MicroRNA-133b调节FGFR1-ERK1/2-SOX2信号通路对裸鼠肺癌NCI-H1975细胞移植瘤生长的影响

Influence of microRNA-133b on growth of lung cancer cell transplanted tumor in nude mice by regulating FGFR1-ERK1/2-SOX2 signaling pathway

摘要:

目的 探讨microRNA-133b（miR-133b）对裸鼠肺癌NCI-H1975细胞移植瘤生长的抑制作用以及对成纤维细胞生长因子受体1-细胞外信号调节激酶1/2-性别决定区Y-box蛋白2信号通路（FGFR1-ERK1/2-SOX2）的影响。方法 qRT-PCR检测人肺成纤维细胞、肺癌细胞株miR-133b表达。miR-133b过表达NCI-H1975细胞。将NCI-H1975细胞分为对照组、mimic NC组、miR-133b mimic组、miR-133b mimic+pcDNA3.1组、miR-133b mimic+pcDNA3.1 FGFR1组。CCK-8法检测NCI-H1975细胞增殖抑制率，Transwell实验观察NCI-H1975细胞侵袭、迁移情况。复制裸鼠移植瘤模型并分组，将裸鼠分为对照组、mimic NC组、miR-133b mimic组、miR-133b mimic+AZD4547组，观察各组裸鼠肿瘤体积与重量，HE染色观察各组裸鼠肿瘤组织变化，TUNEL检测肿瘤组织细胞凋亡情况，免疫组织化学法观察裸鼠肿瘤组织Ki-67、Cyclin D1、VEGF-A的表达，Western blotting检测各组肿瘤组织FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量。结果 与人肺成纤维细胞HLF-α比较，肺癌细胞株NCI-H1975、A427、NGE-1、A549中miR-133b mRNA相对表达量降低（P <0.05），其中以NCI-H1975细胞中miR-133b mRNA相对表达量最低。miR-133b mimic组miR-133b mRNA相对表达量较对照组和mimic NC组升高（P <0.05）。miR-133b可通过负调控FGFR1抑制肺癌NCI-H1975细胞增殖和迁移。miR-133b mimic组移植瘤重量较对照组降低、体积缩小，miR-133b mimic+AZD4547组移植瘤重量较miR-133b mimic组降低、体积缩小（P <0.05）。miR-133b mimic组空泡样变性程度较对照组、mimic NC组减轻（P <0.05），miR-133b mimic+AZD4547组空泡样变性程度较miR-133b mimic组减轻（P <0.05）。miR-133b mimic组肿瘤组织细胞凋亡率较对照组升高（P <0.05），miR-133b mimic+AZD4547组肿瘤组织细胞凋亡率较miR-133b mimic组升高（P <0.05）。miR-133b mimic组VEGF-A、Cyclin D、Ki-67阳性细胞比例较对照组降低（P <0.05），miR-133b mimic+AZD4547组VEGF-A、Cyclin D、Ki-67阳性细胞比例较miR-133b mimic组降低（P <0.05）。miR-133b mimic组FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量较对照组降低（P <0.05），miR-133b mimic+AZD4547组FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量较miR-133b mimic组降低（P <0.05）。结论 miR-133b过表达可能通过抑制FGFR1-ERK1/2-SOX2轴，抑制裸鼠肺癌NCI-H1975细胞移植瘤生长。

Abstract:

Objective To investigate the inhibitory effect of miR-133b on the growth of lung cancer NCI-H1975 cells subcutaneously transplanted tumor in nude mice and its influence on fibroblast growth factor receptor 1 (FGFR1)-extracellular signal-regulated kinase 1/2 (ERK1/2)-sex-determining region Y-box protein 2 (SOX2) signaling pathway.Methods Human lung fibroblasts HLF-α and lung cancer cells NCI-H1975, A427, NGE-1, and A549 were cultured in vitro, the expression level of miR-133b was detected by qRT-PCR, and the cell line with the lowest expression of miR-133b was selected as the research cell line; NCI-H1975 cells were divided into control group, mimic group, miR-133b mimic group, miR-133b mimic+pcDNA3.1 group, and miR-133b mimic+pcDNA3.1 FGFR1 group. The proliferation inhibition rate of NCI-H1975 cells was detected by CCK-8, and the invasion and migration of NCI-H1975 cells were measured by Transwell chamber method. MiR-133b mimic and mimic NC were transfected into NCI-H1975 cell line, and a certain amount of NCI-H1975 cells were taken for routine culture. NCI-H1975 cells in logarithmic growth phase were digested and injected into BALB/C nude mice. After tumor formation, nude mice were injected with serum-free medium, mimic NC, miR-133b mimic, and miR-133b mimic+FGFR1 inhibitor (AZD4547), respectively, as control group, mimic NC group, miR-133b mimic group, and miR-133b mimic+ AZD4547 group, respectively. The tumor volume and weight in each group were observed; HE staining was performed to observe the change of tumor tissue; TUNEL was performed to measure tumor cell apoptosis; immunohistochemistry was performed to measure the expression of Ki-67, CyclinD1, and VEGF-A in mouse tumor tissue; western blot was performed to determine the levels of FGFR1-ERK1/2-SOX2 pathway-related proteins in tumor tissues of each group.Results Compared with human lung fibroblasts HLF-α, the levels of miR-133b in lung cancer cell lines NCI-H1975, A427, NGE-1, and A549 were decreased (P < 0.05), and the expression of miR-133b in NCI-H1975 cell line was lowest; miR-133b negatively regulates FGFR1 to inhibit proliferation and metastasis of lung cancer NCI-H1975 cells. Compared with the control group, the tumor tissue of nude mice in the miR-133b mimic group had less vacuolar degeneration, and the tumor weight, tumor volume, tumor cell apoptosis rate, positive expression rates of Ki-67, Cyclin D1, VEGF-A, and the levels of FGFR1, p-ERK1/2/ERK1/2, and SOX2 decreased at the time of sacrifice (P < 0.05); compared with the miR-133b mimic group, the degree of vacuolar degeneration of nude mice in the miR-133b mimic+AZD4547 group was reduced, the tumor weight and volume, tumor cell apoptosis rate, positive expression rates of Ki-67, Cyclin D1, VEGF-A, the levels of FGFR1, p-ERK1/2/ERK1/2, and SOX2 decreased at the time of sacrifice (P < 0.05).Conclusion Overexpression of miR-133b may inhibit the growth of lung cancer NCI-H1975 cells subcutaneously transplanted tumor in nude mice by inhibiting the FGFR1-ERK1/2-SOX2 axis.