ABSTRACT
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.
ABSTRACT
OBJECTIVE@#To investigate potential human leucocyte antigen (HLA)-A2-restricted epitope peptides of glypican-3 (GPC3) and determine the cytotoxicity of peptide-specific cytotoxic T lymphocytes (CTLs) against hepatocellular carcinoma (HCC) cells.@*METHODS@#The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3 SMMC 7721 and HepG2 cells was detected using IFN-γ based enzyme-linked immunospot and lactate dehydrogenase release assays in vitro.@*RESULTS@#A total of six peptides were identified for bindings to HAL-A2 and the GPC3 522-530 and GPC3 229-237 peptides with HLA-A*0201 molecules displayed high binding affinity and stability. The CTLs induced by the GPC3 522-530 or positive control GPC3 144-152 peptide responded to the peptide by producing IFN-γ, which were abrogated by treatment with anti-HLA-A2 antibody. The GPC3 522-530-specific CTLs responded to and killed SMMC 7721 and HepG2 cells, instead of GPC3-silenced SMMC 7721 or HepG2 cells. GPC3 522-530-specific CTLs response to HCC cells was blocked by anti-HLA-A2 antibody.@*CONCLUSIONS@#The GPC3 522-530 peptide contains antigen-determinant and its specific CTLs can effectively kill HCC in a HLA-A2-restricted and peptide-dependent manner. Our findings suggest that this peptide may be valuable for development of therapeutic vaccine.
ABSTRACT
Objective To investigate potential human leucocyte antigen (HLA)-A2-restricted epitope peptides of glypican-3 (GPC3) and determine the cytotoxicity of peptide-specific cytotoxic T lymphocytes (CTLs) against hepatocellular carcinoma (HCC) cells. Methods The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3