Let-7d通过靶向调控Rhotekin基因抑制骨肉瘤细胞的增殖、迁移和侵袭

目的 探讨let-7d在骨肉瘤组织中的表达，并研究let-7d及其靶基因对人骨肉瘤细胞U2OS增殖、迁移和侵袭的影响。方法 收集2010年至2015年于我科行手术切除的25例骨肉瘤患者的肿瘤组织和癌旁组织（距肿瘤组织边缘>5 cm），并用qPCR检测let-7d的表达情况。构建稳定过表达let-7d的U2OS细胞，用qPCR验证let-7d过表达情况，以转染pCDH空病毒载体的U2OS细胞作为对照组细胞，并分别采用CCK-8实验、划痕实验和Transwell实验检测过表达let-7d对U2OS细胞增殖、迁移和侵袭能力的影响。通过微RNA靶基因预测软件和双荧光素酶实验明确let-7d的下游靶基因，检测过表达let-7d的U2OS细胞中靶基因的表达水平，并通过小干扰RNA技术研究抑制靶基因表达对U2OS细胞增殖、迁移和侵袭的影响。结果 骨肉瘤组织中let-7d表达水平低于癌旁组织（P<0.01）。与人成骨细胞hFOB1.19相比，U2OS细胞中let-7d表达水平下调（P<0.01）。与对照组相比，过表达let-7d能抑制U2OS细胞的增殖、迁移和侵袭（P均<0.05）。微RNA靶基因预测软件和双荧光素酶实验结果显示，Rhotekin（RTKN）基因是let-7d的直接靶基因，且过表达let-7d导致U2OS细胞中RTKN mRNA表达水平较对照组降低（P<0.01）。干扰RTKN表达能抑制U2OS细胞增殖、迁移和侵袭（P均<0.05）。结论 Let-7d通过靶向调控RTKN抑制骨肉瘤细胞的增殖、迁移和侵袭，可作为骨肉瘤治疗一个新的靶点。

Let-7d inhibits proliferation, migration and invasion of osteosarcoma cells by targeting Rhotekin gene

Objective To investigate the expression level of let-7d in osteosarcoma tissues, and to explore the effects of let-7d and its target gene on proliferation, migration and invasion of human osteosarcoma cell line U2OS cells. Methods Tumor and adjacent tumor tissues of 25 patients with osteosarcoma undergoing exairesis in our department were collected from 2010 to 2015. The distance from adjacent tumor tissues to tumor margin was greater than 5 cm. The expressions of let-7d were detected by qPCR in the tumor and adjacent tumor tissues. A U2OS cell model stably overexpressing let-7d was constructed, and the expression of let-7d was confirmed by qPCR. The U2OS cells transfected with pCDH empty virus vector were used as control cells. CCK-8 assay, scratch assay and Transwell assay were used to determine the proliferation, migration and invasion of the cell after transfection, respectively. A downstream target gene of let-7d was identified based on microRNA target gene prediction software and luciferase activity assay, and the expression of the target gene was detected in U2OS cells overexpressing let-7d. Effects of the target gene on the cell proliferation, migration and invasion were analyzed by small interfering RNA technology. Results The expression level of let-7d was significantly lower in the osteosarcoma tissues than that in the adjacent tumor tissues (P<0.01). The expression level of let-7d was significantly lower in U2OS cells compared with human osteoblast cell line hFOB1.19 cells (P<0.01). Compared with the control group, overexpression of let-7d significantly reduced the proliferation, migration and invasion of U2OS cells (all P<0.05). MicroRNA target gene prediction software and luciferase activity assay showed that Rhotekin (RTKN) gene was a direct target gene of let-7d. Compared with the control group, overexpression of let-7d significantly reduced mRNA expression level of RTKN in the U2OS cells (P<0.01). Down-regulation of RTKN significantly inhibited the proliferation, migration and invasion of U2OS cells (all P<0.05). Conclusion Let-7d inhibits proliferation, migration and invasion of osteosarcoma cells by targeting RTKN, which indicates let-7d may be a novel candidate biological therapeutic target for osteosarcoma.