Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2020 Oct 07;55(10):944-951
Authors: Zhou SK, Gao F, Zhong ZS, Yao H
Abstract
Objective: To evaluate the role of exosomal long non-coding RNA (lncRNA) colon cancer associated transcript-2(CCAT2) in angiogenesis in nasopharyngeal carcinoma(NPC). Methods: Human umbilical vein endothelial cells (HUVECs) were divided into CNE2 supernatant coculture group and NP69 supernatant coculture group. The proliferation ability of HUVEC in each group was examined by CCK8. The lncRNA CCAT2 level in HUVEC was detected using real-time fluorescent quantitative PCR (qRT-PCR). HUVECs were divided into coculture group of serum in patients with NPC and coculture group of serum in healthy donors. The proliferation ability of HUVEC in each group was examined by CCK8. The lncRNA CCAT2 level in each group was detected using qRT-PCR. Exosomes from CNE2,NP69 supernatant were obtained by ultracentrifugation. HUVECs were divided into coculture group of CNE2's supernatant exosomes and coculture group of NP69's supernatant exosomes. Biological experiments like CCK8 were conducted to compare the angiogenesis ability of HUVEC in each group. shRNA transfection was used in HUVEC to suppress the expression of lncRNA CCAT2. Biological experiments like CCK8were conducted to compare the HUVEC's function with different expression of lncRNA CCAT2. CNE2 was transfected with shRNA, the total supernatant exosomal RNA was extracted, and the difference of exosomal lncRNA CCAT2 in CNE2 supernatant with low expression and normal expression of lncRNA CCAT2 was detected by qRT-PCR. The above mentioned two groups of exosomes were cocultured with HUVEC respectively. The difference of lncRNA CCAT2 expression in each group's HUVEC was detected by qRT-PCR and the function of HUVEC was examined with migration assay. GraphPad Prism 8 software was used for graphing and statistical analysis. Results: Compared with those in the NP69 supernatant coculture group, HUVECs in the CNE2 supernatant coculture group showed a higher proliferation ability and higher expression of lncRNA CCAT2(1 vs. 1.40±0.01, t=42.23, P=0.000). Compared with those in serum of healthy donors, HUVECs in serum of patients with NPC showed a higher proliferation ability and the expression of lncRNA CCAT2 increased (1 vs. 1.25±0.03, t=14.43,P=0.001). Compared with those in the coculture group of NP69 cells' supernatant exosomes, HUVEC in the coculture group of CNE2 cells' supernatant exosomes showed a higher proliferation ability and there were more cells that had passed the well during the migration experiment (53.12±2.13 vs. 154.74±4.17, t=37.73, P=0.000) and more knots formed in the tube-formation experiment(10.72±1.02 vs. 53.65±3.21, t=22.63, P=0.000). Compared with those in the sh-NC group, HUVEC in the sh-CCAT2 group showed a lower proliferation ability, and there were less cells that had passed the cell during the migration experiment(401.34±22.15 vs.138.25±6.85, t=23.19, P=0.000) and less MVC in the experiment of subcutaneous stroma embolization in nude mice(41.00±0.32 vs. 27.15±0.23, t=61.53, P=0.000). Compared with the supernatant exosomes in the sh-NC exosome group, the expression of lncRNA CCAT2 was lower in the supernatant exosomes of sh-CCAT2 group (1 vs. 0.65±0.02, t=30.31, P=0.000). Compared with those of the exosomes of sh-NC exosome group, the expression of lncRNA CCAT2 was lower in the HUVEC cocultured with exosomes of the sh-CCAT2 exosome group (1 vs. 0.73±0.01, t=46.77, P=0.000) and the exosomes of sh-CCAT2 exosome group showed a lower migration ability (389.73±26.34 vs. 190.54±8.36, t=12.47, P=0.001) after cocultured with HUVEC. Conclusion: Nasopharyngeal carcinoma-derived exosomal lncRNA CCAT2 could promote angiogenesis.
PMID: 33036509 [PubMed - as supplied by publisher]
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