Roles and molecular mechanism of microRNA-491 on proliferation, invasion and migration of cervical cancer cells
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摘要:目的 探讨微小RNA-491(miR-491)在宫颈癌(CC)细胞增殖、侵袭及迁移中的作用及分子机制。方法 收集CC患者的58对癌组织及癌旁组织标本；采用实时荧光定量反转录聚合酶链反应(qRT-PCR)检测CC组织和癌旁组织(对照组)中miR-491和爪蟾驱动蛋白样蛋白2的靶向蛋白(TPX2)的表达水平。将CC细胞系中的Hela和HT-3细胞随机分为NC组[磷酸盐缓冲液(PBS)处理]、miR-491组(过表达miR-491处理)、si-TPX2组(抑制TPX2)、TPX2组(过表达TPX2)及miR-491+TPX2组(同时过表达miR-491和TPX2)。采用细胞活力和克隆形成实验测定miR-491对CC细胞增殖能力的影响；采用双荧光素酶报告实验验证miR-491和 TPX2 的靶向关系；采用划痕和侵袭实验检测miR-491对CC细胞侵袭和迁移能力的影响；构建裸鼠成瘤模型验证miR-491和TPX2对肿瘤生长的影响。结果 miR-491在CC组织和细胞中的表达降低，而TPX2的表达升高，差异有统计学意义(P<0.05)。过表达miR-491能够抑制CC细胞的细胞增殖。双荧光素酶报告结果表明，与NC组比较, miR-491模拟物可以降低PmirGLO-TPX2-3′UTR WT的荧光素酶活性，差异有统计学意义(P<0.05)。细胞活力和凋亡检测结果发现, miR-491能够通过调控TPX2抑制细胞增殖来增高细胞凋亡率。进一步的划痕和transwell检测结果发现, miR-491过表达能够抑制细胞的迁移和侵袭，而TPX2过表达能够促进细胞迁移和侵袭。肿瘤体积和质量检测结果发现, miR-491能够抑制肿瘤生长，而TPX2过表达能够促进肿瘤生长且这种促进作用能够被miR-491逆转。苏木精-伊红染色(HE)检测发现, miR-491组的肿瘤组织出现部分坏死，而TPX2组肿瘤细胞增殖明显。结论 miR-491能够通过调控TPX2的表达影响CC进程，为未来CC生物标志物的选择及治疗靶点提供理论依据。Abstract:Objective To investigate the roles and molecular mechanism of microRNA-491 (miR-491) in the proliferation, invasion and migration of cervical cancer (CC) cells and its molecular mechanism.Methods A total of 58 pairs of cancer tissue and paracancerous tissue samples from CC patients were collected. The expression levels of miR-491 and Xenopus kinesin-like protein 2 target protein (TPX2) in CC tissues and paracancerous tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Hela and HT-3 cells of CC cell line were randomly divided into NC group[phosphate buffer (PBS) treatment], miR-491 group (overexpression of miR-491 treatment), si-TPX2 group (inhibition of TPX2), TPX2 group (overexpression of TPX2) and miR-491+TPX2 group (simultaneous overexpression of miR-491 and TPX2). Cell viability and clone formation assays were used to determine the effect of miR-491 on the proliferation ability of CC cells. The targeting relationship between miR-491 and TPX2 was verified by dual-luciferase reporter assay. Scratch and invasion assays were used to detect the effect of miR-491 on the invasion and migration of CC cells. A nude mouse tumorigenic model was constructed to verify the effects of miR-491 and TPX2 on tumor growth.Results The results of the study showed that the expressions of miR-491 in CC tissues and cells were significantly reduced, while the expressions of TPX2 were significantly increased, and the differences were statistically significant (P<0.05). Overexpression of miR-491 could inhibit the cell proliferation of CC cells. The results of the dual luciferase report showed that miR-491 could significantly reduce the luciferase activity of PmirGLO-TPX2-3′UTR WT compared with the NC group (P<0.05). The test results of cell viability and apoptosis found that miR-491 mimics can significantly inhibit cell proliferation and increase the rate of apoptosis by regulating TPX2. Further scratches and transwell detection revealed that the miR-491 overexpression inhibited cell migration and invasion, while overexpression of TPX2 could promote cell migration and invasion. In addition, this effect was reversed by the overexpression of miR-491. From the results of tumor volume and weight detection, it is found that miR-491 could inhibit tumor growth, while overexpression of TPX2 can promote tumor growth, and its promotion effect was reversed by miR-491. Further detection of hematoxylin and eosin staining(HE) showed that the tumor tissues in the miR-491 group showed partial ring necrosis, while the tumor cell proliferation effect in the TPX2 group was obvious.Conclusion This study confirms that miR-491 can affect the progress of CC by regulating the expression of TPX2, which provides a theoretical basis for the selection of cervical cancer biomarkers and therapeutic targets in the future.
