摘要： 目的 探讨miR-9-5P对脉络膜黑色素瘤细胞增殖、凋亡及迁移、侵袭的调控作用，并探寻其发挥作用的靶基因CDH1，CTNNA1和ITGA6。方法 体外给予脉络膜黑色素瘤细胞转染阴性对照（NC）和miR-9-5p mimics。采用RTCA实时增殖实验检测细胞的增殖情况；EDU实验进一步检测细胞的增殖变化；流式细胞技术检测细胞的凋亡变化；Transwell实验观察细胞迁移和侵袭能力的变化情况；Targetscan生物信息学网站分析预测miR-9-5P的靶基因，并通过GO分类和KEGG富集分析找到与迁移和侵袭相关靶基因；Western blotting检测CDH1蛋白表达情况。结果 RTCA实时增殖实验结果显示，miR-9-5p能够抑制脉络膜黑色素瘤细胞MUM-2B（t=6.925，P < 0.05）及MUM-2C（t=4.762，P < 0.05）的增殖；EDU实验结果显示，miR-9-5p能够抑制脉络膜黑色素瘤细胞MUM-2B（t=4.779，P < 0.05）和MUM-2C（t=4.514，P < 0.05）的增殖；流式细胞检测结果表明，miR-9-5p能够促进脉络膜黑色素瘤细胞MUM-2B（t=7.385，P < 0.05）和MUM-2C（t=4.893，P < 0.05）的凋亡；Transwell实验结果显示，miR-9-5p能够抑制细胞的迁移和侵袭；生物信息学预测及分析得出与细胞迁移侵袭相关的3个miR-9-5p的靶基因CDH1，CTNNA1和ITGA6；Western blotting证明CDH1是miR-9-5p的靶基因。结论 miR-9-5p能抑制脉络膜黑色素瘤细胞的增殖、迁移和侵袭，促进脉络膜黑色素瘤细胞的凋亡，并通过调节CDH1，CTNNA1和ITGA6表达发挥调控作用。

miR-9-5P靶向CDH1、CTNNA1和ITGA6对脉络膜黑色素瘤细胞功能的影响

应曼曼, 张晓楠, 刘然, 宁宏

中国医科大学附属第一医院眼科, 沈阳 110001

收稿日期:2019-11-18出版日期:2020-10-30发布日期:2020-10-13
通讯作者:宁宏E-mail:xiaoxiao1998@21.cn.com
作者简介:应曼曼(1991-),女,医师,硕士.
基金资助:辽宁省自然科学基金（20180551171）


关键词: 脉络膜黑色素瘤, miR-9-5p, CDH1, CTNNA1, ITGA6
Abstract: Objective To investigate the effect of miR-9-5p on choroidal melanoma cell line proliferation,apoptosis,migration,and invasion and study the effect of miR-9-5p on the above function by targeting CDH1,CTNNA1,and ITGA6. Methods Choroidal melanoma cell lines MUM-2B and MUM-2C were transfected with negative control (NC) and miR-9-5p mimics in vitro. RTCA was used to detect the proliferation of cells. Furthermore,EdU assay was conducted to detect proliferation capacity and flow cytometry was implemented to examine cell apoptosis. Transwell assays were conducted to observe cell migration and invasion. TargetScan,a bioinformatics web server,was used to predict the target genes of miR-9-5p,and several target genes influencing migration and invasion were found through GO classification and KEGG enrichment analysis;Western blotting was used to detect the expression level of CDH1. Results RTCA assays showed that miR-9-5p could inhibit the proliferation of MUM-2B (t=6.925,P < 0.05) and MUM-2C (t=4.762, P < 0.05). EdU assays also showed that miR-9-5p could inhibit the proliferation of MUM-2B (t=4.779,P < 0.05) and MUM-2C (t=4.514,P < 0.05). The results of flow cytometry showed that miR-9-5p could promote the apoptosis of MUM-2B (t=7.385,P < 0.05) and MUM-2C (t=4.893,P < 0.05). Transwell assays showed that miR-9-5p could suppress cell migration and invasion. Bioinformatics prediction and analysis revealed that CDH1,CTNNA1,and ITGA6 were the target genes of miR-9-5p influencing cell migration and invasion. The results of western blotting indicated that CDH1 is the target gene of miR-9-5p. Conclusion miR-9-5p can inhibit the proliferation,migration,and invasion of choroidal melanoma cells,and promote the apoptosis of choroidal melanoma cells. It regulates migration and invasion of choroidal melanoma cells by regulating the expressions of CDH1,CTNNA1,and ITGA6.
Key words: choroidal melanoma, miR-9-5p, CDH1, CTNNA1, ITGA6
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