| Aims:To study the effect of mi R-675-5p which sheared by long non-coding RNA H19 on development and progress of Nasopharyngeal Cancer(NPC) and whether mi R-675-5p regulating invasion and metastasis of NPC through targeting SFN(14-3-3?). To validate the relationship of H19, mi R-675-5p and SFN in NPC and their relationship with invasion and metastasis of NPC.Methods:To detect 14-3-3? protein level in different differentiated cell line,the protein levels of 14-3-3? in normal nasopharynx epithelial cells NP69 and varied nasopharyngeal carcinoma cells 6-10 B and 5-8F were detected using Western blotting. At same time, to find out the relationship between 14-3-3? protein level and the expression levels of H19 and mi R-675-5p, the expression levels of H19 and mi R-675-5p in normal nasopharynx epithelial cells NP69 and varied nasopharyngeal carcinoma cells 6-10 B and 5-8F were quantified by real-time PCR. mi R-675-5p mimic and inhibitor were transfected into NPC 6-10 B to over-express and down-express mi R-675-5p, mi R-675-5p mimic negative control and inhibitor negative control were transfected into NPC 6-10 B as control groups,The effect of over-expresion and down-expression mi R-675-5p on expression level of 14-3-3? protein wsa detected by using Western blotting. The 3'-UTRsegments of SFN containing mi R-675-5p binding sites were amplified by PCR and the luciferase activity in the transfected cells wsa assayed to detect weather SFN is the derect target of mi R-675-5p.The effect of over-expresion and down-expression mi R-675-5p on invasiveness and migration capability of 6-10 B cell was evaluated using wound healing test and Transwell test.Results:1. the protein levels of 14-3-3? in normal nasopharynx epithelial cells NP69 and varied nasopharyngeal carcinoma cells 6-10 B and 5-8F were detected by western blot, there were significantly higher 14-3-3? level in NP69 cell than 6-10 B and 5-8F cells(p(27)0.05), and the protein expression levels in Low-Metastatic nasopharyngeal carcinoma cell 6-10 B wsa higher than High-Metastatic nasopharyngeal carcinoma cell 5-8F.This show that the protein levels of 14-3-3? was associated with the development of nasopharyngeal carcinoma. 2. the expression levels of H19 and mi R-675-5p in normal nasopharynx epithelial cells NP69 and varied nasopharyngeal carcinoma cells 6-10 B and 5-8F were quantified by real-time PCR. H19 and mi R-675-5p were significantly higher in 6-10 B and 5-8F cells than NP69 cell(p(27)0.05).And the expression levels in Low-Metastatic nasopharyngeal carcinoma cell 6-10 B wsa lower than High-Metastatic nasopharyngeal carcinoma cell 5-8F.This shows that the expression levels of H19 and mi R-675-5p was associated with the development of nasopharyngeal carcinoma. 3. The expression level of 14-3-3? protein in 6-10 B cell transfected with mi R-675-5p mimic(mimic group), mimic negative control(NC group) anduntreated 6-10 B cell(blank group)were detected by Western blot, the level of 14-3-3? protein were significantly lower in mimic group, but there were no significant difference between NC group and blank group. 4. The expression level of 14-3-3? protein in 6-10 B cell transfected with mi R-675-5p inhibitor(inhibitor group), inhibitor negative control(NC group) and untreated 6-10 B cell(blank group)were detected by Western blot, the level of 14-3-3? protein were significantly higher in inhibitor group, but there were no significant difference between NC group and blank group. 5. Dual-luciferase reporter assay system was used to verivfied wether SFN was the target m RNA of mi R-675-5p,And the result show that The 3'-UTR segments of SFN containing mi R-675-5p binding sites. SFN was the target gene of mi R-675-5p. 6. Transwell test verified that in mimic group, there are 55.4±4.03 cells in each high-power field and the number is bigger than which in NC group(24.5±6.08 cells in each high-power field) and blank group(23.7±6.86 cells in each high-power field). The invasion abilty of mimic group was significantly enhanced. But The invasion abilty between NC group and blank group has no significant difference(p>0.05). The invasive cells of inhibitor group are 55.4±4.03 in each high-power field and the number is lower than NC group and blank group(p(27)0.05). The invasion abilty between NC group and blank group has no significant difference(p>0.05). 7. Scratch test verified that the distance of cell migration is 0.37±0.016 mm in mimic group and the distance is longer than which in NCgroup(0.19±0.015 mm) and blank group(0.18±0.009 mm). The migration abilty of mimic group was significantly enhanced(p(27)0.05). But The migration abilty between NC group and blank group has no significant difference(p>0.05). The migration distance of inhibitor group is 0.06±0.012 mm and the distance is shorter than NC group(0.20±0.019 mm) and blank group(0.19±0.021 mm).The migration ability of inhibitor group is sianificantly decreased(p(27)0.05). The migration abilty between NC group and blank group has no significant difference(p>0.05).Conclusion: Mi R-675-5p inhibited the expression level of 14-3-3? and increased invasion and migration capability in nasopharyngeal carcinoma cell by targeting to SFN. SFN is the target gene of mi R-675-5p. |
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