宫颈癌HeLa细胞外泌体对细胞迁移和侵袭的影响及其Wnt/β-catenin信号通路机制

doi:10.13481/j.1671-587x.20200421

摘要/Abstract

摘要： 目的：探讨宫颈癌HeLa细胞外泌体对细胞迁移、侵袭及Wnt/β-连环蛋白（β-catenin）信号通路的影响，阐明其外泌体在宫颈癌发生发展过程中的可能作用机制。方法：取对数生长期宫颈癌HeLa细胞，采用超速离心法分离宫颈癌HeLa细胞外泌体，透射电镜下观察外泌体形态表现，Western blotting法检测外泌体标志蛋白CD63和CD81的表达，鉴定分离出来的外泌体。将HeLa细胞分为外泌体组（Exo组）和对照组，2组细胞分别用含和不含HeLa细胞外泌体的培养基培养。共聚焦显微镜下观察HeLa细胞摄取外泌体情况，其中外泌体采用PKH26荧光标记，HeLa细胞用DAPI荧光进行核染色，Transwell小室实验检测细胞侵袭能力，细胞划痕实验测定细胞迁移能力，Western blotting法检测细胞中Wnt1、β-catenin和T细胞因子4（TCF4）蛋白表达水平。结果：透射电镜下观察，HeLa细胞中分离的外泌体为是圆形或椭圆形、直径为40~100 nm的囊泡状小体，且富含CD63和CD81蛋白。共聚焦显微镜下观察，Exo组HeLa细胞膜表面有外泌体附着，而对照组HeLa细胞膜表面无外泌体附着。Transwell小室实验，Exo组HeLa细胞中侵袭细胞数明显高于对照组（t=17.894，P<0.01）。细胞划痕实验，Exo组HeLa细胞迁移距离明显长于对照组（t=9.689，P<0.01）。Western blotting法检测，与对照组比较，Exo组HeLa细胞中Wnt/β-catenin信号通路相关蛋白Wnt1、β-catenin和TCF4蛋白表达水平均明显升高（t=13.159，P<0.01；t=15.478，P<0.01；t=7.734，P<0.01）。结论：宫颈癌HeLa细胞来源的外泌体可促进宫颈癌细胞的侵袭和迁移能力，其机制可能与HeLa细胞来源外泌体可促进Wnt/β-catenin信号通路的激活及下游TCF4基因的表达有关。

关键词: 宫颈肿瘤, 外泌体, 细胞迁移, 细胞侵袭, Wnt/β-连环蛋白信号通路, T细胞因子4

Abstract: Objective: To investigate the effects of cervical cancer HeLa cell exosomes on the migration, invasion and Wnt/β-catenin signaling pathway of the cells, and to elucidate the possible mechanism of the exosomes in the occurrence and development of cervical cancer. Methods: The cervical cancer HeLa cells in the logarithmic growth phase were selected. The cervical cancer HeLa cell exosomes were isolated by ultracentrifugation. The morphology of isolated exosomes was observed under transmission electron microscope(TEM). Western blotting method was used to detect the expressions of exosomal marker proteins CD63 and CD81, and the isolated exosomes were identified. The HeLa cells were divided into exosomes group(Exo group) and control group. The cells in two groups were cultured separately with medium containg HeLa cell exosomes or not. The uptake of exosomes by HeLa cells was observed with confocal microscope, the exosomes were labeled with PKH26 fluorescence, and the nuclei of HeLa cells were stained with DAPI fluorescence. Transwell chamber was used to measure the cell invasion ability.Cell scratch assay was used to measure the cell migration ability.Western blotting method was used to measure the expression levels of Wnt1, β-catenin and T cell factor 4 (TCF4) proteins in the cells. Results: The TEM results showed cylindrical or oval vesicle bodies with a diameter of 40-100 nm and rich in CD63 protein and CD81 protein. The confocal microscope results showed that there was exosomal attachment on the membrane surface of HeLa cells in Exo group,but there was no exosomal attachment on the membrane surface of the HeLa cells in control group; The Transwell assay results showed that the number of invasion HeLa cells in Exo group was significantly higher than that in control group(t=17.894,P<0.01);the scratch assay results showed that the migration distance of the cells in Exo group was significantly longer than that in control group(t=9.689,P<0.01).The Western blotting results showed the expression levels of Wnt/β-catenin signaling pathway-related proteins Wnt1, β-catenin and TCF4 in the HeLa cells in Exo group were significantly higher than those in control group(t=13.159,P<0.01;t=15.478,P<0.01;t=7.734,P<0.01). Conclusion: The HeLa cell-derived exosomes can promote the invasion and migration abilities of cervical cancer cells. The mechanism may be related to that the HeLa cell-derived exosomes can promote the activation of Wnt/β-catenin signaling pathway and the downstream TCF4 gene expression.

Key words: cervical cancer, exosomes, migration, invasion, Wnt/β-catenin signaling pathway, T cell factor 4

中图分类号:

R737.33

刘晶, 王景, 葛静, 冯艳萍, 房桂英, 王旭, 杨艳红, 李林. 宫颈癌HeLa细胞外泌体对细胞迁移和侵袭的影响及其Wnt/β-catenin信号通路机制[J]. 吉林大学学报(医学版), 2020, 46(04): 798-803.

LIU Jing, WANG Jing, GE Jing, FENG Yanping, FANG Guiying, WANG Xu, YANG Yanhong, LI Lin. Effect of HeLa cell exosomes on migration and invasion and its mechanism of Wnt/ β-catenin signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 798-803.

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