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Keywords:
- cervical cancer /
- microRNA-491 /
- cell proliferation /
- cell migration /
- tissue necrosis /
- cell vitality
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图 1 CC组织和癌旁组织中miR-491和TPX2的表达情况
A: qRT-PCR检测CC组织中miR-491的表达情况; B: qRT-PCR检测CC细胞系中miR-491的表达情况; C: qRT-PCR检测TPX2 mRNA在CC组织中的表达情况; D: miR-491和TPX2 mRNA的表达的相关性分析。
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图 2 miR-491对细胞增殖能力的影响
A: qRT-PCR验证各组miR-491的表达情况; B: CCK-8检测验证miR-491对细胞增殖的影响; C: 克隆形成实验验证miR-491对细胞增殖的影响。与NC组比较, *P<0.05, **P<0.01。
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图 3 miR-491与TPX2的靶向关系验证
A: qRT-PCR验证TPX2的表达情况; B: TPX2在细胞中的蛋白表达情况; C: 双荧光素酶报告实验验证miR-491与TPX2的靶向关系。
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图 4 miR-491通过TPX2调控Hela细胞的增殖、迁移及侵袭
A、B: 验证各组TPX2的表达情况; C: CCK-8检测各组细胞活力; D: 流式细胞仪检测各组细胞凋亡情况; E: 划痕实验验证各组细胞迁移情况; F: transwell实验验证各组细胞侵袭能力。
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图 5 miR-491通过TPX2调控HT-3细胞的增殖、迁移及侵袭
A、B: 验证各组TPX2的表达情况; C: CCK-8检测各组细胞活力; D: 流式细胞仪检测各组细胞凋亡情况; E: 划痕实验验证各组细胞迁移情况; F: traswell实验验证各组细胞侵袭能力情况。
表 1 临床样本信息
特征 miR-491表达情况 P 低表达(n=41) 高表达(n=17) 性别 男 23 9 0.825 9 女 18 8 年龄/岁 < 60 22 9 0.960 2 ≥60 19 8 TNM分期 Ⅰ~Ⅱ期 21 11 0.347 2 Ⅲ~Ⅳ期 20 6 T分期 T1~T2期 9 10 0.006 5 T3~T4期 32 7 淋巴结转移 无 33 9 0.032 6 有 8 8 
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表 2 各基因引物序列情况
基因名称 引物 序列 miR-491 上游 5′- ACACTCCAGCTGGGAGTGGGGAACCCTT-3′ 下游 5′-TGGTGTCGTGGAGTCG-3′ TPX2 上游 5′- ACCTTGCCCTACTAAGATT-3′ 下游 5′- AATGTGGCACAGGTTGAGC-3′ U6 上游 CTCGCTTCGGCAGCACA 下游 AACGCTTCACGAATTTGCGT GADPH 上游 5′- GGGTGTGAACCACGAGAAAT-3′ 下游 5′-ACTGTGGTCATGAGCCCTTC -3′
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