Huang Hui-Ping, Liu Wen-Jun, Guo Qu-Lian, Bai Yong-Qi
Department of Pediatrics, The First Affiliated Hospital of Sichuan Medical University, Luzhou, Sichuan 646000, P.R. China.
Int J Mol Med. 2016 Mar;37(3):669-78. doi: 10.3892/ijmm.2016.2480. Epub 2016 Feb 4.
Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF‑PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (P<0.05). The distribution of cells in the cell cycle was also altered. Specifically, more cells were present in the G0/G1 phase compared to the S phase (P<0.05). In addition, the apoptotic rate was significantly higher in cells transfected with HOXA5‑specific siRNA (P<0.05). In conclusion, high expression levels of HOXA5 mRNA and protein in children with ALL indicate that HOXA5 is closely associated with childhood ALL. In addition, HOXA5-specific siRNA effectively silences HOXA5 gene expression and induces apoptosis and cell-cycle arrest in Jurkat cells, thus inhibiting cell proliferation.
急性淋巴细胞白血病（ALL）是儿童中一种常见的恶性肿瘤，发病率较高，约占白血病病例的80%。尽管患者的治疗已有改善，但频繁复发导致预后不良。本研究的目的是确定HOXA5是否可作为白血病基因治疗的靶点，以便提供一种新的治疗方法。根据临床研究目的从骨髓中提取单核细胞。在对急性期ALL进行检测后，在ALL缓解组（每组25例）和对照组[20例，免疫性血小板减少症（ITP）]中检测HOXA5的相对mRNA和蛋白表达。通过RNA干扰（RNAi）进行基因沉默，以研究小干扰RNA（siRNA）转染至Jurkat细胞后沉默HOXA5的效果。使用脂质体将HOXA5特异性siRNA转染至Jurkat细胞。实验分为实验组（HOXA5靶向siRNA的脂质体转染）、阴性对照组（用阴性对照siRNA进行细胞脂质体转染）和对照组（仅加入等量的细胞和培养基）。使用蛋白质免疫印迹法和定量荧光聚合酶链反应（QF-PCR）检测各细胞组中HOXA5的相对mRNA表达和蛋白分布。使用流式细胞术确定细胞周期中的细胞分布和凋亡细胞率。ALL急性期HOXA5在mRNA和蛋白水平的表达明显高于缓解期ALL组和对照组。在用HOXA5特异性siRNA转染的细胞中，HOXA5在mRNA和蛋白水平的表达显著降低（P<0.05）。细胞周期中的细胞分布也发生了改变。具体而言，与S期相比，G0/G1期的细胞更多（P<0.05）。此外，用HOXA5特异性siRNA转染的细胞凋亡率明显更高（P<0.05）。总之，ALL患儿中HOXA5 mRNA和蛋白的高表达水平表明HOXA5与儿童ALL密切相关。此外，HOXA5特异性siRNA有效沉默HOXA5基因表达，并诱导Jurkat细胞凋亡和细胞周期停滞，从而抑制细胞增殖。
