烯酯酰辅酶A水解酶1过表达对小鼠低淋巴道转移潜能肝癌细胞株Hca-P生物学行为的影响

Effect of Echl overexpression on biological behavior of mouse hepatocarcinoma Hca-P cells in vitro

目的探讨烯酯酰辅酶A水解酶1（Eehl）过表达对小鼠低淋巴道转移潜能肝癌细胞株Hca-P体外增殖生长、迁移和侵袭能力的影响，及其Echl表达水平与肝癌淋巴道转移潜能的关系。方法将pcDNA3．1（＋）-Echl重组质粒和空载体pcDNA3．1（＋）分别稳定转染Hca-P细胞，通过半定量逆转录聚合酶链反应和酶联免疫吸附试验检测目的基因及其蛋白表达水平；在获得过表达Echl的P‰。细胞株和实验对照组P空载体细胞株后，采用CCK．8法检测细胞分裂增殖能力，Transwell迁移实验和Transwell侵袭实验评估Echl过表达对Hca-P细胞迁移和侵袭能力的影响。结果PEch1，组和P空载体组细胞中EchlmRNA相对表达水平分别为3．21±0．43和1．44±0．03，差异有统计学意义（P=0.029）。PEch1组、P空载体组和未经处理的Hca-P组（P组）细胞中Ecbl蛋白表达水平分别为0．140±0．005、0．088±0．003和0．078±0．006，差异有统计学意义（P〈0．05）。PEch1，组的细胞增殖能力明显高于P空载体组和P组（P〈0．05）。Transwell迁移实验显示，PEch1组、P空载体组和P组穿膜细胞数分别为（143．00±7．25）个、（95．73±3．88）个和（106．67±3．54）个，PEch1组与P空载体组和P组比较，差异均有统计学意义（均P〈0．05）。Transwell侵袭实验显示，PEch1组、P空裁体组和P组穿膜细胞数分别为（77．20±5．46）个、（46．34±4．35）个和（49．80±5．21）个，PEch1组与P空载体组和P组比较，差异均有统计学意义（均P〈0．05）。结论Ech1过表达可以显著促进Hca-P细胞增殖，增强其迁移和侵袭能力。Ech1有望成为临床基因治疗肝癌的靶点之一。

Objective To investigate the effect of enoyl coenzyme A hydratase-1 （Echl） on the proliferation and invasion ability of mouse hepatocarcinoma Hca-P cells in vitro. Methods Recombinant PeDNA3.1 （ ＋ ） -Eehl gene and PeDNA3.1 （ ＋ ） were transfected into Hca-P cells by cationic liposomes introduction. Clone of PEohl cells that stably expressing Echl and clone of control Proctor cells were screened by G418. The Echl expression was identified subsequently by reverse transcriptase-polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbent assay （ ELISA）, respectively. The malignant behaviors of the cell lines were compared by proliferation, invasion and migration test. Results The cell line Hea-P cells stably expressing Echl gene was constructed. The relative expression of Echl mRNA in the PEch1 group was 3.21±0.43 and in the Pvector group was 1.44 ± 0.03, with a significant difference between the two groups （P = 0.029）. The results of ELISA revealed that the expression of Echl protein was 0. 140± 0.005 in the PEchl group, 0. 088 ±0. 003 in the Pvector group, and 0. 078 ± 0. 006 in the Hca-P group, showing a significant difference between the PEch1 group and the P and Hca-P groups （ P 〈 0.05 ）. Transwell migration test showed that the number of penetrated cells in the PEchl group was 143. 00 ± 7.25 cells, significantly higher than that of the Pvector group （95.73 ±3.88 cells） and un-treated Hea-1 group （106.67 ±3.54 cells, both P 〈 0.05 ）. The Transwell invasion assay showed that the number of penetrated cells was 77.20 ＋ 5.46 cells in the PEch1 group, significantly higher than 46.34± 4.35 cells in the Pvector group and 49.80 ± 5.21 cells in the un-treated Hca-I group （ both P 〈 0.05 ）. Conclusions The results showed that overexpressed Echl in Hea-P cells may significantly inerease the cell proliferation in a time-dependent manner. The up-regulation of Eehl may increase to some extent the migration and invasion capacity of Hca-P cells. The efforts aiming at up-regulation of Echl expression may become a therapeutic target in the treatment of henatocarcinoma.