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  • 邵晓雯,秦锦龙,宋力雯,等.微小RNA-7-5p调控三叶因子3对宫颈癌细胞增殖和迁移的影响[J].同济大学学报(医学版),2019,30(3):292-297,304.    [点击复制]
  • SHAO Xiao-wen,QIN Jin-long,SONG Li-wen,et al.Effect of microRNA-7-5p on migration and invasion of cervical cancercells through regulation of trefoil factor gene[J].同济大学学报(医学版),2019,30(3):292-297,304.   [点击复制]
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微小RNA-7-5p调控三叶因子3对宫颈癌细胞增殖和迁移的影响
邵晓雯,秦锦龙,宋力雯,高丹凤,刘琼花,成佳景
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(同济大学附属第十人民医院妇产科,上海 200072;江苏大学附属澳洋医院妇产科,江苏 张家港 215600)
摘要:
目的 研究三叶因子3(TFF3)基因在宫颈癌组织和正常组织中的表达情况,同时探讨微小RNA-7-5p(miR-7-5p)调控TFF3对人宫颈癌细胞增殖和迁移能力的影响。方法 实时聚合酶链反应检测TFF3在30例宫颈癌及其邻近正常组织中的表达;采用脂质体转染法将TFF3-siRNA、miR-7-5p转染入宫颈癌HeLa及SiHa细胞中。采用CCK-8、Transwell实验和平板克隆实验检测瞬时转染TFF3-siRNA及miR-7-5p mimics对宫颈癌细胞增殖、迁移及克隆形成能力的影响;并利用双荧光素酶报告实验检测miR-7-5p与TFF3的相互关系。结果 TFF3在73.33%(22/30)宫颈癌组织中表达量明显高于其邻近正常组织(P<0.001)。与阴性对照组比较,TFF3 siRNA转染宫颈癌细胞株HeLa和SiHa 3~5d后,细胞增殖明显受到抑制(P<0.05)。Transwell细胞迁移实验表明: HeLa和SiHa阴性对照组迁移细胞分别为(170±15)个/高倍镜视野和(155±10)个/高倍镜视野,TFF3干扰组迁移细胞分别为(70±20)个/高倍镜视野和(54±8)个/高倍镜视野,迁移能力被显著抑制(P<0.05)。克隆形成实验表明: HeLa和SiHa阴性对照组克隆形成数分别为(160±34)个和(183±17)个,TFF3干扰组细胞克隆数分别为(45±15)个和(33±12)个,形成能力明显减弱(P<0.05)。荧光素报告系统表明miR-7-5p可抑制含野生型TFF3 3′UTR序列的荧光素酶活性(P<0.01),但对含突变型TFF3 3′UTR的荧光素酶活性影响不显著(P>0.05)。与阴性对照组比较,miR-7-5p mimics转染宫颈癌细胞株HeLa和SiHa 4~5d后,增殖明显受到抑制(P<0.05)。Transwell细胞迁移实验表明: HeLa和SiHa阴性对照组迁移细胞为(364±48)个/高倍镜视野和(411±27)个/高倍镜视野,miR-7-5p mimics转染组迁移细胞为(165±15)个/高倍镜视野和(100±208)个/高倍镜视野,迁移能力被显著抑制(P<0.05)。克隆形成实验表明: HeLa和SiHa阴性对照组克隆形成数为(83±11)个和(129±21)个,miR-7-5p mimics转染组细胞克隆数为(25±7)个和(14±8)个,形成能力明显减弱(P<0.05)。结论 TFF3基因的异常高表达可能会对宫颈癌的发生发展起促进作用,miR-7-5p抑制宫颈癌细胞TFF3的表达或可为将来宫颈癌的靶向治疗提供一定的实验基础。
关键词:  微小RNA  三叶因子3  宫颈癌  细胞增殖  细胞迁移
DOI:10.16118/j.1008-0392.2019.03.006
投稿时间:2018-11-15
基金项目:国家自然科学基金青年项目(81502451)
Effect of microRNA-7-5p on migration and invasion of cervical cancercells through regulation of trefoil factor gene
SHAO Xiao-wen,QIN Jin-long,SONG Li-wen,GAO Dan-feng,LIU Qiong-hua,CHENG Jia-jing
(Dept. of Obstetrics and Gynecology, Tenth People’s Hospital, Tongji University, Shanghai 200072, China;Dept. of Obstetrics and Gynecology,Aoyang Hospital, Jiangsu University, Zhangjiagang 215600, Jiangsu Province, China)
Abstract:
Objective To investigate the effect of microRNA-7-5p (miR-7-5p) on proliferation and invasion of human cervical cancer cells and its relation to trefoil factor 3 gene (TFF3). Methods The expression of TFF3 in cervical cancer and para-carcinoma tissues were detected by real-time polymerase chain reaction (qRT-PCR).The cervical cancer HeLa and SiHa cells were transfected with TFF3 siRNA. The proliferation, migration and colony formation capacity of transfected cells and negative control were measured by CCK-8 assays, Transwell migration and colony assays, respectively. The interaction between TFF3 and miR-7-5p was validated through the dual luciferase reporter assay. Results The expression of TFF3 was increased in 73.33% (22/30) of cervical cancer tissue samples compared to adjacent normal tissues (P<0.001). Compared to negative control, the proliferation rate of HeLa and SiHa cells with TFF3 knockdown on day 3~5 significantly suppressed (P<0.05). Transwell migration assay showed the penetrated HeLa and SiHa control cells were (170±15) cells/HP and (155±10) cells/HP, while TFF3-knockdown cells were (70±20) cells/HP and (54±8) cells/HP respectively (P<0.05). Colony formation assay showing that the negative control HeLa and SiHa cells were able to form (160±34) and (183±17) colonies, while TFF3-inhibited cells formed (45±15) and (33±12) colonies (P<0.05). The luciferase assay showed that miR-7 inhibited luciferase activity of plasmids with wild TFF3 3′UTR while had no effect on the plasmids with mutant TFF3 3′UTR (P>0.05). Compared to negative control, the proliferation of miR-7-5p mimics-transfection Hela and SiHa cells was suppressed at d 4-5 (P<0.05). Transwell assay showed that penetrated control Hela and SiHa cells were (364±48) cells/HP and (411±27) cells/HP, while the penetrated TFF3-knockdown cells were (165±15) cells/HP and (100±208) cells/HP, respectively (P<0.05). Conclusion It is suggested that TFF3 may contribute to the malignant progression of cervical cancers and miR-7-5p/TFF3 may serve as a potential therapeutic target for patients with cervical cancer.
Key words:  microRNA  trefoil factor 3  cervical cancer  cell proliferation  cell migration

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