Effects Of SARI Overexpression On The Proliferationand Apoptosis Of CNE2 Nasopharyngeal Carcinoma Cells And Its Possible Molecular Mechanisms

Objective:To construct the eukaryotic expression vector of SARI gene.To verify the effects of SARI overexpression on biological properties of CNE2 nasopharyngeal carcinoma cell line and explore the underlying mechanisms.Methods:?1?The full length cDNA sequence of SARI gene was cloned and purified and then digested by Bg1 II and Xba I enzymes.SARI cDNA was then connected into the pDsRed2-C-RFP vector 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 Bg1 II and Xba I double enzyme digestion and nucleotide sequencing;transduction of pDsRed2-SARI-RFP vector into CNE2 cells was enabled;expression of SARI-RFP fusion protein was observed by fluorescence microscope and Western blotting.?2?The effects of SARI overexpression on biological properties of CNE2 cell lines were estimated by cell counting kit 8?CCK-8?,Transwell assay,wound healing,activity test of Caspase 3,DNA ladder electrophoresis and Western blot,respectively.?3?Western blot was employed to observe the expression levels of apoptosis related proteins,some markers about the endogenous?mitochondria?apoptotic pathway.Results:?1?The full length cDNA sequence of SARI gene was cloned successfully.The sequence of the recombined vector were completely correct,pDsRed2-SARI-RFP eukaryotic expression vector was constructed successfully.Expression of SARI-RFP fusion protein was observed.?2?CCK-8 test?Transwell analyses and wound healing showed that the cell proliferation as well as invasion and migration in pDsRed2-SARI-RFP group of CNE2 cells were inhibited remarkably compared with that of two control groups?P<0.05?.The activity of Caspase 3 in pDsRed2-SARI-RFP group of CNE2 cells was higher then pDsRed2-C-RFP group to induce apoptosis?P<0.05?,and the DNA ladder test presented the similar apoptosis results.?3?Western blot displayed that the expression of Bcl-2 protein in the pDsRed2-SARI-RFP group of CNE2 cells decreased,while Bex and Cytochrome C expression increased,comparing two control groups.In addition,both PARP and Caspase-9 were increased to activate Actived-Caspase3.Conclusion:?1?The pDsRed2-SARI-RFP eukaryotic expression vector was constructed successfully.?2?Overexpression of SARI gene exhibited antitumor effect by inhibiting cell proliferation and inducing cell apoptosis in CNE2nasopharyngeal carcinoma cell lines.?3?The molecular mechanism could be that SARI might activate the endogenous?mitochondria?apoptotic pathway.