MicroRNA-203抑制骨肉瘤细胞增殖和迁移的机制研究

Mechanism of microRNA-203 inhibiting proliferation and migration of osteosarcoma cells

摘要:

摘要：目的  探讨microRNA-203（miR-203）对骨肉瘤细胞增殖和迁移的影响及机制。方法  通过逆转录聚合酶链反应（RT-PCR）测定miR-203在正常成骨细胞hFOB及骨肉瘤细胞系中的表达，将MG-63细胞系分成miR-203组、Scramble组、RAB22A组、NC组，Lipofectamine 2000分别转染miR-203 mimics、Scrambles、pcDNA3.1-RAB22A、空白对照质粒；通过噻唑蓝（MTT）实验测定增殖能力，细胞划痕实验测定迁移能力；通过Target Scan和双荧光素酶实验预测及验证miR-203与RAB22A基因的靶向调节关系；RT-PCR测定RAB22A mRNA表达水平；Western blot测定E-cadherin、N-cadherin、Vimentin蛋白表达水平。结果  miR-203在骨肉瘤细胞系SAOS-2、SOSP-9607、U2OS及MG-63中较hFOB细胞系表达降低。MTT显示，在培养48和72 h后，miR-203组相对细胞数少于Scramble组（P <0.05）；培养24 h后，Scramble组细胞相对划痕面积小于miR-203组（P <0.05）；双荧光素酶实验示，miR-203与RAB22A基因存在靶向调节关系。RAB22A组相对划痕面积小于NC组（P <0.05）；MTT显示，在培养24、48和72 h后，RAB22A组相对细胞数多于NC组（P <0.05）；RAB22A组与NC组比较，E-cadherin下调表达，N-cadherin上调表达，Vimentin上调表达。结论  miR-203通过下调RAB22A基因表达，抑制上皮间质转化，进而抑制骨肉瘤细胞增殖与迁移。

关键词:

miR-203;RAB22A;骨肉瘤;增殖;迁移

Abstract:

Abstract: Objective To investigate the potential role of miR-203 in the regulation of the proliferation and migration of osteosarcoma cells, as well as the regulatory mechanism. Methods The expression levels of miR-203 in the osteosarcoma cells and the normal osteoblasts were measured by RT-PCR. The MG-63 cell line was divided into miR-203 group transfected with miR-203 mimics, scramble group transfected with scrambles, RAB22A group transfected with pcDNA3.1-RAB22A plasmid and NC group transfected with blank plasmid by Lipofectamine 2000. The cell migration ability was measured by wound healing assay. Cell number was examined by MTT assay. The target relationship between miR-203 and RAB22A gene was predicted and verified by Target Scan software and double luciferase assay. The expression level of RAB22A mRNA was measured by RT-PCR. The expression levels of E-cadherin, N-cadherin, Vimentin and RAB22A protein were measured by Western blot. Results Compared with the hFOB cells, the expression of miR-203 was significantly low in the osteosarcoma cell lines SAOS-2, SOSP-9607, U2OS and MG-63. MTT method showed that the number of MG-63 cells in the miR-203 group was significantly smaller than that in the scramble group after cultured for 48 and 72 h (P < 0.05). After cultured for 24 h, the relative scratch area of the scramble group was significantly smaller than that of the miR-203 group (P < 0.05). TargetScan software and double luciferase assay verified the target relationship between miR-203 and RAB22A gene. The relative scratch area in the RAB22A group was significantly smaller than that in the NC group (P < 0.05). MTT method showed that the number of the MG-63 cells was significantly increased in the RAB22A group compared with the NC group after cultured for 24, 48 and 72 h (P < 0.05). Compared with the NC group, E-cadherin expression was down-regulated, N-cadherin and Vimentin expressions were up-regulated in the RAB22A group. Conclusions MiR-203 inhibits proliferation and migration of osteosarcoma cells by inhibiting RAB22A gene expression and epithelial-mesenchymal transition.

Key words:

microRNA-203; RAB22A; osteosarcoma; proliferation; migration