微小RNA-221靶向组织金属蛋白酶抑制因子2介导Akt/mTOR信号通路对非小细胞肺癌移植瘤小鼠模型的影响

Effect of microRNA-221 targeting tissue inhibitor of metalloproteinase-2 to mediate Akt/mTOR signaling pathway on non-small cell lung cancer transplanted tumor mouse model

目的 探讨微小RNA(miR)-221对非小细胞肺癌(NSCLC)移植瘤模型小鼠肿瘤细胞增殖、迁移及侵袭的影响,初步分析其通过靶向组织金属蛋白酶抑制因子2(TIMP-2)调控Akt/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对NSCLC模型小鼠肿瘤细胞可能的作用机制。方法 将A549细胞分为对照组、模拟物(mimic)组、TIMP-2组和Mimic+TIMP-2组。Mimic组和TIMP-2组分别转染miR-221 mimic以及TIMP-2过表达质粒。Mimic+TIMP-2组同时转染miR-221 mimic和TIMP-2过表达质粒。对照组转染等量的阴性对照质粒。转染后,将各组的细胞在左前肢经皮下注射,从而构建相应的4组NSCLC小鼠模型。通过免疫组织化学染色检测增殖蛋白(Ki67)水平以测定其对细胞增殖的影响。通过Western blotting检测和比较各组基质金属蛋白酶2(MMP-2)和N-钙黏蛋白(N-cadherin)的蛋白表达评估细胞的迁移和侵袭能力。通过Real-time PCR或Western blotting检测骨髓和肿瘤组织中miR-221、TIMP-2和Akt/mTOR通路的水平。结果 当共同转染野生型(WT)-TIMP-2和miR-221 mimic后,细胞中的相对荧光素酶活性显著降低(P<0.05)。与对照组比较,mimic组的肿瘤组织质量、体积、Ki67、MMP-2和N-cadherin表达水平,miR-221和Akt/mTOR通路水平显著升高,而TIMP-2 mRNA和蛋白水平显著降低(P<0.05)。与对照组比较,TIMP-2组的TIMP-2 mRNA和蛋白水平显著升高,而其余指标显著降低(P<0.05)。Mimic+TIMP-2组的肿瘤组织质量、体积、Ki67、MMP-2和N-cadherin蛋白表达水平及miR-221、Akt/mTOR通路水平显著低于mimic组且显著高于TIMP-2组,而TIMP-2 mRNA和蛋白水平显著高于mimic组且显著低于TIMP-2组(P<0.05)。结论 MiR-221可能通过靶向抑制TIMP-2蛋白介导Akt/mTOR信号途径,促进NSCLC细胞增殖、迁移和侵袭能力。

Objective To explore the effects of miR-221 on tumor cell proliferation, migration and invasion in non-small cell lung cancer(NSCLC) xenograft model mice, and to preliminarily analyze its possible mechanism of regulating Akt/mammalian target of rapamycin(mTOR) signaling pathway by targeting tissue inhibitor of metalloproteinase-2(TIMP-2) on tumor cells in non-small cell lung cancer(NSCLC) through tumor-bearing nude mice. Methods The A549 cells were divided into control group, mimic group, TIMP-2 group and mimic+TIMP-2 group. The mimic group and TIMP-2 group were transfected with miR-221 mimic and TIMP-2 overexpression plasmids, respectively. The mimic+TIMP-2 group was simultaneously transfected with miR-221 mimic and TIMP-2 overexpression plasmids. The control group was transfected with the same amount of negative control plasmid. After transfection, the cells of each group were injected subcutaneously into the left forelimb to construct the corresponding 4 groups of NSCLC mouse models. The proliferation-related protein(Ki67) was detected by immunohistochemical staining to detected the effect of cell proliferation ability. Matrix metalloproteinase-2(MMP-2) and N-cadherin proteins in each group were tested by Western blotting to assess and compare the abilities of migration and invasion. The levels of miR-221, TIMP-2 and Akt/mTOR pathways in bone marrow and tumor tissues were detected by Real-time PCR and Western blotting. Results When co-transfected with wild type(WT)-TIMP-2 and miR-221 mimic, the relative luciferase activity in the cells reduced significantly(P<0.05). Compared with the control group, the tumor mass, volume, Ki67, MMP-2 and N-cadherin protein expression levels, miR-221 and Akt/mTOR pathway levels were increased significantly, while the levels of TIMP-2 mRNA and protein were significantly reduced in the mimic group(P<0.05). Compared with the control group, the levels of TIMP-2 mRNA and protein in the TIMP-2 group increased significantly, while the other indicators decreased significantly(P<0.05). Tumor tissue mass, volume, Ki67, MMP-2, N-cadherin, miR-221 and Akt/mTOR pathway levels in mimic+TIMP-2 group were significantly lower than those in the mimic group and significantly higher than those in the TIMP-2 group, while TIMP-2 mRNA and protein levels were significantly higher than those in the mimic group and significantly lower than those in the TIMP-2 group(P<0.05). Conclusion In the NSCLC transplanted tumor mouse model, miR-221 may mediate the Akt/mTOR pathway by targeting the expression of TIMP-2 protein to promote cell proliferation, migration and invasion.