EGFR通过调控CXCR4/CXCL12信号通路对恶性外周神经鞘膜瘤细胞增殖、侵袭和迁移能力的影响

The effect of EGFR on the proliferation，invasion and migration of malignant peripheral nerve sheath tumor cells by regulating the CXCR4/CXCL12 signaling pathway

摘要:

目的：探讨表皮生长因子受体（epidermal growth factor receptor，EGFR）的表达对恶性外周神经鞘膜瘤（malignant peripheral nerve sheath tumors，MPNST）细胞增殖、侵袭和迁移能力的影响，以及对C-X-C型趋化因子受体4/C-X-C型趋化因子配体12（C-X-C motif chemokine receptor 4/C-X-C motif chemokine ligand 12，CXCR4/CXCL12）信号通路的调节机制。方法：收集2017年6月至2019年6月在广州中医药大学第二临床医学院进行手术切除病灶的50例MPNST患者肿瘤组织作为观察组，50例皮肤型神经纤维瘤组织（dermal neurofibroma，DNF）作为对照组，免疫组化检测组织样本中的EGFR表达。细胞实验分为正常组（Normal组）：不加任何药物，常规培养；对照组（Control组）：细胞转染150 nmol/L EGFR-siRNA-NC后，常规培养；实验组（EGFR-siRNA组）：细胞转染150 nmol/L EGFR-siRNA后，常规培养；5-乙炔基-2-脱氧尿嘧啶核苷（5-ethynyl-2’-deoxyuridine，EdU）标记实验检测各组细胞的增殖能力；Transwell小室实验检测各组细胞的迁移和侵袭能力；裸鼠皮下种植瘤模型检测各组细胞的体内生长与转移能力；基因芯片技术与实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）分析确定沉默EGFR基因后作用的靶基因；Western blot检测各组细胞中EGFR、CXCR4、CXCL12的表达。结果：免疫组化结果显示，对照组的EGFR阳性表达率为（2.35±0.32）%，MPNST患者肿瘤组织中的EGFR阳性表达率为（78.94±12.25）%，差异具有统计意义（t=123.573，均P=0.000）；与Normal组相比，EGFR-siRNA组细胞的增殖能力（t=53.147，P=0.000）、侵袭与转移能力（t=26.947、34.638，P=0.000），体内生长与转移能力（t=15.683、22.197，均P=0.000）明显降低。基因芯片和RT-qPCR分析确定CXCR4、CXCL12是EGFR作用的靶基因。与Normal组相比，EGFR-siRNA组细胞中EGFR、CXCR4、CXCL12的表达明显下降（t=2.579、2.673、2.945，均P=0.000）。结论：在MPNST中，EGFR能调控CXCR4/CXCL12信号表达，调控肿瘤的生物学恶性行为，沉默EGFR表达，癌细胞的增殖、侵袭、迁移能力明显下降。

Abstract:

Objective：To investigate the effect of epidermal growth factor receptor（EGFR） expression on the proliferation，invasion and migration of malignant peripheral nerve sheath tumors（MPNST） cells，and its regulation mechanism on C-X-C motif chemokine receptor 4/C-X-C motif chemokine ligand 12（CXCR4/CXCL12） signaling pathway. Methods：The tumor tissues of 50 cases of MPNST who were surgically removed from the lesions in our hospital from June 2017 to June 2019 were collected as the observation group，and 50 cases of dermal neurofibroma（DNF） were collected as the control group. Immunohistochemistry was used to detect the expression of EGFR. Cell experiments were divided into 3 groups：Normal group（cells were regularly cultured without external intervention）；Control group（cells were transfected with 150 nmol/L EGFR-siRNA-NC and then regularly cultured）；exper－imental group（EGFR-siRNA group，cells were transfected with 150 nmol/L EGFR-siRNA and then regularly cultured）. Besides，5-ethynyl-2’-deoxyuridine（EdU） assay was used to detect the proliferation ability of each group of cells；the Transwell chamber test was used to detect the migration and invasion ability of each group of cells；the nude mouse subcutaneous tumor model was used to detect the growth and migration ability in vivo of each group of cells；gene chips and real-time quantitative polymerase chain reaction（RT-qPCR） was used to determine the target genes of the silence of EGFR；Western blot was used to detect the expression levels of EGFR，CXCR4 and CXCL12 in each group of cells. Results：The immunohistochemical results showed that compared with the control group’s（2.35±0.32）%，the positive expression rate of EGFR in the tumor tissues of MPNST patients was （78.94±12.25）%，with significant differences（t=123.573，P=0.000）. Compared with the Normal group，the cell proliferation（t=53.147，P=0.000），invasion and migration ability（t=26.947，t=34.638，both P=0.000），the growth and migration ability in vivo（t=15.683，t=22.197，both P=0.000） of the EGFR-siRNA group were significantly reduced. Gene chip and RT-qPCR analysis confirmed that CXCR4 and CXCL12 were the target genes of EGFR. Compared with the Normal group，the expression of EGFR，CXCR4 and CXCL12 in the cells of the EGFR-siRNA group were decreased significantly（t=2.579，t=2.673，t=2.945，all P=0.000）. Conclusion：In MPNST，EGFR can regulate the expression of CXCR4/CXCL12 signaling pathway and regulate the biological malignant behavior of tumors. Silencing the expression of EGFR can significantly reduce the proliferation，invasion and migration of cancer cells.