Yue Zhi-Xia, Gao Rui-Qi, Gao Chao, Liu Shu-Guang, Zhao Xiao-Xi, Xing Tian-Yu, Niu Jing, Li Zhi-Gang, Zheng Hu-Yong, Ding Wei
1Department of Medical Genetics and Developmental Biology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069 China.
Beijing Key Laboratory of Pediatric Hematology Oncology, National Key Discipline of Pediatrics, Ministry of Education, MOE Key Laboratory of Major Diseases in Children, Hematology Oncology Center, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, 100045 China.
Cancer Cell Int. 2018 Jul 27;18:106. doi: 10.1186/s12935-018-0600-5. eCollection 2018.
Cajal body (CB) is a nucleic organelle where small nuclear ribonucleoproteins undergo modification, maturation, splicing and/or assembly. Coilin is the marker structural protein of CBs. The expression level and cellular localization of coilin is sensitive to chemotherapeutic reagents, such as cisplatin. The gene of cyclin-dependent kinase inhibitor 1B (p27) is located with a high incidence translocation region of leukemic chromosomes, and its expression was of prognosis values in a variety of adult leukemia types. The exact profile and associated functions of coilin, as well as p27, in children's acute lymphoblastic leukemia (ALL) is obscure.
Bone marrow samples from 144 patients with ALL were collected. The expression levels of coilin and p27 were detected by qRT-PCR. The patient cohort was divided into low and high groups of coilin and p27 respectively. The prognosis and clinicobiological characteristics of different groups were investigated, especially focused on the treatment outcome. Leukemia cells of Reh or RS4;11 were exposed to different concentrations of DNR, prior to the detection for morphological changes of coilin by immunofluorescence. In Reh cells, lentivirus sh-coilin was used to silence coilin expression. Western blotting was used to detect coilin and p27 expression; flow cytometry was used for cell cycle and apoptosis assay; MTS method was used for measuring cell viability to examine the drug sensitivity of DNR.
In this study, we found that daunorubicin was able to induce significant morphological changes of CBs in Reh and RS4;11 cells. Knockdown the expression of coilin increased the sensitivity to daunorubicin and inhibited the expression of p27 in Reh cells, and led to increased apoptosis. Importantly, not only the levels of coilin and p27 mRNA expression at initial diagnosis ALL children are markedly higher than those at complete remission (CR), but also both coilin and p27 expression in the relapsed patients was observed significantly higher comparing to the continuous CR patients. The 4-year EFS and RFS indicated that low levels of both coilin and p27 group favored better prognosis (p < 0.05).
Our results indicated that consideration of coilin and p27 levels could be a prognostic reference for predicting the outcome of pediatric ALL patients, especially for disease recurrence. Reduction of coilin expression was sufficient to increase the sensitivity of leukemic cells to daunorubicin treatments, and during which possibly involved functions of p27 in cell cycle regulation and its effects on cell apoptosis.
卡哈尔体（CB）是一种核细胞器，小核核糖核蛋白在此进行修饰、成熟、剪接和/或组装。卷曲螺旋蛋白是CB的标志性结构蛋白。卷曲螺旋蛋白的表达水平和细胞定位对顺铂等化疗试剂敏感。细胞周期蛋白依赖性激酶抑制剂1B（p27）基因位于白血病染色体的高频易位区域，其表达在多种成人白血病类型中具有预后价值。在儿童急性淋巴细胞白血病（ALL）中，卷曲螺旋蛋白以及p27的确切特征和相关功能尚不清楚。
收集144例ALL患者的骨髓样本。通过qRT-PCR检测卷曲螺旋蛋白和p27的表达水平。将患者队列分别分为卷曲螺旋蛋白和p27的低表达组和高表达组。研究不同组的预后和临床生物学特征，尤其关注治疗结果。将Reh或RS4;11白血病细胞暴露于不同浓度的柔红霉素后，通过免疫荧光检测卷曲螺旋蛋白的形态变化。在Reh细胞中，使用慢病毒sh-卷曲螺旋蛋白沉默卷曲螺旋蛋白的表达。采用蛋白质免疫印迹法检测卷曲螺旋蛋白和p27的表达；采用流式细胞术进行细胞周期和凋亡检测；采用MTS法检测细胞活力以检测柔红霉素的药物敏感性。
在本研究中，我们发现柔红霉素能够诱导Reh和RS4;11细胞中CB发生显著的形态变化。敲低卷曲螺旋蛋白的表达可增加对柔红霉素的敏感性，并抑制Reh细胞中p27的表达，导致凋亡增加。重要的是，不仅初诊ALL儿童的卷曲螺旋蛋白和p27 mRNA表达水平明显高于完全缓解（CR）时，而且复发患者的卷曲螺旋蛋白和p27表达均显著高于持续CR患者。4年无事件生存期（EFS）和无复发生存期（RFS）表明，卷曲螺旋蛋白和p27低水平组的预后较好（p < 0.05）。
我们的结果表明，考虑卷曲螺旋蛋白和p27水平可作为预测儿童ALL患者预后的参考指标，尤其是对于疾病复发。卷曲螺旋蛋白表达的降低足以增加白血病细胞对柔红霉素治疗的敏感性，在此过程中可能涉及p27在细胞周期调控中的作用及其对细胞凋亡的影响。
