EphA1/EphrinA反馈环调控内皮祖细胞促肝细胞癌血管生成潜能

The effect of regulated EphA1/EphrinA1 signaling axis on endothelial progenitor cells to promote their angiogenesis potency in hepatocellular carcinoma

目的:研究体内外调控内皮祖细胞系EPCsEphA1/SiRNA-Tet中EphA1/EphrinA1信号通路对其细胞生物学行为及活体内促肝细胞癌血管生成潜能的影响。方法:体内外用不同浓度强力霉素调控EPCsEphA1/SiRNA-Tet中EphA1基因的表达,分为调控组Dox(+)(培养液中分别含强力霉素1、10和100μg/mL)和非调控组Dox(-);用Western blot法检测对EphA1/EphrinA1信号通路的影响;CCK8法、细胞划痕实验及细胞侵袭实验检测对细胞增殖、运动及侵袭能力的影响;绘制肝癌裸鼠移植瘤生长曲线结合瘤体免疫组织化学染色分析调控EPCsEphA1/SiRNA-Tet中EphA1基因的表达对其促血管生成潜能的影响。结果:体外实验中用10μg/mL强力霉素能最大程度抑制EphA1/EphrinA1信号通路活性,EphA1及其配体EphrinA1蛋白表达分别为0.293±0.029、0.603±0.038,与Dox(-)组的0.943±0.041、0.960±0.062比,差异均有统计学意义(t=12.940,4.864;P<0.001,0.008)。细胞生物学行为分析显示调控EphA1基因表达对EPCsEphA1/SiRNA-Tet增殖能力并无影响;10μg/mL强力霉素调控时能最有效地抑制EPCsEphA1/SiRNA-Tet运动及侵袭能力,与Dox(-)组比差异均有统计学意义(t=4.435,2.467;P=0.002,0.039)。体外100μg/mL强力霉素可最大程度诱导活体内EPCsEphA1/SiRNA-Tet中EphA1基因表达下调,在第6周移植瘤体积为(543.8±24.6)mm3,比Dox(-)组体积(924.5±81.8)mm3明显减少(t=4.909,P=0.001)。免疫组织化学染色显示100μg/mL强力霉素调控组微血管为(21.6±3.6)个,明显少于Dox(-)组的(37.0±4.1)个(t=2.823,P=0.024)。结论:不同浓度强力霉素可在体内外精确调控内皮祖细胞中EphA1/EphrinA1信号轴的活性,进而达到调控内皮祖细胞促肝癌血管生成潜能的目的。

Objective:To study the effect of regulated EphA1/EphrinA1 signaling pathways in endothelial progenitor cells(EPCs)on their cell biological behavior and promotion of angiogenesis potency in hepatocellular carcinoma.Methods:Different concentrations of doxycycline were used to regulate the EphA1 gene express in EPCs line EPCsEphA1/SiRNA-Tet in vitro and in vivo.Western blot was used to test the EphA1/EphrinA1 signaling pathway variation;CCK8 method,wound healing and cell invasion assay were used to test the changes in cell proliferation,migration and invasion ability;xenograft tumor growth curve and immunohistochemical staining of tumor microvascular were used to test their promote angiogenesis potency in hepatocellular carcinoma.T test was used to compare parameters between the groups.Results:In vitro assay it was showed that 10μg/mLdoxycycline could inhibit EphA1/EphrinA1 signaling pathway activity to the maximum extent.Compared withβ-actin,the gray value of EphA1 and its ligand EphrinA1 protein expressions were 0.293±0.029 and 0.603±0.038 respectively,being statistically different(t=12.940,4.864;P<0.001,0.008)from Dox(-)group 0.943±0.041 and 0.960±0.062.The results of cell biological behavior showed that regulated EphA1 gene expression in EPCs had no effected on their proliferation ability.Compared with Dox(-)group,10μg/mL doxycycline regulation could inhibit EPCs’motility and invasion ability(t=4.435,2.467;P=0.002,0.039)to the maximum extent.100μg/mL doxycycline induction in vitro could decrease to capacity EphA1 gene expression in EPCs in vivo.Compared with tumor volume(924.5±81.8)mm3 in Dox(-)group,the regulated group tumor volume(543.8±24.6)mm3 of nude mice was decreased at the end of sixth week(t=4.909,P=0.001)and compared with Dox(-)group(37.0±4.1),the microvascular density in regulated group 21.6±3.6 was significantly decreased(t=2.823,P=0.024).Conclusion:Different concentrations of doxycycline can precisely regulate the activity of EphA1/EphrinA1 signaling axis in EPCs in vitro and in vivo,thus controlling the promoted angiogenesis potency in hepatocellular carcinoma.