Li Bo, Guo Lixing, Zhang Yuping, Xiao Yankai, Wu Mingjuan, Zhou Lingling, Chen Shaohua, Yang Lijian, Lu Xiang, Li Yangqiu
Institute of Hematology, Medical College, Jinan University, Guangzhou, China.
Department of Hematology, Guangzhou First Municipal People's Hospital Affiliated to Guangzhou Medical College, Guangzhou, China.
J Hematol Oncol. 2016 Mar 31;9:32. doi: 10.1186/s13045-016-0261-6.
A previous study has demonstrated a significantly increased CD3ζ gene expression level in aplastic anemia (AA). However, the mechanism underlying the upregulated CD3ζ mRNA expression level and that of T cell activation signaling molecules in AA patients remains unclear. Thus, we investigated the expression levels of the CD3ζ, CD28, CTLA-4, and Cbl-b genes, the SNP rs231775 in the CTLA-4 gene, and the distribution of the CD3ζ 3'-UTR splice variant in AA patients.
CD3ζ 3'-UTR splice variants were identified in peripheral blood mononuclear cells (PBMCs) from 48 healthy individuals and 67 patients with AA [37 cases of severe aplastic anemia (SAA) and 30 cases of non-sever aplastic anemia (NSAA)] by RT-PCR. CD3ζ, CD28, CTLA-4, and Cbl-b gene expression was analyzed by real-time quantitative PCR. The SNP rs231775 in CTLA-4 gene was analyzed by PCR-RFLP.
CD3ζ and CD28 expression was significantly higher, while CTLA-4 and Cbl-b expression was significantly lower in AA patients compared with healthy individuals. Significantly higher CD3ζ expression was found in the NSAA subgroup compared with the SAA subgroup. 64 % of the AA samples had the same genotype (WT(+)AS(+)CD3ζ 3'-UTR); 22 % of the AA patients had a WT(+)AS(-)CD3ζ 3'-UTR genotype, and 14 % of the AA patients had a WT(-)AS(+)CD3ζ 3'-UTR genotype. The CD3ζ expression level of WT(-)AS(+) subgroup was the highest in the SAA patients. A significantly higher frequency of the GG genotype (mutant type, homozygous) of SNP rs231775 in CTLA-4 gene was found in the AA patients. Positive correlation between the CTLA-4 and Cbl-b gene expression levels was found in healthy individuals with the AA and AG genotypes, but not in the AA patients.
This is the first study analyzing the expression characteristics of the CD28, CTLA-4, and Cbl-b genes in AA. Our results suggest that aberrant T cell activation may be related to the first and second signals of T cell activation in AA. The GG genotype of SNP rs231775 in CTLA-4 gene might be associated with AA risk in the Chinese population. The characteristics of CD3ζ 3'-UTR alternative splicing may be an index for evaluating the T cell activation status in AA patients, particularly in SAA patients.
先前的一项研究表明再生障碍性贫血（AA）患者中CD3ζ基因表达水平显著升高。然而，AA患者中CD3ζ mRNA表达水平上调以及T细胞活化信号分子表达上调的潜在机制仍不清楚。因此，我们研究了AA患者中CD3ζ、CD28、CTLA-4和Cbl-b基因的表达水平、CTLA-4基因中的单核苷酸多态性rs231775以及CD3ζ 3'-UTR剪接变体的分布情况。
通过逆转录聚合酶链反应（RT-PCR）在48名健康个体和67例AA患者[37例重型再生障碍性贫血（SAA）和30例非重型再生障碍性贫血（NSAA）]的外周血单个核细胞（PBMC）中鉴定CD3ζ 3'-UTR剪接变体。通过实时定量聚合酶链反应分析CD3ζ、CD28、CTLA-4和Cbl-b基因表达。通过聚合酶链反应-限制性片段长度多态性分析CTLA-4基因中的单核苷酸多态性rs231775。
与健康个体相比，AA患者中CD3ζ和CD28表达显著更高，而CTLA-4和Cbl-b表达显著更低。与SAA亚组相比，NSAA亚组中CD3ζ表达显著更高。64%的AA样本具有相同的基因型（WT(+)AS(+)CD3ζ 3'-UTR）；22%的AA患者具有WT(+)AS(-)CD3ζ 3'-UTR基因型，14%的AA患者具有WT(-)AS(+)CD3ζ 3'-UTR基因型。WT(-)AS(+)亚组的CD3ζ表达水平在SAA患者中最高。在AA患者中发现CTLA-4基因的单核苷酸多态性rs231775的GG基因型（突变型，纯合子）频率显著更高。在具有AA和AG基因型的健康个体中发现CTLA-4和Cbl-b基因表达水平呈正相关，但在AA患者中未发现。
这是第一项分析AA中CD28、CTLA-4和Cbl-b基因表达特征的研究。我们的结果表明异常的T细胞活化可能与AA中T细胞活化的第一和第二信号有关。CTLA-4基因中rs231775单核苷酸多态性的GG基因型可能与中国人群的AA风险相关。CD3ζ 3'-UTR可变剪接的特征可能是评估AA患者尤其是SAA患者T细胞活化状态的一个指标。
