| Objective:To study the effect of miR-597 on development and progression of Nasopharyngeal Carcinoma(NPC)cells and the effect of miR-597 on the epithelial mesenchymal transition(EMT),migration and invasion of NPC cells by regulating 14-3-3σ.Methods:Through bioinformatics analysis,found the most likely target miRNA of SFN.The expression level of 14-3-3σ protein in different metastatic potential NPC cell lines 6-10 B,5-8F and different differentiation of NPC cell lines CNE1 and CNE2 were detected by Western blotting.At the same time,the expression of miR-597 in the four NPC cell lines was detected by qRT-PCR.The 3’-UTR segments of SFN containing miR-597 binding sites were amplified by qRT-PCR and the luciferase activity in the transfected cells was assayed to detect weather SFN is the direct target gene of miR-597.We transfected 6-10 B cells with miR-597 mimic,miR-597 mimic NC,miR-597 inhibitor,miR-597 inhibitor NC and verified the expression of 14-3-3σ and the EMT related proteins E-cadherin and Vimentin by western blotting.The changes of migration and invasion ability of NPC cells 6-10 B before and after transfection of miR-597 mimic and inhibitor were determined by wound healing assay and Transwell assay.Results: 1.The target miRNAs of SFN were predicted and analyzed by miRwalk online software,the results showed that: 5 target miRNAs meet the conditions of mirSVR ≤-0.1: miR-220 c,miR-597,miR-362-3p,miR-329,and miR-922.Among them,miR-597 had the best sequence conservation and the strongest stability,miR-597 was the most likely target miRNA binding to SFN.2.Prediction of binding sites of miR-597 and SFN in TargetScan 6.2 showed that the binding site was located in the SFN gene at bases 489–495 combined with the sequence 5’-GUGACAC-3’,which is consistent with the partial sequence of miR-597.3.The 14-3-3σ protein was significantly downregulated in the poorly differentiated NPC cell line(CNE2)compared with its expression in the well-differentiated NPC cell line(CNE1)(P < 0.05).The expression level of14-3-3σ was higher in the low metastatic NPC cell line 6-10 B than in the high metastatic potential NPC 5-8F cell line(P<0.05).4.The expression of miR-597 was significantly higher in the poorly differentiated CNE2 cell line than in the well-differentiated line CNE1(P<0.05),and it was higher in high metastatic 5-8F cells than in low metastatic 6-10 B cells(P<0.05).5.Dual-luciferase reporter assay system was used to verivfied whether SFN was the target gene of miR-597,and the result show that The 3’-UTR segments of SFN containing miR-597 binding sites.SFN was the target gene of miR-597.6.The expression of 14-3-3σ protein in miR-597 mimic was lower than that in miR-597 mimic NC and untransfected group(P<0.05),while the expression of14-3-3σ in miR-597 inhibitor was higher than that in miR-597 inhibitor NC and untransfected group(P<0.05).7.Western blotting shows changes in the expression levels of the EMT related proteins,Compared with the miR-597 mimic NC and the untransfected groups,miR-597 mimic significantly downregulated E-cadherin and upregulated Vimentin(P < 0.05).Consistently,miR-597 inhibitor transfection upregulated E-cadherin and downregulated Vimentin(P<0.05).9.The Wound healing assay verified that the distance of cell migration in miR-597 mimic is longer than which in miR-597 mimic NC and untransfection group(P < 0.05).The migration distance of miR-597 inhibitor is shorter than miR-597 inhibitor NC and untransfection group(P<0.05).10.Transwell migration assay verified that the number of migration cells in miR-597 mimic is more than miR-597 mimic NC and untransfection group(P<0.05).The number of migration cells in miR-597 inhibitor is lower than miR-597 inhibitor NC and untransfection group(P(27)0.05).11.Transwell invasion assay verified that the number of invasion cells in miR-597 mimic is more than miR-597 mimic NC and untransfection group(P<0.05).The number of invasion cells in miR-597 inhibitor is lower than miR-597 inhibitor NC and untransfection group(P(27)0.05).Conclusion: 1.The protein 14-3-3σ expression is down regulated in NPC,and miR-597 was highly expressed in NPC,and they are associated with the metastasis and differentiation of NPC cell lines.2.In NPC cell lines,over-expression of miR-597 can inhibit the expression level of 14-3-3σ protein.And inhibiting the expression level of miR-597 can increase the expression level of 14-3-3σ protein.There was correlation between the expression of miR-597 and 14-3-3σ.3.miR-597 can bind to 3’-UTR of SFN.SFN is the target gene of miR-597.miR-597 promotes migration and invasion of NPC cells by negative regulating of SFN. |
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