Screening FLOT2-Interacting Proteins In Nasopharyngeal Carcinoma Cells By Yeast Two-Hybrid

Nasopharyngeal carcinoma (NPC) is a kind of malignant cancer derived from nasopharyngeal-epithelium with high incidence in Southeast Asia, Southern China and Africa. Dietary pattern, enviromental factor, Epstein-Barr virus (EBV) infection and genetic variation are closely related to its occurrence and development. The clinical characteristics of NPC are early cervical lymph node metastasis and high rate of distant metastasis. Current data have shown that cervical lymph node metastasis is the first symptom when the patients are diagnosed and some of them have distant metastasis. The palindromia incidence of NPC patients which have distant metastasis is higher than that of patients with no distant metastasis.Flotillin-2(Flot2) was first discovered in the process of axon regeneration of retinal ganglion cells in goldfish. It mainly locates in cellular inner surface, and exists in many cell types. It is not only the basic scaffold for protein-proein interaction (PPI), but also participates in various cellular processes such as signal transduction, neuranagenesis, endocytosis and lymphocytic activation.In our previous work, several genes differentially expressed in5-8F and6-10B cells were discovered and identified through suppression subtractive hybridization (SSH) by using--the cDNA of the two cell lines which derived from the same SUNE-1cell line. A promising NPC metastasis-related gene,-Flot2, was chosen for further study. When the expression of-Flot2was inhibited by RNA interference in5-8F cells, a serial of changes in biological characteristics-were found. For example, the cells were arrested in G1phase, and showed much lower cell proliferation, motility and invasion abilities, suggesting that Flot2may be involved in the metastasis of NPC.Based on the previous work, we established a cDNA library of5-8F cell line and selected interacting proteins of Flot2using yeast-two-hybrid method. The experimental results were as follows: First, the open reading frame (ORF) sequence of Flot2was amplified from the5-8F cells by RT-PCR. Subsequently, the expression vector of Flot2(designated as pMD18-Flot2) was constructed by inserting the Flot2ORF into the pGBKT7vector. Then the pGBKT7-Flot2vector was transferred into Y2HGold yeast cells by PEG\LiAc. Meanwhile, we established the cDNA library of5-8F by LD-PCR and transferred pGADT7-Rec and cDNA library into yeast cells.Positive clones were screened by yeast mating and the plasmids of yeast cells were obtained. Experimental results showed that there were36positive clones and5interacting proteins of Flot2were discovered (PLCD3, PCDH7, PTPRJ, PCBP and copin Ⅱ). Lastly4interacting proteins (PLCD3, PCDH7, PTPRJ and PCBP) were further confirmed by co-transforming the bait vector and each of the prey vectors into Y2HGold cells.In summary, the cDNA library of5-8F cell line for yeast hybridization was constructed; the Y2HGold cells stably expressing Flot2were established;4interacting proteins of Flot2were successfully screened and identified using yeast two-hybrid method. These results provide solid basis for further study of the function and mechanism of Flot2in NPC invasion and metastasis.