The Effects Of Vimentin On Invasion And Metastasis In Nasopharyngeal Carcinoma Via Regulating Annexin A1 And S100A9

Objective: Established a nasopharyngeal carcinoma cell line with upregulated and down-regulated Vimentin gene,detecting the expression of Annexin A1 and S100A9 protein,and observing the changes of invasion and migration ability after up-regulated or down-regulated Vimentin gene in nasopharyngeal carcinoma cells.And preliminarily investigating the effect of Vimentin on the invasion and migration of nasopharyngeal carcinoma cells in vitro by regulating the expression of Annexin A1 and S100A9,providing laboratory data for the molecular mechanism of distant metastasis of nasopharyngeal carcinoma.Methods:1.Constructed a Vimentin overexpressed plasmid vector and a Vimentin interference plasmid vector;2.Transfected he vimentin gene into the 6-10 B nasopharyngeal carcinoma cell line which has low expression and low metastasis potential of Vimentin gene by liposomal transfection.To establish the 6-10B-Vim nasopharyngeal carcinoma cell line with high expression of Vimentin gene and the empty vector control cell line 6-10B-Vec(vector).3.Used Small molecule interfering RNA(siRNA)technology to introduce siRNA into 5-8F nasopharyngeal carcinoma cell line which has high expression and high metastasis potential of Vimentin.To establish the 5-8F-si-Vim nasopharyngeal carcinoma cell line with down-regulated the expression of Vimentin and empty vector control cell line 5-8FNC(Negetive Control).4.Detected The mRNA and protein expressions of Vimentin in the overexpression plasmid vector transfection group 6-10B-Vim,the interference vector transfection group 5-8F-si-Vim and the corresponding control groups by Q-PCR and Western blot experiments,respectively,to confirm the transfection efficiency.5.Detected the expression of Annexin A1 and S100A9 in each nasopharyngeal carcinoma cell line when Vimentin gene expression was up-regulated or down-regulated by Western blot assay.6.Detected the effect of up-regulation and down-regulation of Vimentin gene expression on invasion and migration ability in nasopharyngeal carcinoma cell lines in vitro by scratch test and Transwell chamber test.Results:1.Successfully constructed Vimentin overexpressed plasmid vector and Vimentin interference vector;2.Transient transfection Vimentin expressed genes,tested overexpression plasmid vector transfection group 6-10B-Vim cells and empty vector control group 6-10B-Vec the expression of Vimentin mRNA and protein in cells through Q-PCR and Western blot experiments,respectively.Q-PCR results showed that compared with the control cells,Vimentin overexpression plasmid vector transfection group 6-10B-Vim cells the Vimentin mRNA expression level significantly raised(P<0.001);Western blot results showed that Vimentin protein level in 6-10B-Vim cells was significantly up-regulated compared with that in control cells(P<0.001).3.Next,instantaneous transfection of Vimentin si RNA interference gene,the mRNA and protein expression of Vimentin in 5-8F-si-Vim cells of the interference vector transfection group and 5-8F-NC cells of the blank vector control group were detected by Q-PCR and Western blot.Q-PCR results showed that Vimentin mRNA expression was significantly downregulated in 5-8F-si-Vim cells transfected with interfering vectors compared with control cells(P<0.001).Western blot results showed that Vimentin protein level in 5-8F-si-Vim cells was significantly downregulated compared with control cells(P<0.001).4.Western blot assay was performed to detect the expression of Annexin A1 and S100A9 proteins in four groups nasopharyngeal carcinoma cells after Vimentin expression was up-regulated and downregulated(6-10B-Vim,6-10B-Vec,5-8F-si-Vim and 5-8F-NC).After the expression of Vimentin was down-regulated,the expression level of S100A9 was significantly down-regulated(P<0.05),while the expression level of Annexin A1 was significantly up-regulated(P<0.05).5.Transwell chamber and scratch assay were performed to detect the invasion and migration ability of nasopharyngeal carcinoma cells in vitro after up-regulation and down-regulation of Vimentin gene expression.The results showed that the invasion and migration ability of nasopharyngeal carcinoma cells in vitro was significantly enhanced after up-regulation of Vimentin expression(P<0.01),while the invasion and migration ability of nasopharyngeal carcinoma cells in vitro was significantly decreased after down-regulation of Vimentin expression(P<0.01).Conclusion:1.Altered the expression of Vimentin in nasopharyngeal carcinoma cells affects the expression of Annexin A1 and S100A9 proteins.2.Up-regulation the expression of Vimentin in nasopharyngeal carcinoma cells can increase their invasion and migration ability.3.Vimentin may affect the invasion and migration of nasopharyngeal carcinoma cells by regulating the expression of Annexin A1 and S100A9 proteins.