体外不完全射频消融后肝癌细胞上皮-间质转化相关长链非编码RNA差异表达

Differential expression profile of EMT-related LncRNAs in hepatocellular carcinoma cells after incomplete radiofrequency ablation in vitro

目的研究上皮-间质转化(EMT)相关长链非编码RNA(LncRNA)在不完全射频消融(RFA)肝细胞癌(HCC)病灶的差异表达谱变化。方法采用Huh7细胞47℃水浴加热,获取体外模拟的不完全RFA HCC细胞。镜下及免疫印迹检测Huh7-H细胞EMT改变,Transwell检测细胞迁移与侵袭能力,CCK-8检测细胞增殖能力。采用Lnc Path^(TM) Human EMT Array芯片分析Huh7-H和Huh7差异表达的EMT相关LncRNA,RT-PCR验证。结果镜下显示Huh7-H呈现EMT形态学改变。免疫印迹检测显示Huh7-H细胞上皮表型标志分子E-cadherin表达降低,间质表型标志分子N-cadherin、vimentin表达增加。Transwell迁移及侵袭实验显示,Huh7-H、Huh7穿膜细胞数分别为(138.0±11.8)和(61.0±5.2)、(82.7±39.4)和(33.3±7.8),Huh7-H细胞迁移及侵袭能力明显增强(P<0.05)。CCK-8检测显示Huh7-H细胞增殖能力明显增强(P<0.05)。Lnc Path^(TM) Human EMT Array芯片筛选出3个差异表达LncRNA(P≤0.05,变化倍数≥1.5),即2条LncRNA(FUNDC2P4、RPL27P7)下调,1条LncRNA(MTND4LP14)上调。RT-PCR验证HUH7-H组FUNDC2P4、RPL27P7、MTND4LP14基因表达丰度分别是HUH-7组的0.137、0.869、1.037倍。临床标本验证LncRNA FUNDC2P4在癌旁组织表达高于HCC组织,HCC组织高于RFA术后残余HCC组织。结论筛选出不完全RFA后HCC细胞低表达的EMT相关LncRNA FUNDC2P4,为深入探讨该基因功能及分子机制提供了基础。

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 simulated 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, HuhT-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 an＇ay 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 1.037 times 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 EMT- related 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. （J Intervent Radiol, 2017, 26： 823-829）