RITA体外选择性抑制BAP1缺失的皮肤黑色素瘤细胞的生长

RITAselectively inhibits proliferation of BAP1-deficient cutaneous melanoma cells in vitro

目的构建抑癌基因BAP1敲除的皮肤黑色素瘤细胞模型,筛选对BAP1缺失的皮肤黑色素瘤细胞具有选择性抑制活性的小分子化合物。方法选择表达野生型BAP1的皮肤黑色素瘤细胞利用CRISPR-Cas9系统敲除BAP1基因,获得与亲代细胞同基因型的BAP1敲除克隆,通过MTT法筛选化合物库中对BAP1敲除细胞具有选择性抑制活性的小分子化合物。通过挽救实验,明确BAP1敲除细胞对化合物的敏感性是否与BAP1蛋白缺失直接相关。流式细胞术检测化合物对细胞周期和凋亡的影响。Western blotting检测化合物对蛋白表达的影响。结果化合物库中的p53激活剂RITA选择性抑制BAP1敲除细胞的细胞活力。过表达野生型BAP1能够逆转BAP1敲除细胞对RITA的敏感性,而过表达去泛素化酶失活的突变型BAP1(C91S)没有挽救作用。与BAP1野生型对照细胞相比,BAP1敲除细胞对RITA引起的细胞周期阻滞和凋亡更为敏感(P<0.0001)。BAP1敲除增加了细胞p53蛋白的基础水平,而RITA处理后进一步增加p53蛋白表达(P<0.0001)。结论BAP1缺失导致皮肤黑色素瘤细胞对p53激活剂RITA敏感。BAP1的去泛素化酶活性与细胞对RITA的敏感性直接相关。BAP1敲除导致p53蛋白增加可能是细胞对RITA敏感的一个关键原因。

Objective To screen for small molecular compounds with selective inhibitory activity against cutaneous melanoma cells with BAP1 deletion.Methods Cutaneous melanoma cells expressing wild-type BAP1 were selected to construct a BAP1 knockout cell model using CRISPR-Cas9 system,and small molecules with selective inhibitory activity against BAP1 knockout cells were screened from a compound library using MTT assay.Rescue experiment was carried out to determine whether the sensitivity of BAP1 knockout cells to the candidate compounds was directly related to BAP1 deletion.The effects of the candidate compounds on cell cycle and apoptosis were detected with flow cytometry,and the protein expressions in the cells were analyzed with Western blotting.Results The p53 activator RITA from the compound library was shown to selectively inhibit the viability of BAP1 knockout cells.Overexpression of wild-type BAP1 reversed the sensitivity of BAP1 knockout cells to RITA,while overexpression of the mutant BAP1(C91S)with inactivated ubiquitinase did not produce any rescue effect.Compared with the control cells expressing wild-type BAP1,BAP1 knockout cells were more sensitive to RITA-induced cell cycle arrest and apoptosis(P<0.0001)and showed an increased expression of p53 protein,which was further increased by RITA treatment(P<0.0001).Conclusion Loss of BAP1 results in the sensitivity of cutaneous melanoma cells to p53 activator RITA.In melanoma cells,the activity of ubiquitinase in BAP1 is directly related to their sensitivity to RITA.An increased expression of p53 protein induced by BAP1 knockout is probably a key reason for RITA sensitivity of melanoma cells,suggesting the potential of RITAas a targeted therapeutic agent for cutaneous melanoma carrying BAP1-inactivating mutations.