人ACSL4基因真核表达载体的构建及其生物学功能研究

Construction of eukaryotic expression vector of human ACSL4 gene and its biological function

目的构建带Flag标签的长链脂酰辅酶A合成酶4(acyl-CoA synthetase long chain family member 4,ACSL4)基因的真核表达载体,并对其表达产物Flag-ACSL4的生物学功能进行初步鉴定。方法通过PCR技术从人乳腺文库中扩增出ACSL4基因,并将其克隆到Flag载体中,经酶切和测序验证成功后,转染到乳腺癌ZR75-1细胞中,再通过Western印迹检测其表达情况,并通过细胞增殖检测、划痕、Transwell和铁死亡敏感性实验,测定细胞的增殖能力及ACSL4对乳腺癌细胞铁死亡的影响。结果从人乳腺文库中扩增得到大小为2136 bp的DNA片段,成功克隆到Flag载体上,经测序与目的序列完全一致,转染乳腺癌ZR75-1细胞后基因表达成功。细胞增殖实验表明,与空载体细胞相比,转染Flag-ACSL4的乳腺癌细胞增殖较快。划痕实验显示,转染Flag-ACSL4的乳腺癌ZR75-1细胞较空载体细胞迁移性增强。Transwell实验显示,转染Flag-ACSL4的乳腺癌ZR75-1细胞较空载体细胞侵袭能力增强。铁死亡敏感性实验表明,ACSL4可使铁死亡诱导剂RSL3(Ras-selective-lethal compound 3)诱导的细胞死亡敏感性显著增强。结论成功构建了带Flag标签的人ACSL4真核表达载体,为进一步研究ACSL4在脂肪酸代谢和在肿瘤发生发展中的功能打下良好的基础。

Objective To construct a eukaryotic expression vector of the acyl-CoA synthetase long chain family member4(ACSL4)gene with the Flag tag and to identify the biological functions of its expression product Flag-ACSL4.Methods The ACSL4 gene was amplified from the human breast library by PCR and cloned into the Flag vector.After the sequencing was made completely consistent with the target sequence,it was transfected into ZR75-1 cells,and the expression product was detected by Western blotting.The proliferation ability of cells and the effect of ACSL4 on iron death(ferroptosis)of breast cancer cells were determined by cell proliferation assay,scratch test,Transwell test and ferroptosis sensitivity test.Results The 2136 bp DNA fragment was amplified from the human breast library and successfully cloned into the Flag vector.The sequencing was completely consistent with the sequence,and the gene was successfully expressed after being transfected into breast cancer ZR75-1 cells.Cell proliferation assay showed that breast cancer cells transfected with FlagACSL4 proliferated faster than those transfected with an empty vector.The scratch test showed that breast cancer ZR75-1 cells transfected with Flag-ACSL4 had higher mobility than no-load cells.Transwell assay showed that breast cancer ZR75-1 cells transfected with flag-ACSL4 had better invasion ability than no-load cells.Ferroptosis sensitivity experiments showed that ACSL4 could significantly enhance the sensitivity of ferroptosis inducer RSL3(ras-selective-lethal compound3)to cell death.Conclusion The eukaryotic expression vector of ACSL4 with Flag label is successfully constructed,which can facilitate subsequent study on the function of ACSL4 in fatty acid metabolism and tumor development.