Preliminary Studies On Caveolin-1, A Candidate Gene Related To Nasopharyngeal Carcinoma Metastasis

Nasopharyngeal carcinoma (NPC) is an endemic cancer with high incidence in Southeast Asia and Southern China, which obviously shows an ethnic aggregation and geographic distribution. Nasopharyngeal carcinogenesis is a multi-stage process involving interaction among multiple factors. Epstein-Barr virus (EBV), certain chemical carcinogens and genetic factors are thought to be closely associated with NPC. NPC often metastasizes to lymph-nodes nearby at very early stage. Local recurrences and remote metastasis occur frequently in patients with NPC. However, the molecular mechanism underlying the metastasis of NPC remains poorly understood.Caveolin-1 (Cav-1) is the main structural protein of caveolae, plasma membrane invaginations that have been implicated in vesicular transport, cholesterol homeostasis, and the regulation of signal transduction and tumorigenesis. Cav-1 is highly expressed in terminally differentiated or quiescent cells, including adipocytes, endothelia, and smooth muscle cells. Previous evidence also suggested that Cav-1 might have a role in negative regulator of cell proliferation and acted as a candidate tumor suppressor gene. But many groups demonstrated that Cav-1 upregulated during tumor metastasis which could promote tumor progression.Based on pSUPER.retro retrovirus vector system, a short hairpin RNA expression vector targeting Cav-1, shCav-1, was constructed and confirmed by sequencing. Subsequently, the shCav-1 vector was transfected into 5-8F cell line, a NPC cell line with tumorigenicity and high metastasis ability, by LipofecTAMINE. After two weeks of puromycin selection, the drug-resistant clones were picked out and expanded. Subsequently, RT-PCR assay was performed and the results showed that the expression level of Cav-1 was significantly inhibited (>80%) in shCav-1 transfected 5-8F cells. Western Blot analysis further confirmed that the protein expression level of Cav-1 was also significantly down-regulated (>95%) in shCav-1 transfected 5-8F cells. All these data showed that we had successfully established stable shCav-1-transfected 5-8F cells (5-8F-shCav-1).To illustrate the possible influences of Cav-1 knockdown on cell cycle distribution of 5-8F-shCav-1 cells, flow cytometry (FCM) analysis was carried out. The FCM results indicated that G0/G1 phase was blocked in 5-8F-shCav-1 cells.The proliferation ability of 5-8F-shCav-l cells was measured by cloning efficiency assay and MTT assay. Compared with pSUPER.retro transfected 5-8F cells (5-8F-pSUPER.retro), 5-8F-shCav-1 cells proliferated much more slowly indicating knockdown of Cav-1 could reduce proliferation ability of 5-8F-shCav-1 cells.Wound healing assay and transfilter cell (matrigel) invasion assay were performed to investigate the mobility and invasion ability of 5-8F-shCav-l cells, respectively. The results showed that the knockdown of Cav-1 could decrease the mobility and invasion ability of 5-8F cells. Tumorigenicity assay in nude mice demonstrated that the knockdown of Cav-1 could reduce tumorigenicity and metastasis ability of 5-8F cells in vivo.In summary, we have established stable shCav-1-5-8F cells in which Cav-1 transcription was silenced sucessfuly by RNAi. Based on the changes on biological characteristics of 5-8F-shCav-1 cells, we suggest that Cav-1 might be a potential candidate gene related to NPC metastasis.