Functional Role Of ARA55 Gene In Human CNE2 Nasopharyngeal Carcinoma Cells

Objective: The current study aims to: 1. construct the eukaryotic expression vector of ARA55 gene and evaluate the effects of ARA55 overexpression on biological properties of CNE2 nasopharyngeal carcinoma cell lines; 2. clarify the functional role of ARA55 in TGFβ1-mediated epithelial-to-mesenchymal transitions(EMT) as well as invasion and metastasis in CEN2 cells.Methods: 1. The full length cDNA sequence of ARA55 gene was cloned, and the PCR product was was purified and digested by Hind III and Xho I enzymes. ARA55 cDNA and pCMV-C-EGFP were integrated with the aid of the T4 DNA ligase, followed by transformation, clone picking and amplification, respectively. The combined plasmids were extracted and sent for sequencing; identification of the recombinant plasmid was performed using double enzyme digestion and nucleotide sequencing; transduction of pCMV-ARA55-EGFP vector into CNE2 cells was enabled using ZLip2000 trancfection regent; expression of ARA55-EGFP fusion protein was observed by fluorescence microscope and Western blotting; the effects of ARA55 overexpression on biological properties of CNE2 cell lines were estimated by cell counting kit 8(CCK-8), wound healing, Transwell assay, Annexin V PE/7-AAD fluorescence test, DNA ladder electrophoresis and Western blotting, respectively. 2. Expression of ARA55 in CEN2 cells was induced by TGFβ1 with certain concentration, and Western blotting was employed in the evaluation of the alterations of certain EMT related proteins as N-cadherin and Claudin-1; si RNA-ARA55 was transfected into CNE2 cells with the aid of X-treme GENE si RNA transfection regent. the functional role of ARA55 induction on biological properties of CNE2 cell lines were estimated by CCK-8, wound healing and Transwell analyses.Results: 1. Results of double enzyme digestion and nucleotide sequencing indicated that pCMV-ARA55-EGFP eukaryotic expression vector was constructed successfully; 2. Data from CCK-8, wound healing and Transwell analyses revealed that cell proliferation as well as migration and invasion in pCMV-ARA55-EGFP group were suppressed and decreased when vs. pCMV-C-EGFP mock control(P<0.05 or 0.01). FCM and Annexin V PE/7-aminoactinomycin D showed that cells transfected with pCMV-ARA55-EGFP exhibited higher apoptosis ratios(%) when compared with the apoptosis ratio of cells infected with pCMV-C-EGFP, and the DNA ladder test presented the similar results. Besides, immunoblotting also displayed that the expression of Bcl-2 protein in the pCMV-ARA55-EGFP-transfected CEN2 cells decreased, while Cytochrome C expression increased, all with P<0.05 when comparing the pCMV-C-EGFP-transfected cells with the untreated CNE2 cells. In addition, both Caspase-3 and Caspase-9 were activated by presenting their spliceosomes; 3. Expression of ARA55 was induced after co-incubation with TGFβ1, accompanied by the up-regulation of N-cadherin and down-regulation of Claudin-1. Importantly, cell viability was promoted and the migration and invasion of CNE2 cells were strengthened(P<0.05 or 0.01); on the other hand, silence of ARA55 by si RNA could decrease the proliferation as well as migration and invasion of CEN2 cells(P<0.05 or 0.01).Conclusions: 1. The pCMV-ARA55-EGFP eukaryotic expression vector was successfully constructed; 2. ARA55 overexpression could inhibit proliferation and promote apoptosis in CEN2 cells; 3. ARA55 participates in TGFβ1-induced epithelial-mesenchymal transition in CNE2 cells.