miR-383-3p对鼻咽癌6-10B细胞生物学功能的影响及其可能作用机制

目的:探讨miR-383-3p对鼻咽癌6-10B细胞功能的影响及其可能作用的靶基因。方法:采用脂质体介导法将miR-383-3p模拟物（miR-383-3p mimic）、miR-383-3p抑制物（miR-383-3p inhibitor）以及对照模拟物（NC）转染6-10B细胞，MTT 法检测各组6-10B细胞的增殖能力，Annexin V-FITC/PI双染法检测各组6-10B细胞的凋亡情况，Transwell实验检测各组6-10B细胞的迁移能力，双荧光素酶报告基因实验确定miR-383-3p与高迁移率族蛋白A2(high mobility group A2,HMGA2)的靶向作用情况，qRT-PCR检测不同鼻咽癌细胞中miR-383-3p及各组6-10B细胞中miR-383-3p和HMGA2 mRNA表达水平，Western blot检测各组6-10B细胞中水通道蛋白5（aquaporins 5,AQP5)、环磷腺苷效应元件结合蛋白（cAMP-response element binding protein,CREB)、p-CREB以及HMGA2蛋白表达水平，免疫荧光染色检测各组6-10B细胞中AQP5和核因子-κB（nuclear factor-kappa B,NF-κB)的荧光强度。结果:与NP69细胞相比，miR-383-3p在鼻咽癌细胞CNE1、CNE2、HONE1、HNE-1、C666-1、6-10B中的表达量均明显下降（P＜0.05或P＜0.001）；与NC组相比，miR-383-3p mimic 组能够显著抑制鼻咽癌6-10B细胞的增殖和迁移并诱导细胞凋亡（P＜0.05），而miR-383-3p inhibitor组6-10B细胞增殖和迁移能力明显升高，细胞凋亡水平则明显下降（P＜0.05）；HMGA2是miR-383-3p的靶基因，与NC组相比，miR-383-3p mimic 组HMGA2 mRNA 和蛋白的表达水平下降（P＜0.05），而miR-383-3p inhibitor组明显升高（P＜0.05）；与NC组相比，miR-383-3p mimic 组AQP5和p-CREB蛋白表达量明显升高（P＜0.05），而miR-383-3p inhibitor组明显下降（P＜0.05）；与NC组相比，miR-383-3p mimic组AQP5荧光信号较强，而 NF-κB荧光信号较弱,miR-383-3p inhibitor组检测结果与此相反。结论:miR-383-3p可能通过负调控HMGA2的表达来抑制鼻咽癌细胞的增殖与迁移，进而促进鼻咽癌细胞凋亡，该作用可能与AQP5/CREB途径的信号通路有关。

Effect of miR-383-3p on the biological function of nasopharyngeal carcinoma 6-10B cells and its possible mechanism

Objective:To investigate the effect of miR-383-3p on the function of 6-10B cells in nasopharyngeal carcinoma and its possible target genes.Methods:miR-383-3p mimic（miR-383-3p mimic group),miR-383-3p inhibitor（miR-383-3p inhibitor group) and control mimic（NC group) were respectively transfected into 6-10B cells by liposome mediated method.The proliferation of 6-10B cells in each group was detected by MTT assay.The apoptosis of 6-10B cells in each group was detected by Annexin V-FITC/PI double staining.The migration of 6-10B cells in each group was detected by Transwell chamber method.The prime enzyme reporter gene was used to detect the targeting relationship between miR-383 and HMGA2.qRT-PCR was used to detect the expression levels of miR-383-3p and HMGA2 mRNA in miR-383-3p and 6-10B cells in different nasopharyngeal carcinoma cells.The expression levels of AQP5,CREB,p-CREB and HMGA2 in 6-10B cells of each group were detected by Western blot.The fluorescence intensity of AQP5 and NF-κB in 6-10B cells of each group was detected by immunofluorescence.Results:Compared with NP69 cells,the expression levels of miR-383-3p in nasopharyngeal carcinoma cells CNE1,CNE2,HONE1,HNE-1,C666-1,6-10B were significantly decreased（P＜0.05 or P＜0.001).Compared with the NC group,the miR-383-3p mimic group significantly inhibited the proliferation and migration of nasopharyngeal carcinoma 6-10B cells and induced apoptosis（P＜0.05),while the miR-383-3p inhibitor group proliferated and migrated.The ability was significantly higher than that of the NC group,and the level of apoptosis was significantly decreased（P＜0.05).HMGA2 was a target gene of miR-383-3p.Compared with NC group,the expression level of HMGA2 mRNA and protein in miR-383-3p mimic group decreased（P＜0.05),while that in miR-383-3p inhibitor group increased significantly(P＜0.05).Compared with NC group,the expression of AQP5 and p-CREB protein in miR-383-3p mimic group was increased（P＜0.05),and that in miR-383-3p inhibitor group was significantly lower than that in NC group（P＜0.05).Compared with the NC group,the relative expressions of AQP5 and p-CREB protein in miR-383-3p mimic group were significantly increased（P＜0.05),but decreased significantly in miR-383-3p inhibitor group（P＜0.05).Compared with the NC group,the miR-383-3p mimic group had stronger AQP5 fluorescence signals,while the NF-κB fluorescence signal was weaker,and the result of the miR-383-3p inhibitor group was opposite.Conclusion:miR-383-3p may inhibit the proliferation and migration of nasopharyngeal carcinoma cells by targeting negative regulation of HMGA2 expression,and then promote the apoptosis of nasopharyngeal carcinoma cells,which may be related to the regulation of AQP5 expression and CREB signaling pathway.