Suresh Rahul, Picard Daniel, Lo Rita, Beaulieu Jamie, Remke Marc, Diaz Roberto Jose
Montreal Neurological Institute, McGill University, Montreal, Québec, Canada.
Division of Pediatric Neuro-Oncogenomics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Neurooncol Adv. 2021 Apr 23;3(1):vdab064. doi: 10.1093/noajnl/vdab064. eCollection 2021 Jan-Dec.
Alterations in actin subunit expression have been reported in multiple cancers, but have not been investigated previously in medulloblastoma.
Bioinformatic analysis of multiple medulloblastoma tumor databases was performed to profile mRNA levels. Western blot was used to verify protein expression in established medulloblastoma cell lines. Immunofluorescence microscopy was performed to assess ACTC1 localization. Stable cell lines with ACTC1 overexpression were generated and shRNA knockdown of ACTC1 was accomplished. We used PARP1 cleavage by Western blot as a marker of apoptosis and cell survival was determined by FACS viability assay and colony formation. Cell migration with overexpression or knockdown of ACTC1 was determined by the scratch assay. Stress fiber length distribution was assessed by fluorescence microscopy.
mRNA expression is highest in SHH and WNT medulloblastoma among all subgroups. ACTC1 protein was confirmed by Western blot in SHH subgroup and Group 3 subgroup cell lines with the lowest expression in Group 3 cells. Microscopy demonstrated ACTC1 co-localization with F-actin. Overexpression of ACTC1 in Group 3 cells abolished the apoptotic response to Aurora kinase B inhibition. Knockdown of ACTC1 in SHH cells and in Myc overexpressing SHH cells induced apoptosis, impaired colony formation, and inhibited migration. Changes in stress fiber length distribution in medulloblastoma cells are induced by alterations in ACTC1 abundance.
Alpha-cardiac actin (ACTC1) is expressed in SHH medulloblastoma. Expression of this protein in medulloblastoma modifies stress fiber composition and functions in promoting resistance to apoptosis induced by mitotic inhibition, enhancing cell survival, and controlling migration.
已有报道称肌动蛋白亚基表达改变在多种癌症中存在，但此前尚未在髓母细胞瘤中进行研究。
对多个髓母细胞瘤肿瘤数据库进行生物信息学分析以分析mRNA水平。使用蛋白质印迹法验证已建立的髓母细胞瘤细胞系中的蛋白质表达。进行免疫荧光显微镜检查以评估ACTC1的定位。构建了ACTC1过表达的稳定细胞系，并完成了ACTC1的短发夹RNA敲低。我们使用蛋白质印迹法检测PARP1裂解作为细胞凋亡的标志物，并通过流式细胞术活力测定和集落形成来确定细胞存活率。通过划痕试验测定ACTC1过表达或敲低时的细胞迁移情况。通过荧光显微镜评估应力纤维长度分布。
在所有亚组中，SHH和WNT髓母细胞瘤中的mRNA表达最高。蛋白质印迹法在SHH亚组和3组亚组细胞系中证实了ACTC1蛋白的存在，其中3组细胞中的表达最低。显微镜检查显示ACTC1与F-肌动蛋白共定位。3组细胞中ACTC1的过表达消除了对极光激酶B抑制的凋亡反应。在SHH细胞和Myc过表达的SHH细胞中敲低ACTC1可诱导细胞凋亡、损害集落形成并抑制迁移。髓母细胞瘤细胞中应力纤维长度分布的变化是由ACTC1丰度的改变引起的。
α-心肌肌动蛋白（ACTC1）在SHH髓母细胞瘤中表达。该蛋白在髓母细胞瘤中的表达改变了应力纤维的组成，并在促进对有丝分裂抑制诱导的凋亡的抗性、增强细胞存活和控制迁移方面发挥作用。
