Objective To investigate the differential expression profile of epithelial mesenchymal transition (EMT) related long chain noncoding RNA (LncRNA) in hepatocellular carcinoma (HCC) cells after incomplete radiofrequency ablation (RFA) in vitro.Methods Incomplete RFA was simnulated in vitro by using Huh7 cells in water bath at 47℃.EMT change was detected by microscopy and Western blot.Cell invasion and migration were detected by transwell assays.Cell proliferation was determined by cell counting kit-8 (CCK-8) assay.Differential expression profile of EMT-related LncRNAs between Huh7-H and Huh7 were analyzed by LncPath human EMT array,and it was validated by RT-PCR.Results Under microscopy,Huh7-H presented characteristic EMT morphological changes.Western blot analysis showed that the expression of E-cadherin in Huh7-H cells was decreased,while the expressions of N-cadherin and Vimentin were increased.Transwell assay test indicated that cell migration and invasion were increased in Huh7-H cell when compared with Huh7 cell (61.0±5.2 vs 138.0±11.8 and 33.3±7.8 vs 82.7±39.4,respectively),the abilities of Huh7-h cell in migration and invasion were evidently strengthened (P＜0.05).CCK-8 assay shawed that the proliferation ability of Huh 7-H was obviously higher than that Huh 7 (P＜0.05).LncPath human EMT array screened out 3 differential expressed LncRNAs (P≤0.05 and fold changes ≥ 1.5),including two down-regulated LncRNAs (FUNDC2P4,RPL27P7) and one up-regulated LncRNA (MTND4LP14).RT-PCR validation revealed that the expressions of FUNDC2P4,RPL27P7 and MTND4LP14 in Huh7-H cells were 0.137,0.869 and 1.037times of that in Huh7 cells,respectively.Clinical specimen validation showed that the expression of LncRNA FUNDC2P4 in peficancerous tissue was higher than that in HCC tissue,and the expression of LncRNA FUNDC2P4 in HCC tissue was higher than that in the residual HCC tissue after RFA.Conclusion EMTrelated LncRNA FUNDC2P4 in HCC cells with low expression after incomplete RFA is successfully screened out,which provides basis for the further investigation of the function and molecular mechanism of this gene.

OBJECTIVE: To investigate the effect of exosomes derived from Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) cells on lymphangiogenesis and lymph node metastasis of NPC. METHODS: Exosomes from NP69 cells and EBV-positive HK1 (HK1-EBV) cells were obtained by ultracentrifugation and identified by Western blotting and nanoparticle tracking analysis. Dio dye phagocytosis test was performed to observe exosome uptake by lymphatic endothelial cells. Lymphatic endothelial cells were treated with exosomes from nasopharyngeal epithelium (NP69), HK1-EBV, and C666-1 cells or exosome-free supernatant of HK1-EBV and C666-1 cells, and tube formation and migration of the cells were observed. In a nude mouse model of popliteal lymph node metastasis of NPC, the effects of normal saline, NP69 cell-derived exosomes, HK1-EBV cell-derived exosomes, exosome-free supernatant of HK1-EBV cells, and HK1-EBV exosome-free supernatant protein on lymphangiogenesis and lymph node metastasis of the tumor were observed. RESULTS: The exosomes obtained by ultracentrifugation contained abundant exosome-specific proteins and showed a normal size range. The exosomes from NPC cells and NP69 cells could be taken up by lymphatic endothelial cells. Compared with the blank control and exosomes form NP69 cells, exosomes derived from HK1-EBV and C666-1 cells significantly promoted tube formation and migration of lymphatic endothelial cells ( CONCLUSIONS Exosomes from EBV-positive NPC cells can significantly promote lymphangiogenesis and lymph node metastasis of NPC.
Animals ; Cell Line, Tumor ; Endothelial Cells ; Epstein-Barr Virus Infections ; Exosomes ; Herpesvirus 4, Human ; Humans ; Lymphangiogenesis ; Lymphatic Metastasis ; Mice ; Mice, Nude ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms