Li Liyan, Liu Hui, Wang Honglei, Liu Zhaoyun, Chen Yingying, Li Lijuan, Song Jia, Wang Guojin, Fu Rong
Department of Hematology, Tianjin Medical University General Hospital, Tianjin 300052, P.R. China.
Exp Ther Med. 2019 Jun;17(6):4536-4546. doi: 10.3892/etm.2019.7475. Epub 2019 Apr 10.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal proliferative disease of hematopoietic stem cells. Various gene mutations, including the phosphatidylinositol glycan anchor biosynthesis class A (PIG-A) gene, may contribute to the proliferation of PNH clones. In order to explore the mechanism of PNH clone proliferation, a study was performed on 13 patients with PNH who underwent whole exome sequencing. The frequency of mutations in these patients was explored, and an additional 30 patients with PNH were selected for analysis of cluster of differentiation 59-negative (CD59) cells. The mRNA expression of 13 genes, which were selected based on their high frequency in patients with PNH and the fact that they met four screening conditions, was determined in these CD59 cells. Cell proliferation, apoptosis and cell cycle were evaluated upon knocking down the recombinant signal binding protein of immunoglobulin κJ region (RBPJ) gene in 5 patients . The detection rate of PIG-A gene mutation was 61.54% (8/13), and additional mutations in somatic genes were detected, including RBPJ, zinc finger protein 717, polycomb repressive complex 2 subunit and tet methylcytosine dioxygenase. Upon screening according to the mutation frequency and expression level, the present study focused on the RBPJ gene. The expression level of RBPJ in CD59 cells was apparently higher than that in CD59 cells and normal controls which was significantly correlated with clinical data. Furthermore, the expression of RBPJ in PNH primary cells could be effectively inhibited by small interfering RNA-RBPJ. Once the expression of RBPJ decreased remarkably, the apoptotic rate increased gradually, while cell proliferation activity decreased with transfection time and cells were blocked in G0/G1 phase. In conclusion, mutations and abnormal expression of the RBPJ gene may participate in the abnormal proliferation of PNH clones.
阵发性睡眠性血红蛋白尿症（PNH）是一种获得性造血干细胞克隆增殖性疾病。包括磷脂酰肌醇聚糖锚定生物合成A类（PIG-A）基因在内的各种基因突变可能导致PNH克隆的增殖。为了探究PNH克隆增殖的机制，对13例接受全外显子测序的PNH患者进行了一项研究。探究了这些患者的突变频率，并另外选择30例PNH患者分析分化簇59阴性（CD59）细胞。在这些CD59细胞中测定了基于在PNH患者中的高频率以及满足四个筛选条件而选择的13个基因的mRNA表达。在5例患者中敲低免疫球蛋白κJ区重组信号结合蛋白（RBPJ）基因后评估细胞增殖、凋亡和细胞周期。PIG-A基因突变的检出率为61.54%（8/13），还检测到体细胞基因的其他突变，包括RBPJ、锌指蛋白717、多梳抑制复合物2亚基和四甲基胞嘧啶双加氧酶。根据突变频率和表达水平进行筛选后，本研究聚焦于RBPJ基因。RBPJ在CD59细胞中的表达水平明显高于CD59细胞和正常对照中的表达水平，且与临床数据显著相关。此外，小干扰RNA-RBPJ可有效抑制PNH原代细胞中RBPJ的表达。一旦RBPJ的表达显著下降，凋亡率逐渐升高，而细胞增殖活性随转染时间降低，细胞被阻滞在G0/G1期。总之，RBPJ基因的突变和异常表达可能参与PNH克隆的异常增殖。
