The Expression Of Flotillin-2and Preliminary Investigation On Its Molecular Mechanism In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a kind of malignant tumor with high incidence in Southeast Asia and Southern China. Nasopharyngeal carcinogenesis involves interactions among multiple factors, including Epstein-Barr virus infection, chemical carcinogen, genetic and epigenetic variation.The NPC cell lines5-8F and6-10B were derived from the same NPC cell line SUNE-1. Although sharing almost the same genetic background, the two NPC cell lines have different metastatic capability, based on which our group has built a subtractive cDNA library of these two cell lines by suppressive subtractive hybridization (SSH) technique previously. Multiple genes up-regulated in5-8F cells have been identified. A promising NPC metastasis-related gene, Flotillin-2(FLOT2), was chosen for further study.FLOT2was initially discovered in the process of axon regeneration of retinal ganglion cells in goldfish. It is involved in many physiological processes including axon regeneration, neuron differentiation, endocytosis, activation of T lymphocytes, recruitment of membrane proteins, tumorigenesis and metastasis of melanoma, etc.In our previous work, by RNA interference (RNAi), we have successfully established cell lines in which FLOT2expression is significantly inhibited by transfection of shFLOT2vector. The shFLOT2transfected cells (5-8F-shFLOT2cells) show decreased proliferation, invasion, mobility and metastatic abilities and increased percentage of G1phase cells, suggesting that FLOT2may correlate with NPC metastasis.In this study, we further detected mRNA expression levels of FLOT2in a variety of NPC cell lines. Meanwhile, we investigated the expression level of FLOT2protein in chronic nasopharyngitis and NPC tissues by immunohistochemistry. Subsequently, we extracted RNA from blank vector transfected5-8F cells (5-8F-vector cells) and5-8F-shFLOT2cells, then performed gene expression microarray and bioinformatic analysis, and further verified partial microarray results preliminarily. Finally, we analyzed the promoter region of FLOT2by bioinformatic softwares and detected the expression level of FLOT2and its promoter activity in5-8F and6-10B cells at different time points after they were treated by BIO, an activator of Wnt signaling pathway.RT-PCR results showed that strong expression of FLOT2mRNA was detected in5-8F, HNE-1, HNE-2, HNE-3, CNE-1, CNE-2, HONE-1, HK-1and C666-1cell lines.6-1OB cell line displayed the lowest FLOT2mRNA expression level among all the NPC cell lines, but its level was still slightly higher than that in normal nasopharyngeal epithelial cell line NP69. Immunohistochemical results showed that from chronic nasopharyngitis to non-metastatic NPC and metastatic NPC, the expression level of FLOT2increased successively (P<0.001).Using gene expression microarray and bioinformatic analysis, we found that, after FLOT2was inhibited by RNAi, p53signaling pathway was reactivated, TGF-(3signal pathway was suppressed, and the expression levels of numerous cell adhesion and cell cycle related genes significantly changed. RT-PCR and/or qRT-PCR detection comfirmed that E-cadherin and p21, two genes negatively related to NPC metastasis and cell cycle progression, respectively, were significantly up-regulated in5-8F-shFLOT2cells.The2000bp region upstream of the transcription start site of FLOT2was analyzed by bioinformatic softwares and the results demonstrated that it contained several candidate promoter regions and binding sites for transcription factors of Wnt signaling pathway. We generated a luciferase reporter construct containing the Wnt signaling pathway transcription factor binding sites. Then the reporter vector was transfected into5-8F and6-10B cells. After stimulation by BIO for48hours, both6-10B and5-8F cells showed higher luciferase activity and FLOT2gene expression level increased remarkably.From the above results, we can make the following conclusions. FLOT2expression level is significantly up-regulated in NPC cell lines and tissues, especially in metastatic NPC cell line and tissues. The changes of microarray expression data in5-8F-shFLOT2cells suggests that FLOT2may play an important role in NPC occurrence and metastasis, partially owing to abnormal p53and TGF-β signaling pathways and aberrant expression of Klf4, E-cadherin and p21in NPC. FLOT2gene expression can be up-regulated by Wnt signaling pathway through regulating FLOT2gene promoter.