The Effect Of LMP1 On MiR-200 Family And ZEB1 In Nasopharyngeal Carcinoma

Objective To detect the expression levels of miR-200 (miR-200a,miR-200b, miR-200c, miR-141 and miR-429) family, LMP1, ZEB1 and E-cadherin in nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) and analysed the relationship among them to investigate the mechanism of invasion and metastasis in NPC.Methods 1. ZEB1, LMP1 and E-cadherin protein expression were detected in NPC tissues and nasopharyngitis tissue by immunohistochemistry and the expression relevance of those three protein was also analyzed.2. pIRES2-Zs-Green1-LMP1 plasmid was used to transfect CNE1 and CNE2 cells and then Quantitative Real-time PCR (qRT-PCR) and Western Blotting were used to detect the expression of LMP1 in the cells above.3. qRT-PCR was also used to detect the expression of miR-200s, LMP1,ZEB1 and E-cadherin mRNA in CNE1 and CNE2 transfected with pIRES2-Zs-Greenl-LMPl plasmid.Western blotting was used to examine the protein expression levels of LMP1, ZEB1 and E-cadherin after transfection pIRES2-Zs-Greenl-LMPl plasmid in CNE1 and CNE2 cell lines.4. SPSS 16.0 software was used to do statistical analysis, mean ± standard deviation showed measurement data, independent samples t test (homogeneity of variance) or corrected t-test (equal variances not assumed) was used to compare the two groups difference ,comparison of count data rates was used Fisher’s exact test (total number of cases <40 cases), Pearson correlation analysis or Spearman rank correlation analysis was used to deal with correlation analysis. P <0.05 was considered statistically significant.Results: 1. The positive expression frequency of LMP1,2EB1 and E-cadherin in the NPC tissues was respectively 53.8%, 71.8%, 20.5% ,which was significantly higher than those in nasopharyngitis tissues(p <0.05 ). LMP1 protein expression was positively correlated with ZEB1 protein expression (r =0.563, p <0.05), and was negatively correlated with E-cadherin protein expression (r = -0.421,p<0.05),while there was a negative correlation between ZEB1 and E-cadherin (r = -0.387,p <0.05).2. Transfection Results: Compared with pre-transfection ,the expression of LMP1 in CNE1/LMP1 cells and CNE2/LMP1 cells were both up-regulated detected by qRT-PCR and WB , indicating that transfection experiments were successful.3. qRT-PCR results:① Expression of LMP1 and ZEB1 mRNA were higher while E-cadherin mRNA was lower in pIRES2-Zs-Greenl-LMP1 plasmid transfected CNE1 and CNE2 than those in non-transfected cells(p<0.05).② The expression of miR-200a ,miR-200c and miR-141 were lower,while miR-200b expression levels was higher in the pIRES2-Zs-Greenl-LMT1 transfected CNE1 cells in which miR-141 was significantly down-regulated than the control group (p<0.05).③ In the pIRES2-Zs-Greenl -LMP1 transfected CNE2 cells ,the expression of miR-200a ,miR-200c and miR-141 were lower than the control group in which miR-141 significantly down-regulated.4. WB results:In CNE1 and CNE2 cells transfected with pIRES2-Zs-Green1-LMP1 plasmids, the protein expression of LMP1 and ZEB1 were both up-regulated ,while the protein expression of E-cadherin were both down-regulated (p <0.05).Conclusion:1. In NPC tissues, the protein expression of LMP1 was positively correlated with The protein expression of ZEB1 , while the latter was negatively correlated with the protein expression of E-cadherin.2. In NPC cells, LMP1 may mediate the down-regulation of miR-200a ,the up-expression of ZEB1 to inhibit the expression of E-cadherin, thereby promoting the invasion and metastasis of NPC.