We aimed to understand the molecular mechanism underlying the incidence of Oxaliplatin resistance in colorectal cancer. The Oxaliplatin-resistant (OR) HT29 colorectal cell line was established by long-term exposure to Oxaliplatin. Cell viability and proliferation were determined by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide and direct counting assays, respectively. Transcript level of metallothionein 2A (MT2A) was measured by real-time polymerase chain reaction. Protein levels of MT2A, BRCA1-associated RING domain 1 (BARD1), BRCA1, and β-actin were quantified by immunoblotting. Direct interaction between MT2A with BARD1 and BRCA1 was analyzed by co-immunoprecipitation. Colocalization between of MT2A and BARD1 was determined by immunofluorescence. MT2A was upregulated in OR cells at both transcript and protein levels. Knockdown of MT2A in HT29 OR cells improved sensitivity to Oxaliplatin, while ectopic overexpression of MT2A conferred HT29 cells relative resistance to Oxaliplatin. We further demonstrated that MT2A interacted with and positively regulated BARD1/BRCA1 in colorectal cancer cells. BARD1 overexpression partially restored the compromised Oxaliplatin resistance elicited by MT2A deficiency in terms of both cell proliferation and viability. Our data highlighted the critical contributions of MT2A-BARD1/BRCA1 in Oxaliplatin resistance in colorectal cancer cells.

我们旨在了解大肠癌中奥沙利铂耐药性发生的分子机制。通过长期暴露于奥沙利铂可建立抗奥沙利铂（OR）HT29结肠直肠细胞系。细胞活力和增殖分别通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物和直接计数测定法测定。通过实时聚合酶链反应测量金属硫蛋白2A（MT2A）的转录水平。通过免疫印迹定量测定MT2A，与BRCA1相关的RING域1（BARD1），BRCA1和β-肌动蛋白的蛋白水平。通过共免疫沉淀法分析了MT2A与BARD1和BRCA1之间的直接相互作用。通过免疫荧光测定MT2A和BARD1之间的共定位。在转录和蛋白质水平，OR2细胞中的MT2A均上调。敲低HT29 OR细胞中的MT2A可以提高对奥沙利铂的敏感性，而异位MT2A的过表达赋予HT29细胞对奥沙利铂的相对抗性。我们进一步证明，MT2A与大肠癌细胞中的BARD1 / BRCA1相互作用并受到正向调控。BARD1过表达部分恢复了MT2A缺乏引起的受损的奥沙利铂抗性，无论是在细胞增殖还是活力方面。我们的数据强调了MT2A-BARD1 / BRCA1在结直肠癌细胞中对奥沙利铂耐药的关键作用。BARD1过表达部分恢复了MT2A缺乏引起的受损的奥沙利铂抗性，无论是在细胞增殖还是活力方面。我们的数据强调了MT2A-BARD1 / BRCA1在结直肠癌细胞中对奥沙利铂耐药的关键作用。BARD1过表达部分恢复了MT2A缺乏引起的受损的奥沙利铂抗性，无论是在细胞增殖还是活力方面。我们的数据强调了MT2A-BARD1 / BRCA1在结直肠癌细胞中对奥沙利铂耐药的关键作用。