The Effect Of Overexpression And Silencing-Expression Of SDF2L1 On The Biological Functions In NPC Nasopharyngeal Carcinoma Cell Lines

Purpose: Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in China,especially in Guangdong and Guangxi.In addition,early diagnosis and treatment of nasopharyngeal carcinoma are difficult because of the special and hidden location of nasopharyngeal carcinoma.In recent years,a large number of studies have shown that the inactivation of tumor suppressor genes is closely related to the occurrence and development of nasopharyngeal cancer.Our previous research results found that SDF2L1 gene expression in nasopharyngeal carcinoma tissue is lower than that of the nasopharyngeal carcinoma tissue.The silencing expression of SDF2L1 promotes nasopharyngeal carcinoma cell lines migration and invasion,and leads to the expression of epithelial-interstitial transformation(EMT)associated E-cadherin and matrix metalloproteinases 9 changed in CNE1 nasopharyngeal carcinoma cell lines,which indicates that SDF2L1 may be involved in the occurrence and development of nasopharyngeal carcinoma,and may be associated with the invasion and distant metastasis of nasopharyngeal carcinoma cells.However,the effects of silencing expression and overexpression of SDF2L1 on the biologicalfunctions of different nasopharyngeal cancer cell lines were not studied at the same time.The mechanism of low SDF2L1 expression,target genes regulated downstream,signal pathways involved,and clinical prognosis of patients with nasopharyngeal cancer are still not clear,which requires further study by our research group.The aim of this study was to investigate the effect of overexpression and silencing-expression of SDF2L1 on the biological behavior of in different NPC cell lines.Methods: 1.Detection of the expression of SDF2L1 mRNA in the h uman nasopharyngeal carcinoma cell lines NP69,HONE1,HK1,CNE2 an d 5-8F was performed by quantitative reverse transcription polymerase cha in reaction(qRT-PCR).2.Construct lentviral vectors that containing SDF2L1 overexpression fragment,SDF2L1 gene interference fragment and their corresponding empty fragment,respectively,and then use them to transfe cted HONE1 and 5-8F nasopharyngeal carcinoma cell lines,and select st able drug-resistant strains with puromycin.Among them,the cells transfect ed with SDF2L1 overexpression and interfering fragment were taken as th e experimental group,the cells transfected with empty fragment were take n as the negative control group,and the untransfected HONE1 and 5-8F cell lines were taken as the blank control group.After 72 h of transfection,the transfection efficiency was observed by photographed and calculated by the inverted fluorescence phase contrast microscope.mRNA and protei n expression levels of SDF2L1 in each group were determined by qRT-P CR and Western Blot(WB).3.The effects of overexpression and silencin g-expression of SDF2L1 on the ability of cell migration and invasion in e ach group were studied by scratch test,Transwell migration test and Tran swell invasion test,respectively.4.Cell Counting kit-8(CCK-8)assay was used to detect the growth and proliferation capacity of cells in each gro up.Results: 1.qRT-PCR results showed that the relative expression levels of SDF2L1 mRNA in HONE1 and 5-8F cells were lower than that in NP69 cells(P values were 0.0062 and 0.0351,respectively),and the differences were statistically significant.The relative expression level of SDF2L1 mRNA in CNE2 cells was not significantly different from that in NP69 cells(P> 0.05).SDF2L1 mRNA relative expression in HK1 cells was higher than that in NP69 cells,and the difference was statistically significant(P< 0.05).2.qRT-PCR and WB validated overexpression and silencing-expression of SDF2L1 results showed that the relative expression levels of SDF2L1 mRNA and protein in the overexpression of SDF2L1 experimental groups were significantly higher than that in the negative control group and the blank control group after stable transfection with the lentviral vector carrying the overexpression fragment of SDF2L1,and the differences were statistically significant(P< 0.05).The relative expression levels of SDF2L1 mRNA and protein in the silencing-expression of SDF2L1 experimental groups were significantly lower than that in the negative control group and the blank control group,and the difference was statistically significant(P< 0.05).3.Scratch test results showed that nasopharyngeal carcinoma cells with overexpression of SDF2L1 had significantly lower scratch healing rate compared with the negative control group and the blank control group(P< 0.05).Compared with the negative control group and the blank control group,nasopharyngeal carcinoma cells with silencing-SDF2L1 expression of SDF2L1 showed significantly higher scratch healing rate,and the difference was statistically significant(P< 0.05).The results of Transwell migration and invasion experiment showed that compared with the negativecontrol group and the blank control group,the number of transmembrane cells of nasopharyngeal carcinoma cells with overexpression of SDF2L1 significantly decreased,and their invasion and migration abilities were significantly inhibited,with statistically significant differences(P< 0.05).Compared with the negative control group and the blank control group,the number of transmembrane cells in nasopharyngeal carcinoma cells with silencing-expression of SDF2L1 significantly increased,and their migration and invasion abilities were significantly enhanced,with statistically significant differences(P< 0.05).4.The cell clonal formation experiment showed that the nasopharyngeal carcinoma cells with overexpression of SDF2L1 had a slower growth rate and smaller clonal formation,and the clonal formation rate was significantly lower than that of the negative control group and the blank control group(P< 0.05).Compared with the negative control group and the blank control group,the clone formation rate of nasopharyngeal carcinoma cells with silencing-expressing of SDF2L1 were significantly increased,and the differences were statistically significant(P< 0.05).CCK-8 results showed that there was no statistically significant difference in the OD value measured at 0h in each group(P> 0.05).From the24 th hour,the OD value of cells in the groups with overexpression of SDF2L1 were significantly lower than that in the negative control group and the blank control group(P< 0.05).The OD values at each time points in the silencing-expression of SDF2L1 group were significantly higher than that in the negative control group and the blank control group(P< 0.05).Conclusion: 1.Overexpression of SDF2L1 inhibited the migration and invasion ability of nasopharyngeal carcinoma cells,while silencing expression of SDF2L1 promoted the migration and invasion ability of nasopharyngeal carcinoma cells.2.Overexpression of SDF2L1 can significantly inhibit thegrowth and proliferation of nasopharyngeal carcinoma cells;Silencing SDF2L1 can obviously promoted the growth and proliferation of nasopharyngeal carcinoma cells.