Preliminary Studies On Flotillin-2, A Candidate Gene Related To Nasopharyngeal Carcinoma Metastasis

Nasopharyngeal carcinoma(NPC)is a malignancy derived from epithelium with characteristics of early cervical lymph node metastasis and high incidence rate of distant metastasis.Epstein-Barr virus(EBV) infection,chemical carcinogens,genetic variation or spontaneous mutation are closely associated with its occurrence and development. Current data indicate that about seventy percent of patients have developed cervical lymph node metastasis when they are first diagnosed. Although the level and equipment of radiotherapy have been improving, the 5-year survival rate of NPC patients always retains between 60%and 70%because of differential individual sensitivity to radiotherapy.Distant metastasis and local recurrence are main causes which result in the failure to cure NPC.Flotillin-2(Flot-2)was first isolated from the enriched domain of caveolae/lipid raft of cellular membrane.It is an important component of cellular membrane,and involved in various cellular processes such as membrane trafficking,T cell and B cell activation,regulation of several signaling pathways associated with cell growth and malignant transformation,maintaining the structure and adhesion of epidermal cells and formation of filopodia.According to recent researches,Flot-2 may be able to promote the invasion and metastasis of melanoma cells.In our previous work,by using 5-8F and 6-10B cell's cDNA, multiple genes up-regulated in 5-8F cells have been screened and identified through suppressive subtractive hybridization(SSH).A promising NPC metastasis-related gene,Flot-2,was chosen for further study.The expression of Flot-2 in 5-8F cells was inhibited by RNA interference and a serial of changes in biological characteristics of 5-8F cells were found.For example,cells are arrested in G1 phase,and exhibit a much lower proliferation,motility and invasion ability,suggesting that Flot-2 may be involved in the metastasis of NPC. Based on previous work,we up-regulated the expression of Flot-2 in 6-10B cells via gene transfection and the changes of biological characteristics of transfected 6-10B cells were examined for further elucidating the possible roles of Flot-2 in promoting metastasis of NPC. The exprimental results are as follows:Firstly,the open reading frame(ORF)sequence of Flot-2 was amplified from 5-8F cells by RT-PCR.Subsequently,the eukaryotic expression vector of Flot-2(designated pcFlot-2)was constructed by inserting the Flot-2 ORF into the pcDNA3.1(+)vector.Then the pcFlot-2 vector was transferred into 6-10B cells by Lipofectamine 2000.After G418 selection,we obtained several G418-resistant cell clones.It is demonstrated that the expression of Flot-2 in G418-resistant cell clones is stably up-regulated both at mRNA and protein levels when detected by RT-PCR and Western blotting respectively,showing that the 6-10B cell line stably expressing Flot-2 gene(named 6-10B-Flot-2)has been successfully established.Then we detected the various changes in biological characteristics of 6-10B cells after the expression of Flot-2 was up-regulated.The cellular morphology changed apparently.The appearance of cells became irregular,some of them got spindle shape,and some of them changed into a neural appearance.Cells spreaded obviously and formed visible lamellipodia,where membrane ruffles could be seen.Cells became larger,and the nucleoplasmic ratio decreased.Then cells were observed under a confocal laser-scanning microscope.It appeared that the number and density of microfilament on cell surface increased.It was likely that the microfilament redistributed and expanded around cells to form conspicuous lamellipodia and membrane ruffles.The flow cytometrical analysis showed that compared to the control group,the percentage of 6-10B-Flot-2 cells in G0-G1 phase decreased significantly(48.23%vs 63.28%),but the pencentage of cells in S phase increased apparently.The clonality and proliferation abilities of 6-10B-Flot-2 cells were measured by colony formation assay and MTT analysis,respectively. Compared with 6-10B cells transfected with blank vector,6-10B-Flot-2 cells have a much higher cloning efficiency and proliferated much more quickly(p＜0.05),providing evidence that Flot-2 has an ability to enhance proliferation ability of 6-10B-Flot-2 cells.Wound healing assay,migration assay and matrigel invasion assay were performed to investigate the mobility and invasion ability of 6-10B-Flot-2 cells respectively.The results demonstrate that up-regulation of Flot-2 can increase the motility of 6-10B cells and enhance the invasion ability as well.In vivo,tumorigenic assay in nude mice indicate that up-regulation of Flot-2 can increase the metastatic ability of 6-10B cells.At last we analyzed the possible reasons of the mestastasis-promoting ability of Flot-2 in 6-10B cells,which may include increasing of microfilament on cell surface,reorganization of the actin cytoskeleton,changes in the expression of Cdc42 gene and formation of filopodia,and so on.In summary,the stably transfected 6-10B cell lines with pcFlot-2 has been established and the expression of Flot-2 in 6-10B cells is up-regulated successfully.Based on the changes in biological characteristics of 6-10B-Flot-2 cells,we further comfirm that Flot-2 is a potential metastasis-associated gene in NPC.