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

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q RT-PCR检测人肺成纤维细胞、肺癌细胞株miR-133b表达。miR-133b过表达NCIH1975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NCIH1975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。

Objective To investigate the inhibitory effect of miR-133b on the growth of lung cancer NCIH1975 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-αand 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+pcDNA3.1FGFR1 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.NCIH1975 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+FGFR1inhibitor(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.