Abstract:

Objective　To investigate the anti-apoptotic effect of abnormal Mcl-1 expression in clones of paroxysmal nocturnal hemoglobinuria (PNH). Methods　Thirteen PNH patients admitted to Department of Hematology, Ningde Municipal Hospital of Ningde Normal University from February 2020 to February 2022 were selected as a case group (7 males and 6 females, with a median age of 27 years old), and fifteen healthy subjects were selected as a control group (8 males and 7 females, with a median age of 38 years old). The expressions of Mcl-1 mRNA in abnormal PNH cloned cells and normal cells were detected by real-time quantitative polymerase chain reaction (RT-PCR), and then the proliferation, apoptosis, and cell cycle distribution of abnormal PNH cloned cells were detected before and after transfection of siRNA-Mcl-1. Fisher exact probability method, independent-sample t test, and rank sum test were used. Results　The expressions of Mcl-1 mRNA in the CD59^- cells, the CD59⁺ cells, and the control group were (2.48±0.25), (1.61±0.19), and (1.21±0.08), respectively, that in the CD59^- cells was significantly higher than those in the CD59⁺ cells and the control group (P=0.031 and 0.022), but there was no statistically significant difference between the CD59⁺ cells and the control group (P=0.126). The relative expression of Mcl-1 mRNA in the PNH patients was positively correlated with the number of CD59^- clone cells (r²=0.523, P=0.012). After Mcl-1 gene silence by siRNA, the Mcl-1 mRNA expression was decreased significantly. The apoptosis rate of the siRNA-Mcl-1 transfection group was significantly higher than those of the siRNA-scr transfection group and the blank control group [(43.12±16.33)% vs. (24.07±15.42)% and (21.56±14.85)%], with statistically significant differences (both P<0.05). The proportion of G₀/G₁ phase cells in the siRNA-Mcl-1 transfection group was higher than those in the siRNA-scr transfection group and the blank control group [(93.16±3.06)% vs. (91.52±4.01)% and (90.78±3.62)%], and the proportion of S-phase cells was lower than those in the siRNA-scr transfection group and the blank control group [(4.96±3.21)% vs. (6.86±3.87)% and (7.05±3.59)%], with statistically significant differences (all P<0.05). Conclusions　The siRNA-Mcl-1 can reduce the proliferation and promote the apoptosis of CD59^- cells in PNH patients. Overexpression of Mcl-1 gene may play a role in the anti-apoptosis of PNH clones.

Key words:

Paroxysmal nocturnal hemoglobinuria, CD59^-, CD59⁺, Mcl-1, Anti-apoptosis

摘要：

目的　探讨Mcl-1基因异常表达在阵发性睡眠性血红蛋白尿症（PNH）异常克隆中的抗凋亡作用。方法　选取2020年2月至2022年2月在宁德师范学院附属宁德市医院血液科就诊的13例PNH患者为病例组（男7例，女6例，中位年龄27岁），15例健康体检者为对照组（男8例，女7例，中位年龄38岁）。实时荧光定量多聚核苷酸链式反应（RT-PCR）检测PNH异常克隆细胞与正常细胞中Mcl-1基因mRNA的表达水平，然后敲除PNH患者CD59^-细胞中Mcl-1基因后检测siRNA-Mcl-1转染前后PNH异常克隆细胞的增殖、凋亡及细胞周期分布等生物学变化。统计学方法采用Fisher确切概率法、独立样本t检验、秩和检验。结果　PNH患者CD59^-细胞组、CD59⁺细胞组、对照组的Mcl-1基因mRNA的表达量分别为（2.48±0.25）、（1.61±0.19）、（1.21±0.08），CD59^-细胞组高于CD59⁺细胞组及对照组（P=0.031、0.022），而CD59⁺细胞组和对照组间比较差异无统计学意义（P=0.126）。同时PNH患者Mcl-1基因mRNA的相对表达量与CD59^-克隆细胞数呈正相关（r²=0.523，P=0.012）。PNH患者CD59^-细胞转染靶向siRNA-Mcl-1后，Mcl-1 mRNA表达水平明显下降。siRNA-Mcl-1转染组细胞凋亡率高于转染无义序列组（siRNA-scr转染组）和空白对照组［（43.12±16.33）%比（24.07±15.42）%、（21.56±14.85）%］，差异均有统计学意义（均P<0.05）。siRNA-Mcl-1转染组G₀/G₁期细胞比例高于siRNA-scr转染组和空白对照组［（93.16±3.06）%比（91.52±4.01）%、（90.78±3.62）%］，而S期细胞比例低于siRNA-scr转染组和空白对照组［（4.96±3.21）%比（6.86±3.87）%、（7.05±3.59）%］，差异均有统计学意义（均P<0.05）。结论　siRNA-Mcl-1转染能减弱PNH患者CD59^-细胞的增殖，促进凋亡。Mcl-1基因的过表达可能在PNH克隆抗凋亡中起作用。

关键词:

阵发性睡眠性血红蛋白尿症, CD59^-, CD59⁺, Mcl-1, 抗凋亡