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
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 cervi-cal cancer cells,exosomes and HUVEC co-cultured with exosomes for 48 h was detected by qRT-PCR.Du-al 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 penetra-tion test to determine whether it could promote cervical cancer metastasis.The expression of TJP1 was de-tected by recovery test,qRT-PCR and Western blot.Results The extracted tiny vesicles were indeed cer-vical 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 statisti-cally significant(P＜0.000 1).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 differ-ence was statistically significant(P＜0.000 1).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 num-ber of cells penetrated in the miR-191-5p mimics group was increased and the mean number of cells pene-trated in the miR-191-5p inhibitor group decreased compared with PBS group,and the difference was sta-tistically 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.
万方数据
维普
