miR-181靶向TIMP3调控胆囊癌细胞迁移及侵袭的实验研究

Experimental study on miR-181 regulates the migration and invasion of gallbladder cancer cells by targeting TIMP3

目的研究miR-181靶向金属蛋白酶组织抑制因子3(TIMP3)调控胆囊癌细胞迁移及侵袭的作用。方法培养胆囊癌GBC-SD细胞,转染miR-181 mimic、阴性对照(NC)mimic、TIMP3 siRNA、NC siRNA、表达TIMP3的重组质粒、空白质粒,荧光定量PCR检测miR-181的表达量,Western blot检测TIMP3的表达量,Transwell检测细胞迁移和侵袭活力,生物信息学分析及荧光素酶报告基因实验验证miR-181靶向TIMP3;皮下注射转染miR-181 mimic或NC mimic的GBC-SD细胞后建立荷瘤鼠模型,检测移植瘤质量及miR-181、TIMP3表达量。结果细胞实验显示:转染miR-181 mimic促进细胞迁移及侵袭、抑制细胞中TIMP3的表达及双荧光素酶报告基因的荧光素酶活力;转染TIMP3 siRNA促进细胞迁移及侵袭;转染表达TIMP3的重组质粒后,miR-181 mimic促进细胞迁移、侵袭的作用减弱。动物实验显示:转染miR-181 mimic使移植瘤的质量减轻、移植瘤中TIMP3的表达增加。结论miR-181具有促进胆囊癌细胞迁移和侵袭的作用,靶向抑制TIMP3是介导该作用的分子机制之一。

Objective To study the effect of miR-181 on the migration and invasion of gallbladder cancer cells by targeting tissue inhibitor of metalloproteinase-3(TIMP3).Methods Gallbladder cancer GBC-SD cells were cultured and transfected with miR-181 mimic,NC mimic,TIMP3 siRNA,NC siRNA,recombinant plasmid expressing TIMP3,blank plasmid.The expression of miR-181 was detected by fluorescence quantitative PCR,the expression of TIMP3 was detected by Western blot,the cell migration and invasion activity were detected by Transwell,and miR-18 target TIMP3 was verified by bioinformatics analysis and luciferase reporter gene experiment.Tumorbearing mouse model was established by subcutaneous injection of GBC-SD cells transfected with miR-181 mimic or NC mimic,then the quality of the transplanted tumor and the expression of miR-181,TIMP3 were detected.Results Cell experiments showed that miR-181 mimic could promote cell migration and invasion,inhibit the expression of TIMP3 and luciferase activity of double luciferase reporter gene;TIMP3 siRNA could promote cell migration and invasion;after transfection of recombinant plasmid expressing TIMP3,the effect of miR-181 mimic on cell migration and invasion was weakened.Animal experiments showed that miR-181 mimic could reduce the weight of transplanted tumor and increase the expression of TIMP3.Conclusion miR-181 promotes the migration and invasion of gallbladder cancer cells,and targeting inhibition of TIMP3 is one of the molecular mechanisms.