Objective: To explore the effects of circTNPO1 on the proliferation and metastasis of osteosarcoma (OS) by sponging miR-338-3p. Methods: The expression of circTNPO1 on osteoblasts and multiple OS cell lines were detected by qRT-PCR. CircTNPO1 stable knockdown 143B cell line was constructed by sh-circTNPO1. Cell count kit 8 (CCK-8) assay and wound healing assay were applied to evaluate the proliferation and metastasis of this cell. Luciferase reporter assay was used to explore the binding between circTNPO1 and miR-338-3p. In xenograft tumor model, miR-338-3p inhibitor or its control was injected into the circTNPO1 knockdown tumors. The weight and size of the tumors were evaluated and Ki-67 expression was detected by immunohistochemistry. Results: The RNA expression of circTNPO1 in OS cell lines U2OS, HOS, MG63, 143B, ZOS and ZOSM were 2.73±0.27, 3.18±0.54, 4.33±0.52, 5.75±0.65, 4.50±0.49 and 3.96±0.35, respectively, higher than 1.00±0.09 in hFOB1.19 (P<0.001). CCK-8 assay revealed that after 48 h and 72 h, the absorbance of sh-circTNPO1 #1 was 0.81±0.05 and 1.09±0.06, while sh-circTNPO1 #2 143B cells was 0.84±0.04 and 1.2±0.04, which were sharply reduced compared with the control (1.00±0.06 and 1.49±0.06, P<0.001); after 48 h and 72 h, the absorbance of 143B cells transfected with circTNPO1 #1 and miR-338-3p (0.92±0.06 and 1.32±0.07) were higher than those of cells transfected with sh-circTNPO1 cells and miR NC (0.92±0.06 and 1.32±0.07, P<0.050). Wound healing assay demonstrated that the 24 hour-migration rates of sh-circTNPO1 #1 and sh-circTNPO1 #2 cells were (24.43±2.15)% and (39.70±4.20)% respectively, which were significantly lower than that of the control [(56.51±3.27)%, P<0.010]; the migration rates of sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor were (26.70±2.21)% and (46.10±5.71)%, with a significant difference (P<0.005). In xenograft tumor model, the weight and size of tumors in control, sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor mice were (458.80±158.10) mg, (262.50±82.09) mg, (395.40±137.60) mg and (593.00±228.40) mm(2,) (203.30±144.20) mm(2,) (488.60±208.60) mm(2,) respectively. Compared with control, sh-circTNPO1 tumors were significantly smaller (P<0.01). Injection with miR-338-3p inhibitor significantly reversed both the weight and size of tumors (P<0.05). Conclusion: CircTNPO1 promotes the proliferation and metastasis of OS by sponging miR-338-3p, which could be a new target for OS treatments.
目的： 探讨circTNPO1通过下调miR－338－3p的表达促进骨肉瘤细胞增殖迁移的作用及其分子机制。 方法： 采用实时荧光定量聚合酶链反应检测骨肉瘤细胞中circTNPO1、miR－338－3p、Ki－67和Vimentin的表达水平。构建circTNPO1稳定敲降的143B细胞系，细胞计数试剂盒8实验与细胞划痕实验检测细胞增殖迁移能力，荧光素酶报告基因实验验证circTNPO1与miR－338－3p的关系。建立裸鼠sh－circTNPO1 143B细胞皮下成瘤模型，瘤内注射miR－338－3p抑制剂与miR阴性对照，观察肿瘤重量与体积的变化。免疫组化法检测肿瘤组织中Ki－67的表达。 结果： 骨肉瘤细胞系U2OS、HOS、MG63、143B、ZOS和ZOSM中circTNPO1 mRNA表达水平分别为2.73±0.27、3.18±0.54、4.33±0.52、5.75±0.65、4.50±0.49和3.96±0.35，均高于成骨细胞hFOB1.19(1.00±0.09，均P<0.05)。培养48和72 h后，sh－circTNPO1#1组143B细胞吸光度(A)值分别为0.81±0.05和1.09±0.06，sh－circTNPO1#2组143B细胞A值分别为0.84±0.04和1.2±0.04，均低于对照组(1.00±0.06和1.49±0.06，均P<0.05)。转染48和72 h时，sh－circTNPO1#1+miR－338－3p抑制剂组143B细胞A值分别为0.92±0.06和1.32±0.07，均高于sh－circTNPO1#1+miR阴性对照组(分别为0.82±0.04和1.07±0.08，均P<0.05)。细胞划痕实验显示，sh－circTNPO1#1组和sh－circTNPO1#2组143B细胞迁移率分别为(24.43±2.15)%和(39.70±4.20)%，均低于对照组[(56.51±3.27)%，均P<0.05]。sh－circTNPO1#1+miR－338－3p抑制剂组143B细胞迁移率[(46.10±5.71)%]高于sh－circTNPO1#1+miR阴性对照组[(26.70±2.21)%，P＝0.005]。sh－circTNPO1#1+miR阴性对照组裸鼠肿瘤重量[(262.50±82.09)mg]与体积[(203.30±144.20)mm(3)]均低于对照+miR阴性对照组[分别为(458.80±158.10)mg和(593.00±228.40)mm(3)，均P<0.05]。sh－circTNPO1#1+miR－338－3p抑制剂组裸鼠肿瘤重量[(395.40±137.60)mg]与体积[(488.60±208.60)mm(3)]均高于sh－circTNPO1#1+miR阴性对照组(均P<0.05)。 结论： circTNPO1通过靶向吸附miR－338－3p并下调miR－338－3p的表达促进骨肉瘤细胞的增殖与迁移，circTNPO1下调miR－338－3p可能是参与骨肉瘤进展的新机制。.
Keywords: Cell metastasis; Cell proliferation; Osteosarcoma; circTNPO1; miR-338-3p.