FGFR1对头颈部鳞状细胞癌细胞增殖和凋亡影响机制研究

Effect of FGFR1 on cell proliferation and apoptosis in head and neck squamous cell carcinoma

目的 探讨成纤维生长因子受体1(FGFR1)对人头颈部鳞状细胞癌细胞增殖和凋亡的影响及作用机制。方法 采用定量逆转录聚合酶链反应法及蛋白质印迹法检测人皮肤永生角质形成细胞株HaCaT和人头颈部鳞状细胞癌细胞系UM-SCC-47、Fadu、SCC4中FGFR1 mRNA和蛋白的表达;慢病毒感染UM-SCC-47和Fadu细胞,构建稳定下调FGFR1的细胞系并检测敲低效率;细胞计数盒8(CCK8)实验检测各组细胞的增殖能力;流式细胞术检测细胞周期和凋亡。裸鼠体内实验验证FGFR1对头颈部鳞状细胞癌增殖的影响,蛋白质印迹法检测FGFR1下游重要信号通路PI3K/AKT信号通路相关蛋白(PI3K、AKT、p-AKT)的表达水平。结果 细胞株HaCaT、UM-SCC-47、Fadu和SCC4中FGFR1 mRNA的表达量分别为1.027±0.026、18.290±2.003、16.430±1.188和10.670±0.890,FGFR1蛋白相对表达量分别为0.304±0.026、3.439±0.166、3.757±0.091和1.583±0.092。与HaCaT相比,3株头颈肿瘤细胞中FGFR1 mRNA及蛋白表达水平均上调,差异有统计学意义,均P<0.001。CCK8实验表明,下调FGFR1的UM-SCC-47和Fadu细胞在96 h测定的光密度值分别为1.475±0.038和1.089±0.051,低于阴性对照组(2.106±0.063和1.719±0.132),差异有统计学意义,P值分别为0.001和0.011。相较于对照组,下调FGFR1后UM-SCC-47和Fadu细胞周期主要阻滞在G_(0)/G_(1)期,差异有统计学意义,P值分别为<0.001和0.005。下调FGFR1组UM-SCC-47和Fadu细胞凋亡率分别为(5.063±1.289)%和(5.063±0.958)%,高于对照组的(0.673±0.037)%和(1.603±0.414)%,差异有统计学意义,P值分别为0.027和0.030。体内实验显示,实验组裸鼠荷瘤体积为(240±40.070) mm^(3),低于对照组的(554±70.930) mm^(3),差异有统计学意义,P=0.018;下调FGFR1的移植瘤组织中Ki-67的阳性表达更低。下调FGFR1后UM-SCC-47和Fadu细胞中p-AKT蛋白相对表达量分别为0.871±0.046和0.753±0.025,低于阴性对照组(1.116±0.043和1.054±0.043),差异有统计学意义,P值分别为0.018和0.004。结论 FGFR1在头颈部鳞状细胞癌细胞中高表达,通过激活PI3K/AKT信号通路促进头颈部鳞状细胞癌细胞增殖并抑制细胞凋亡。

Objective To investigate the effect of overexpression of the fibroblast growth factor receptor 1(FGFR1) on proliferation and apoptosis in human head and neck squamous cell carcinoma(HNSCC) cells and explore the underlying mechanism.Methods Quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western Blot assay were used to detect the expression level of mRNA and protein of FGFR1 in HNSCC cells(UM-SCC-47,Fadu,SCC4) and in HaCaT immortalized human keratinocyte cell(non-cancerous normal cells).Next the expression of FGFR1 was down-regulated by lentiviral vector carrying FGFR1 shRNA and the interference efficiency was determined using qRT-PCR and Western blot.CCK8assay was used to detect the effect of cell proliferation.Cell apoptosis and cycle were determined by flow cytometry.The effect of FGFR1on the proliferation of head and neck squamous cell carcinoma was verified in vivo experiment in nude mice.Western blot assay was used to analyze the expression of PI3K,AKT,p-AKT in signaling pathways downstream of FGFR1.Results The expression levels of FGFR1mRNA in cell lines HaCaT,UM-SCC-47,Fadu and SCC4were 1.027±0.026,18.290±2.003,16.430±1.188and 10.670±0.890,respectively.The relative expression levels of FGFR1protein were 0.304±0.026,3.439±0.166,3.757±0.091and 1.583±0.092,respectively.Compared with HaCaT,the expression levels of FGFR1mRNA and protein in the three HNSCC cells were up-regulated,and the difference was statistically significant(all P<0.001).CCK8assay showed that the optical density values of UM-SCC-47and Fadu cells down regulating FGFR1at 96hwere 1.475±0.038and 1.089±0.051,respectively,which were lower than those in the negative control group(2.106±0.063and 1.719±0.132).The difference was statistically significant(P=0.001;P=0.011).Compared with the control group,the cell cycle of UM-SCC-47and Fadu was mainly blocked in G0/G1phase after down regulating FGFR1,and the difference was statistically significant(P<0.001;P<0.005).The apoptosis rates of UM-SCC-47and Fadu cells in the down regulated FGFR1group were(5.063±1.289)%and(5.063±0.958)%respectively,significantly higher than those of the control group(0.673±0.037)%and(1.603±0.414)%and the difference was statistically significant(P=0.027;P=0.030).The tumor volume of the control group(554±70.930)mm3 was significantly greater than FGFR1-shRNA group(240±40.070)mm3 in vivo(P=0.018);The positive expression of Ki-67was lower in the transplanted tumor tissues down regulated FGFR1.After down regulating FGFR1,the relative expression of phosphorylation-AKT in UM-SCC-47and Fadu cells were 0.871±0.046and 0.753±0.025,respectively,lower than those in the negative control group(1.116±0.043and 1.054±0.043),the difference was statistically significant(P=0.018;P=0.004).Conclusion Overexpression of FGFR1promotes proliferation and inhibits apoptosis of HNSCC cells by activating PI3K/AKT signaling pathway.