过表达miR-488-5p 通过靶向TEM8 降低宫颈癌C33A细胞的增殖和迁移能力

目的:探讨miR-488-5p 在宫颈癌组织中的表达及对宫颈癌C33A细胞增殖和迁移能力的影响。方法:收集2017 年3月至2017 年9 月郑州大学附属洛阳中心医院妇科全子宫切除术12 例宫颈癌组织及相应的癌旁组织，qRT-PCR 检测癌组织中miR-488-5p 的表达。利用Lipofectamine 3000 转染miR-488-5p 和miR-NC至宫颈癌C33A细胞，分为实验组和对照组。流式细胞术检测两组细胞周期分布，CCK-8 法检测两组细胞增殖能力，Transwell 检测两组细胞迁移能力。生物信息学软件预测miR-488-5p 可能的靶基因，荧光素酶活性实验验证miR-488-5p 与靶基因的结合作用。qRT-PCR和Western blotting 检测两组细胞中肿瘤内皮标志物8（tumor endothelial marker 8, TEM8）及下游EGFR信号通路相关蛋白的表达水平。结果: 宫颈癌组织中miR-488-5p 相对表达量明显低于癌旁组织（1.33±0.20 vs 3.68±0.45, P<0.01），实验组细胞中miR-488-5p 相对表达量明显高于对照组（25.23±3.11 vs 1.02±0.10, P<0.01)，实验组C33A细胞在G0/G1 期的细胞比例明显高于对照组(P<0.01)；当C33A细胞生长至96 和120 h，实验组细胞增殖水平明显低于对照组(P<0.05)。同时实验组的细胞迁移细胞数明显低于对照组(P<0.01)。miR-488-5p 可直接结合TEM8 mRNA并抑制其表达(P<0.01)。实验组细胞中TEM8 mRNA相对表达量明显低于对照组（0.42±0.06 vs 1.00±0.06, P<0.01)。C33A细胞转染miR-488-5p 48 h 后，TEM8、p-EGFR、p-ERK、p-AKT蛋白表达明显低于对照组（P<0.01）。结论: 宫颈癌组织中miR-488-5p 的表达量下降，过表达miR-488-5p 能够抑制宫颈癌细胞周期的进展，降低宫颈癌细胞的增殖和迁移能力，其机制可能与靶向干扰TEM8 基因的表达有关。

Over-expression of miR-488-5p decreases proliferation and migration ability of cervical cancer C33A cells through TEM8

Objective: To observe the expression of miR-488-5p in cervical cancer tissues and to explore its effect on the proliferation and migration of cervical cancer C33A cells. Methods: 12 pairs of cervical cancer tissues and corresponding paracancer tissues from patients,who underwent total hysterectomy at the Luoyang Central Hospital of Zhengzhou University from March 2017 to September 2017, were collected for this study; and the expression of miR-488-5p was detected by fluorescence quantitative and real-time polymerase chain reaction (qRT-PCR). Lipofectamine 3000 was used to transfect miR-488-5p (experiment group) and miR-NC (control group) into cervical cancer C33A cells. Cell cycle distribution was detected by Flow cytometry. Cell proliferation was assessed by CCK-8 assay and Transwell assay was used to detect cell migration. Bioinformatics software was used to predict the possible target genes of miR-488-5p, and luciferase activity assay was used to verify the binding of miR-488-5p to target genes. The expressions of tumor endothelial marker 8 (TEM8) and downstream EGFR signaling pathway related proteins in two groups were detected by qRT-PCR and Western blotting. Results: The relative expression level of miR-488-5p in cervical cancer tissues (1.33±0.20) was significantly lower than that in paracancer tissues (3.68±0.45) (P<0.01). The relative expression level of miR-488-5p in the experimental group (25.23±3.11)was significantly higher than that in the control group (1.02±0.10) (P<0.01). The percentage of C33A cells at G0/G1 phase in experimental group (53.39±2.48)% was significantly higher than that in control group (39.57±1.21)% (P<0.01). When the culture time extended to 96 h and 120 h, the proliferation ability of C33A cells in experimental group was significantly lower than that in control group (P<0.05), and the number of migrated cells in the experimental group (117.90±18.86) was significantly less than that in the control group (295.10±19.33) (P <0.01). Luciferase activity assay confirmed that miR-488-5p could directly bind with TEM8 and inhibit its expression (P<0.01). The relative expression of TEM8 mRNA in experimental group (0.42±0.06) was significantly lower than that in control group (1.00 ± 0.06) (P<0.01). After transfection with miR-488-5p for 48h, the protein expressions of TEM8, p-EGFR, p-ERK and p-AKT were significantly lower than those in control group (P <0.01). Conclusion: The expression of miR-488-5p in cervical cancer tissues was decreased. Over-expression of miR-488-5p could inhibit the cell cycle progression of cervical cancer cells and reduce the proliferation and migration of cervical cancer cells. The mechanism may be related to the interference of TEM8 gene expression.