摘要
目的筛选参与内皮细胞调控骨肉瘤转移的关键分子——富亮氨酸胶质瘤失活蛋白4(leucine-rich glioma inactivated proteins 4,LGI4),并分析LGI4参与骨肉瘤转移的功能及分子机制。方法建立人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)-骨肉瘤细胞共培养模型,利用全转录组测序方法并结合相关文献筛选表达差异倍数显著的基因,采用实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)和Western blot验证候选基因LGI4的表达变化。转染LGI4过表达质粒建立LGI4过表达骨肉瘤稳定细胞系143B和MTF;实验分为阴性对照组(正常培养的骨肉瘤细胞)、共培养组(HUVECs与骨肉瘤细胞共培养)、共培养基组(HUVECs培养基与骨肉瘤细胞共培养)、载体对照组(转染过表达对照载体)和LGI4组(转染过表达LGI4载体),采用Transwell和划痕实验检测骨肉瘤细胞的迁移、侵袭能力。利用裸鼠体内转移模型验证LGI4对骨肉瘤细胞远处转移的影响;采用生物信息学分析LGI4的下游潜在靶信号通路,并利用相关抑制剂进行回复实验,明确靶信号通路在LGI4调控骨肉瘤转移中的作用。结果人骨肉瘤细胞系143B及MTF与HUVECs共培养后LGI4表达降低(P<0.05);与阴性对照组相比,共培养组和共培养基组的迁移侵袭能力明显增加,而LGI4组的迁移侵袭能力较载体对照组比较明显减弱(P<0.05)。裸鼠体内实验证明LGI4组的转移能力受到抑制;进一步分析发现NOTCH4信号通路是LGI4的下游靶信号通路,功能回复实验证实LGI4通过抑制NOTCH4信号通路参与骨肉瘤转移(P<0.05)。结论内皮细胞通过抑制骨肉瘤细胞LGI4表达激活NOTCH4信号通路促进肿瘤转移。
Objective To screen a key molecule,leucine-rich glioma inactivated protein 4(LGI4),which participates in endothelial cell-mediated osteosarcoma metastasis,and to further analyze its function and molecular mechanism in osteosarcoma metastasis.Methods A co-culture model of human umbilical vein endothelial cells(HUVECs)-osteosarcoma cells was established,and the genes with significant fold change were screened by whole transcriptome sequencing in combination with relevant literature.After,the candidate target gene,LGI4 was verified with real-time quantitative PCR(RT-qPCR)and Western blotting,the expression of LGI4 at mRNA and protein levels was also determined.Osteosarcoma cell line 143 B and MTF LGI4 were transfected with the overexpression plasmid of LGI4 to construct the cells with stable overexpression of LGI4.Then the experiments included 5 groups of osteosarcoma cells,including negative control group(normally cultured),co-culture group(co-cultured with HUVECs),co-culture medium group(treated with the HUVECs medium),vector control group(transfected with control plasmid)and LGI4 group(overexpression of LGI4).Transwell assay and wound healing test were used to detect the migration and invasion abilities of osteosarcoma cells.A tumor metastasis model in nude mice was used to verify the effect of LGI4 overexpressed osteosarcoma cells on distant metastasis in vivo.Bioinformatics analysis was applied to analyze the potential downstream target signaling pathways of LGI4,and recovery experiments were performed with related inhibitors to clarify the role of target signaling pathways in the regulation of osteosarcoma metastasis by LGI4.Results The expression levels of LGI4 were markedly decreased in 143 B and MTF cells after co-culture with HUVECs(P<0.05).Compared with the negative control group,the cell migration and invasion abilities of the co-culture group and co-medium group were significantly enhanced,whereas the cell migration and invasion rates of the LGI4 overexpression group were significantly lower than that of the vector control group(P<0.05).The nude mice metastasis model revealed that the metastasis ability of osteosarcoma cells was suppressed with LGI4 overexpression.Further analysis revealed that NOTCH4 signaling pathway was the downstream target signaling pathway of LGI4,and functional recovery experiments confirmed that LGI4 resulted in osteosarcoma metastasis by inhibiting NOTCH4 signaling pathway(P<0.05).Conclusion Endothelial cells activate NOTCH4 signaling pathway and thus promote tumor metastasis by inhibiting the expression of LGI4 in osteosarcoma cells.
作者
马红敏
郝翔麟
吕杨帆
曹亚
徐嘉忆
董文浩
王亚丽
付万垒
郭乔楠
MA Hongmin;HAO Xianglin;LYU Yangfan;CAO Ya;XU Jiayi;DONG Wenhao;WANG Yali;FU Wanlei;GUO Qiaonan(Department of Pathology,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400037,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2022年第20期2088-2097,共10页
Journal of Army Medical University
基金
国家自然科学基金面上项目(81972519)
关键词
骨肉瘤
LGI4
血管内皮
NOTCH4
osteosarcoma
leucine-rich glioma inactivated protein 4
vascular endothelium
NOTCH4
