摘要
[目的]通过转染miRNA-137至人骨肉瘤细胞MG-63，观察细胞内TWIST蛋白含量变化及其对细胞增殖活性的影响以探讨miRNA-137在骨肉瘤中的作用及机制。[方法]Target Scan预测miRNA-137与TWIST结合靶位；PCR扩增TWIST基因3’UTR区并克隆至荧光素酶报告基因表达载体；化学法合成miRNA-137 mimics，miRNA-137 inhibitor及NC序列，与荧光素酶报告基因表达载体共转染293细胞，d8h后检测荧光素酶活性；转染miRNA-137至MG-63细胞，转染后48h，Real Time-PCR检测转染后细胞内miRNA-137含量，Western—blot检测细胞中TWIST蛋白含量变化；转染后0～72h，CCK-8检测肿瘤细胞增殖活性。[结果]证实了miRNA-137与其靶基因TwIsT的结合位点；miRNA-137mimics转染MG-63细胞，可明显抑制细胞内TWIST蛋白表达，与对照组比较，差异有统计学意义（P〈0．05），而miRNA-137 inhibitor转染细胞后可明显增强胞内TWIST蛋白的表达，与对照组比较差异有统计学意义（P〈0．05））；miRNA-137mimics可明显抑制MG-63细胞对数期增殖活性，而miRNA-137in．hibitor则使细胞增殖活性进一步增强，相同时间点与对照相比较，均有差异（P〈0．05）。[结论】miRNA-137可以通过抑制其靶基因TWIST表达来抑制MG-63细胞增殖活性。
[ Objective] Objective To investigate the role of miRNA - 137 in osteosarcoma and its mechanism of action through transfecting human osteosarcoma cells MG - 63 with miRNA - 137 and observing the change in the content of TWIST protein and its influence on cell proliferation. [ Methods] TargetScan was used to predict the binding site between miRNA - 137 and TWIST. PCR multiplication was performed for the 3＇- UTR of TWIST gene, which was cloned into a luciferase reporter gene expression vector. The miRNA- 137 mimics, miRNA -137 inhibitor, and NC sequence were synthesized chemically and co - transfected into 293 cells with the luciferase reporter gene expression vector. The luciferase activities were measured after 48 hours. MG -63 cells were transfected with miRNA - 137, and at 48 hours after transfection, the content of miRNA - 137 in the transfected cells were measured by real - time PCR, and the change in the content of TWIST protein was measured by Western blot. The proliferative activity of tumor cells was measured by CCK - 8 during 0 - 72 hours after transfection. [ Results] The binding site between miRNA - 137 and the targeting gene TWIST was identified successfully. Compared with the control group, the MG - 63 cells transfected with miRNA - 137 mimics showed significantly suppressed protein expression of TWIST （ P 〈 0. 05 ）, while the cells transfected with miRNA - 137 inhibitor showed significantly enhanced protein expression of TWIST （P 〈 0. 05） . Compared with the control group at each time point, miRNA - 137 mimics significantly inhibited the proliferative activity of MG- 63 cells in the logarithmic phase （P 〈 0.05）, while miRNA- 137 inhibitor significantly enhanced the proliferative activity of these cells （P 〈 0. 05） . [ Conclusion ] The miRNA - 137 inhibits the proliferation activity of MG -63 cells through suppressing the expression of its target gene TWIST.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2016年第17期1608-1613,共6页
Orthopedic Journal of China
