USP41促进三阴性乳腺癌恶性表型及阿霉素耐药的作用机制

Ubiquitin-specific proteases 41 promotes the malignant phenotype and aoxorubicin-resistance by stabilizing RACK1 in triple negative breast cancer

目的明确泛素特异性蛋白酶41(ubiguitin-specific peptidase 41,USP41)在三阴性乳腺癌(triple-negative breast cancer,TNBC)的表达情况,及其与TNBC恶性表型及阿霉素敏感性的相关性和潜在作用机制。方法采用Western blot、qPCR检测USP41在TNBC耐药细胞株及临床组织样本的表达情况。随后确定USP41分子高表达,并通过CCK8、克隆形成实验、Transwell、Western blot及CoIP-MS等细胞生物学方法评价USP41在TNBC恶性表型及阿霉素耐药中的作用及机制。结果USP41在TNBC样本的表达显著高于癌旁组织。USP41在阿霉素耐药细胞株MDA-MB-231/DXR中表达量高出近40倍,IC50值为6.86μM。干扰USP41可显著增加MDA-MB-231/DXR细胞对阿霉素的敏感性。干扰USP41可显著的抑制细胞的细胞增殖、克隆形成及迁移能力,克隆数量降低30%~80%,迁移细胞数量降低超过70%,差异有统计学意义。此外,USP41敲低可提高MDA-MB-231细胞对阿霉素的敏感性,IC50由5.49μM降低到2.36μM和2.56μM。CO-IP结果显示USP41可与活化的蛋白激酶C1受体(receptor of activated protein kinase C1,RACK1)直接相互作用,且RACK1在癌组织的表达显著高于癌旁。干扰RACK1可抑制MDA-MB-231细胞增殖,IC50由9.87μM降低到4.67μM和4.36μM。克隆形成能力下降超过30%,差异有统计学意义。相较于对照组,USP41敲减使细胞迁移能力下降超过70%。结论USP41高表达与TNBC恶性表型及阿霉素耐药相关,且RACK1可能是USP41发挥作用的关键分子。

Objective To investigate the expression of USP41 in triple-negative breast cancer(TNBC)and its correlation with malignant phenotype and adriamycin sensitivity.Methods The expression of USP41 in TNBC resistant cell lines and clinical tissue samples was detected by Western blot and qPCR.Subsequently,the high expression of USP41 molecule was determined,and the role and possible mechanism of USP41 in the malignant phenotype and adriamycin resistance of TNBC were evaluated by cell biological methods such as CCK8,colony formation assay,transwell,Western blot,and CoIP-MS.Results USP41 expression was significantly higher in triple-negative breast cancer samples than in adjacent non-cancerous tissues.USP41 expression was nearly 40-fold higher in the doxorubicin-resistant cell line MDA-MB-231/DXR,with an IC50 value of 6.86μM.Interference with USP41 significantly increased the sensitivity of MDA-MB-231/DXR cells to doxorubicin.Interference with USP41 significantly inhibited cell proliferation,colony formation and migration of cells,with a decrease in the number of clones of 30%-80%and a decrease in the number of migrating cells of more than 70%,and the difference was statistically significant.In addition,USP41 knockdown improved the sensitivity of MDA-MB-231 cells to doxorubicin,with an IC50 decrease from 5.49μM to 2.36μM and 2.56μM.CO-IP results showed that USP41 could directly interact with RACK1,and the expression of RACK1 was significantly higher in cancer tissues than in adjacent non-cancerous tissues.Interference with RACK1 inhibited MDA-MB-231 cell proliferation,with IC50 decreasing from 9.87μM to 4.67μM and 4.36μM.Colony formation capacity decreased by more than 30%and the difference was statistically significant.USP41 knockdown decreased cell migration by more than 70%compared to control.Conclusion High expression of USP41 is associated with malignant surface and adriamycin resistance in TNBC,and RACK1 may be a key molecule in the role of USP41.