nestin on common biological characteristics in NPC 5-8F cellsMTT, FACS Caliber cytometry and transwell were utilized to determine the ability of cell growth, cell cycle, apoptosis and cell migration in vitro. In vivo subcutaneous tumorigenesity in nude mice was used to evaluate the effect of silenced nestin on NPC cells. 4.Detection of effects of suppressing the expression of nestin on cancer stem cells in NPC 5-8F cellsUsing colony formation assay, the tumor sphere formation assay and FACSCaliber cytometry,we detected the proportion of cancer stem cells after silenced nestin in NPC cells.Results1.Measurement of nestin expression level in NPCWestern blot assay showed a protein band with apparent molecular weight of approximate 250 kD and protein expression of nestin was significantly up-regulated in NPC cells (5-8F,6-10B,CNE2)comparing immortalized nasopharyngeal epithelial cells NP69.The expression of nestin was examined by immunofluorescence and the results showed that green fluorescence can be seen in 5-8F,6-10B and CNE2 cells, most expressed in the cytoplasm, little in nucleus. Real-time PCR showed the expression levels of nestin mRNA was 5-8F and CNE2 cells have higher expression of nestin,5-8F cells express the highest.In an immnunohistochemical study, nestin staining was mostly observed in the nucleus and little in the cytoplasm in poorly differentiated squamous cell carcinoma and undifferentiated carcinoma,and 80% of positive cells. There was little specific staining in chronic nasopharyngitis tissues, as negative.2.Successfully constructed recombinant lentiviral vector targeting nestin and established NPC cell lines with nestin silencedThe recombinant lentiviral vector was successfully constructed and verified by PCR and sequencing.The titer of concentrated virus was 4x106TU/ml.5-8F was transfected with recombinant lentivirus of small interference RNA targeting nestin (pGCL-GFP/shnestin)and the negative control lentivirus(pGCL-GFP). After a selection of GFP expression by limiting dilution assay,real-time PCR and Western blot assays were used to detected the efficiency of RNAi targeting nestin.5-8FNESi with stable knock down of nestin and 5-8FNC of empty vector control were established.3.Effects of suppressing the expression of nestin on common biological characteristics in NPC 5-8F cellsCompared to 5-8F control cells,5-8FNESi clonal cells showed to be inhibited markedly in MTT cell proliferation assay (F=139.447, P< 0.001), transwell migration assay (F=298.278, P<0.001) and cell cycle(F=216.467, P<0.001),but increased apoptosis cells(F=603.612, P< 0.001). In vivo subcutaneous tumorigenesity in nude mice showed that tumor weight and volume of 5-8FNESi were significantly decreased comparing with 5-8F (weight:F=19.089, P<0.001; volume:F=14.670, P<0.001).Furthermore, cell cycle analysis showed that the S phase population increased significantly in 5-8FNESi cells in comparison to the 5-8F cells (P<0.001).4.Effects of suppressing the expression of nestin on cancer stem cells in NPC 5-8F cellsWe detected the proportion of cancer stem cells after silenced nestin in NPC cells with colony formation assay, the tumor sphere formation assay and FACSCaliber cytometry.The results were that 5-8FNESi clonal cells showed to be decreased markedly in the ability of colony formation(F=150.845, P<0.001),the number of tumor sphere formation(F=830.622, P<0.001) and the proportion of SP cells(F=205.400, P<0.001).Conclusion1.The expression of nestin is upregulated in NPC cells and tissues comparing immortalized nasopharyngeal epithelial cells NP69.2.We successfully constructed recombinant lentiviral vector targeting nestin and established NPC cell lines with nestin silenced.3.Funtional investigations showed that, after nestin was suppressed in NPC 5-8F cells, the cell proliferation and cell migration in vivo and in vitro were markedly reduced,but cell apoptosis was increased. Furthermore, in 5-8FNESi cells, cell cycleprogression was blocked from S to G2/M phase. They suggest that nestin should be a NPC associated gene.4.After nestin was suppressed in NPC 5-8F cells,the number of NPC cancer stem cells was markedly reduced,which suggest that nestin may be involved in the role of NPC cancer stem cells.