宫颈癌源外泌体miR-191-5p通过靶向TJP1调控血管内皮细胞层通透性促癌细胞转移的分子机制研究

Study on molecular mechanism of cervical carcinoma derived exosome miR-191-5p regulating vascular endothelial cell layer permeability and promoting cancer cell metastasis by targeting TJP1

目的探讨宫颈癌细胞分泌的外泌体miR-191-5p对血管内皮细胞层的通透性影响,为肿瘤血管相关的基因靶点研究提供理论依据。方法提取并鉴定宫颈癌细胞(SiHa/HeLa)培养上清来源的外泌体。将宫颈癌细胞源外泌体与人脐静脉内皮细胞(HUVEC)共培养,检测外泌体的摄取。跨内皮电阻(TEER)测定HUVEC单层细胞形成时间。内皮细胞层通透性测定HUVEC细胞层通透性。qRT-PCR检测宫颈癌细胞、外泌体及与外泌体共培养48 h的HUVEC中miR-191-5p表达。双荧光素酶报告基因实验检测miR-191-5p与TJP1的靶向关系。qRT-PCR、Western blot检测miR-191-5p、TJP1表达。内皮细胞层穿透实验检测miR-191-5p对HUVEC细胞层通透性的影响以及能否促进宫颈癌转移。恢复实验、qRT-PCR、Western blot检测TJP1的表达。结果提取的微小囊泡确是宫颈癌源外泌体。宫颈癌源外泌体可被HUVEC细胞摄取。TEER随时间增加而增大,在培养3 d时细胞形成单层,TEER达到最大值;外泌体共培养的HUVEC细胞层通透性增大,差异有统计学意义(P<0.0001)。miR-191-5p在宫颈癌源外泌体中富集,并被转运至HUVEC细胞中,miR-191-5p与TJP1存在靶向关系。与PBS组比较,miR-191-5p mimics组的miR-191-5p相对表达水平升高,TJP1的相对表达水平下降,而miR-191-5p inhibitor组的miR-191-5p相对表达水平下降,TJP1的相对表达水平升高,差异有统计学意义(P<0.01);与PBS组比较,miR-191-5p mimics组通透性增加,而miR-191-5p inhibitor组通透性减弱,差异有统计学意义(P<0.0001)。在SiHa细胞中,与PBS组比较,miR-191-5p mimics组穿透的平均细胞数增加,miR-191-5p inhibitor组穿透的平均细胞数下降,差异有统计学意义(P<0.05)。在HeLa细胞中,与PBS组比较,miR-191-5p mimics组穿透的平均细胞数增加,miR-191-5p inhibitor组穿透的平均细胞数下降,差异有统计学意义(P<0.05)。恢复实验结果显示,与PBS组比较,miR-191-5p mimics组TJP1的相对表达量下降,差异有统计学意义(P<0.05);与miR-191-5p mimics组比较,pCMV-T+mimics组中TJP1的表达量增加,差异有统计学意义(P<0.01)。结论宫颈癌源外泌体miR-191-5p通过靶向调控TJP1导致血管内皮细胞层通透性增加,进而促进宫颈癌细胞转移。

Objective To study the effect of exosome miR-191-5p secreted by cervical cancer cells on the permeability of the vascular endothelial cell layer,and to provide a theoretical basis for the study of tumor vasculature-related gene targets.Methods Exosomes derived from culture supernatants of cervical cancer cells(SiHa/HeLa)were extracted and characterized.Exosomes derived from cervical cancer cells were co-cultured with human umbilical vein endothelial cells(HUVEC)to detect exosome uptake.The formation time of HUVEC monolayer cells was measured by transendothelial resistance(TEER)assay.Endothelial cell layer permeability assay HUVEC cell layer permeability assay.The expression of miR-191-5p in cervical cancer cells,exosomes and HUVEC co-cultured with exosomes for 48 h was detected by qRT-PCR.Dual luciferase reporter gene assay was performed to detect the targeting relationship between miR-191-5p and TJP1.The expressions of miR-191-5p and TJP1 were detected by qRT-PCR and Western blot.The effect of miR-191-5p on the permeability of HUVEC cell layer was tested by endothelial cell layer penetration test to determine whether it could promote cervical cancer metastasis.The expression of TJP1 was detected by recovery test,qRT-PCR and Western blot.Results The extracted tiny vesicles were indeed cervical cancer-derived exosomes.Cervical cancer-derived exosomes could be taken up by HUVEC cells.Teer wasincreased with time and reached its maximum at 3 day of culture when cells formed a monolayer;the permeability of the HUVEC cell layer co-cultured with exosomes increased,and the difference was statistically significant(P<0.0001).miR-191-5p was enriched in cervical cancer-derived exosomes and was transported to HUVEC cells,and miR-191-5p was targeted to the presence of TJP1.Compared with the PBS group,the relative expression level of miR-191-5p was increased in the miR-191-5p mimics group and decreased in the relative expression level of TJP1,where as the relative expression level of miR-191-5p was decreased in the miR-191-5p inhibitor group and increased in the relative expression level of TJP1,and the difference was statistically significant(P<0.01).Compared with the PBS group,the permeability was increased in the miR-191-5p mimics group and weakened in the miR-191-5p inhibitor group,and the difference was statistically significant(P<0.0001).In SiHa cells,the mean number of cells penetrated were increased in the miR-191-5p mimics group and were decreased in the miR-191-5p inhibitor group compared with PBS group,and the difference was statistically significant(P<0.05).In HeLa cells,the mean number of cells penetrated in the miR-191-5p mimics group was increased and the mean number of cells penetrated in the miR-191-5p inhibitor group decreased compared with PBS group,and the difference was statistically significant(P<0.05).Recovery experiments showed that expression of TJP 1 was decreased in the miR-191-5p mimics group compared with PBS group,with a statistically significant difference(P<0.05);the expression of TJP1 was increased in the pCMV-T+mimics group compared with the miR-191-5p mimics group,with a statistically significant difference(P<0.01).Conclusion The cervical cancer-derived exosome miR-191-5p leads to increased permeability of tvascular endothelial cell layer by targeting and regulating TJP1,which in turn promotes cervical cancer cell metastasis.