Study on the inhibitory effects of miR-140-5p targeting YES1 proto-oncogene
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摘要:
目的 研究miR-140-5p与YES1的靶向调控关系，阐明miR-140-5p通过YES1调控前列腺癌发生发展的作用，并研究miR-140-5p在前列腺癌细胞中的潜在作用机制。 方法 采用实时定量PCR检测前列腺癌组织和细胞系中miR-140-5p的表达，采用CCK-8、细胞克隆实验、迁移和划痕实验测定miR-140-5p上调对前列腺癌细胞增殖和迁移的影响，用荧光素酶报告实验和蛋白印迹实验鉴定miR-140-5p的靶基因。 结果 癌组织中的miR-140-5p表达量相对癌旁组织明显下调，差异有统计学意义(P＜0.05)。与RWPE-1细胞相比，前列腺癌细胞系中miR-140-5p的表达水平较低，差异具有统计学意义(P＜0.05)。CCK-8实验转染后miR-140-5p的细胞增殖率显著下降，细胞克隆实验实验组相比对照组细胞克隆数目偏少，差异均有统计学意义(P＜0.05)。划痕实验转染miR-140-5p细胞系的愈合能力明显减弱(P < 0.05)；Transwell小室实验转染miR-140-5p的细胞系的迁移能力下降(P < 0.05)；说明过表达miR-140-5p可抑制细胞的增殖和迁移。生物信息学分析和荧光素酶报告分析确定YES1为miR-140-5p的潜在靶基因。YES1在前列腺癌细胞中过表达，与miR-140-5p表达呈负相关，差异具有统计学意义(P＜0.05)。 结论 以上实验表明miR-140-5p通过直接靶向YES1在前列腺癌发生发展中起抑制作用，提示miR-140-5p可能是一个新的靶点，用于前列腺癌的诊断和治疗。 -
关键词:
- miR-140-5p /
- 前列腺癌 /
- 迁移 /
- 增殖
Abstract:Objective To study the targeted regulatory relationship between miR-140-5p and YES1 and elucidate the role of miR-140-5p in regulating the occurrence and development of prostate cancer through YES1. The potential mechanism of action of miR-140-5p in prostate cancer cells was also determined. Methods The expression of miR-140-5p in prostate cancer tissues and cell lines was detected via real-time quantitative PCR. The effects of miR-140-5p up-regulation on the proliferation and migration of prostate cancer cells were investigated via cell Counting Kit-8 (CCK-8) assay, cell cloning experiment, and migration and scratch assay. The target gene of miR-140-5p was identified via luciferase reporter assays and Western blotting. Results The expression level of miR-140-5p in cancer tissues was significantly down-regulated compared with that in adjacent tissues (P < 0.05). The expression level of miR-140-5p was significantly lower in prostate cancer cell lines than in RWPE-1 cells (P < 0.05). After transfection via CCK-8 assay, the proliferation rate of cells transfected with miR-140-5p significantly decreased, and the number of clones in the hyroid cell clone experiment group was significantly lower than that in the control group (P < 0.05). The healing ability of cell lines transfected with miR-140-5p in the scratch experiments significantly decreased (P < 0.05). Transwell assay revealed that the migration ability of cell lines transfected with miR-140-5p (P < 0.05) decreased, suggesting that overexpression of miR-140-5p can inhibit cell proliferation and migration. Bioinformatics analysis and luciferase report analysis identified YES1 as the potential target gene of miR-140-5p. YES1 overexpression in prostate cancer cells was negatively correlated with miR-140-5p expression, and the difference was statistically significant (P < 0.05). Conclusion These experiments demonstrated that miR-140-5p can inhibit the development of prostate cancer by directly targeting YES1, suggesting that miR-140-5p may be a new target for the diagnosis and treatment of prostate cancer. -
Key words:
- MiR-140-5p /
- Prostate cancer /
- Migration /
- Proliferation
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图 1 miR-140-5p在组织与细胞系中的表达
注：A为癌组织与癌旁组织中的miR-140-5p相对表达量比较；B为前列腺腺癌细胞系(LNCaP和PC3)和健康人前列腺细胞(RWPE-1)中的miR-140-5p表达量比较；aP＜0.05，bP＜0.05。
图 2 miR-140-5p对前列腺癌细胞系增殖的影响
注：A为PC3细胞转染后miR-140mimics相对表达量；B为CCK-8实验转染后细胞增殖率；aP < 0.05。
图 3 miR-140-5p对前列腺癌细胞系迁移的影响
注：A为划痕实验转染细胞系的愈合能力；B为Transwell小室实验转染细胞系迁移数量；与对照组比较，aP＜0.05。
图 4 YES1是miR-140-5p的潜在靶基因
注：A为荧光素酶报告实验转染细胞系荧光素酶活性；B为转染细胞系其YES1/GAPDH相对比值；与对照组比较，aP＜0.05。
表 1 15例前列腺癌患者miR-140-5p对比(x ±s)
项目 类别 例数 miR-140-5p 统计量 P值 年龄(岁) 50~60 2 0.49±0.07 0.313a 0.757 61~70 5 0.46±0.08 71~79 8 0.48±0.08 Gleason分级 ≤6 3 0.52±0.07 5.274a 0.037 7 6 0.46±0.09 ≥8 6 0.43±0.08 肿瘤直径(cm) ＜3 6 0.48±0.08 1.427b 0.159 ≥3 9 0.46±0.08 组织分化程度 高分化 5 0.49±0.09 7.643a 0.014 中分化 7 0.44±0.07 低分化 3 0.42±0.07 注：a为F值，b为t值。 
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