The Primary Research On The Roles Of ENO1 Gene In The Pathogenesis Of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC) is one of the commonest carcinomas with a high degree of malignant Phenotype in Southern China.especially those of Cantonese origin. Both the geographic pattern and familial aggregation of incidence are two main characteristics of NPC. The carcinogenesis of nasopharyngeal epithelium is a multi-stage, multi-path and multi-mechanism process, which is involved in the effects of several factors including genetic predisposition, environmental carcinogen and Epstein barr virus (EBV).The changes of various environmental factors, EBV infection, genetics and epigenetics cause the overexpression of oncogenes and downregulated or null expression of tumor suppressors in nasopharyngeal epithelium, Which will lead to the transition of epithelium to Precancerous lesion, and finally be transformed to NPC.Carcinogenesis is associated with the participation of expression alteration of multiple genes.The expression change of any single gene does not have the ability todrive the malignant transformation of normal epithelium.In the past few years, although the knowledge of molecular mechanisms about NPC has greatly inereased. Its pathogenesis remains to be clarified.ENO1 gene primarily encodes one of three enolase isoenzymes found in mammals. it is alpha-enolase,an enzyme in the glycolytic pathway,catalysing the formation of phosphoenolpyruvate from 2-phosphoGly-cerate. This gene also encodes a shorter monomeric structural lens protein, was earlier known as the Myc-binding protein-1 (MBP-1). This shorter protein is localized to the cell nuclei, and has been found to bind to an element in the c-myc promoter. Recent researches show that the c-myc protooncogene is a DNA-binding phosphoprotein that plays an important role in cell growth regulation and differentiation. It's expression level is independent predictive factor for suviver time of patients with tumors.In the early study found that of ENO1, ENO1 could inhibit the growth of tumor. But it is interesting that, recent researches show that ENO1 gene is not, as earlier pointed out,an tumor suppressor gene.In contrast, ENO1 is likely to be play a important role in pathogenesis and progression of tumor.Currently many studies about ENO1 gene function focused on liver cancer, non-small cell lung cancer, hurthle cell cancer,melanoma and other tumors.Through proteomics,western blot and immu-nohistochemical techniques that, ENO1 in these tumors cell lines and tissue have high levels of expression. fuhermore, many papers noted that there was significant relationship between levels of autoantibodies ENO1 in the peripheral blood of patients with multiple tumors and such tumor's stage and prognosis. In the study of the mechanism of ENO1, majority studies suggest that ENO1 play a positive role in the energy metabolism of tumor cells, especially in high metabolic cases.compared with non-proliferation cell lines, the rapid proliferation cell lines have high level of ENO1 expression significantly.So speculate that in the process of cell proliferation, ENO1 expression can affect glycolysis, Krebs cycle and the subsequent oxidative phosphorylation enzyme substrate level, thereby affecting the synthesis of ATP, which is importantant for the growth of tumor cells.But till now the relationship between the ENO1 gene and NPC has not been reported. In Professor Fang Weiyi previous work, we used gene chip technology to analyse 8 groups of NPC patients and control samples, two cell lines, results showed that in pathological specimens and cell lines, ENO1 expression were significantly increased. Based on these results, we want to study that the characteristics of ENO1 in NPC cell, and further by changing the expression levels of ENO1 to observe its biological characteristics of NPC cells. To further define the pathogenesis of nasopharyngeal carcinoma lay a good foundation.CONTENTS AND METHODS1.Identification of expression characteristics of ENO1 in NPC and its significance.The expression of ENO1 protein in NPC tissues was detected by immune-ohistochemistry (IHC).2.Effects of ENO1 knock-down on biological behaviors of HONE1 cells.The expression of ENO1 mRNA in 6 NPC cell lines (5-8F,6-1 OB, SUNE1,CNE2,CNE1,HONE1) was detected by Quantitative real time-PCR respectively. The high-expressing ENO1 clones were screened.Structure of ENO1 mRNA was analysed by software to select targeted interference sites. Three siRNA and one negative control siRNA were constructed and chemically synthesized,then be transfected into NPC cell line Phonel by positive ion liposome Lipofectamine2000.Use Quantitative real time-PCR to detect the expression of ENOl mRNA,then estimate the interference efficieny,screening the most effective siRNA.Specific anti-ENO1 short RNA and control vector were constructed respectively, and then were transfected into HONE1 cells. Quantitative real time PCR was used to detect the efficiency of interference of cell clone.after the inhibition of the endogenous ENO1, the growth, cell cycle and invasion of cells were detected by MTT, Transwell, FACS and Boyden chamber assay in vitro respectively.RESULTS1. Identification of expression characteristics of ENO1 in NPC tissues.In an immunohistochemical study, ENO1 staining was mostly observed in the nucleus of carcinoma cells. No specific ENO1 staining was observed in normal adjacent nasopharyngeal epithelial cells and stroma cells in surrounding tissues. ENO1 was found to express in 60.0%(70/120) cases of NPC, higher then 27.5% (11/40) cases of chronic nasopharyngitis tissue samples (χ2=11.424, P=0.001).The relationship between clinicopathological features and ENO1 expression in NPC was analyzed with Pearson Chi-Square test. But there was no significant relationship between expression of ENO1 and lymph node metastasis and postradiotherapy distant metastasis (P> 0.05). Interestingly, we observed that The positive expression rate of ENO1 protein in stage T1+T2+T3was higher than that of in patients with T4 (x2=13.919, P=0.003).2. Effects of ENO1 knock-down on biologieal behaviors of HONE1.Quantitive real-time PCR was used to detect the expression of ENOl in 6 NPC cell lines (5-8F,6-10B, SUNE1,CNE2,CNE1,HONE1).The result shows that the HONE1 line is a high-exprssing clone.siRNA-ENO1 was successfully transfected into NPC cancer cell line HONE1, ENO1-siRNA were successfully transfected into HONE1 after 12h detected by QPCR, ENO1 mRNA levels in HONE1 cells was significantly inhibited, siRNA-2 resulted in the highest inhibiting rate.HONE1-siRNA cells showed a significantly reduced proliferation compared with HONE1 and HONE1-NC cells as determined by in vitro MTT assay (F=4.764, P=0.001). However, the cell cycle distribution detected by flow cytometry has no significant differences between each other. These results indicated knock-down of ENO1 could reduce proliferation of HONE1 cells.The results of in vitro proliferation assay showed that compared to aNegative control HONE1 cell, the proliferation ability of HONE1-siRNA Cells was significantly inereased (F=3.163.P=0.004). Furthermore, invasion assay showed that HONE1-siRNA cells had significantly increased invasiveness as compared with HONE1-NC cells (t=13.758, P=0.000). These results showed knock-down of ENO1 results in a leads to an upregulated invasion of HONE1 cells.Conclusions:l.The expression of ENO1 is upregulated in NPC and correlates statistically with the malignant status of NPC.2. ENO1 promotes the growth of NPC cells in vitro,but inhibited invasion. These results indicated that ENO1 might play a pivotal role in the tumorigenesis and progression of NPC.