B细胞淋巴瘤LITAF基因甲基化状态及其临床意义

Significance of expression and promoter methylation of LITAF gene in B-cell lymphoma

目的探讨B细胞淋巴瘤中LITAF基因启动子区CpG岛异常甲基化情况，研究去甲基化药物对人B细胞淋巴瘤细胞系Raji、Pfeiffer和Daudi细胞中LITAF基因的转录调控作用，寻找肿瘤治疗新靶点。方法收集存档石蜡包埋组织标本105例[弥漫性大B细胞淋巴瘤（DLBCL）54例，小淋巴细胞性淋巴瘤（SLL）15例，黏膜相关淋巴组织结外边缘区淋巴瘤（MALT）8例，滤泡性淋巴瘤（FL）6例，其他22例]，5例良性反应性增生淋巴组织作为正常对照。甲基化特异性PCR（MSP）检测LITAF基因启动子区甲基化状态。运用MSP、Western blot、免疫组织化学方法检测5-氮杂脱氧胞苷处理前后Raji、Pfeiffer和Daudi细胞系的甲基化状态及蛋白表达情况。四甲基偶氮唑盐（MTT）法检测经去甲基化药物处理后细胞生长抑制情况。结果B细胞淋巴瘤中89．5％（94／105）的病例存在LITAF基因启动子区高甲基化，其中3．8％（4／105）的病例发生完全高甲基化，22．9％（24／105）的病例以部分非甲基化为主，对照组5例良性淋巴结增生标本中LITAF基因启动子区均未检测到高甲基化改变。经去甲基化处理后，Raji、Daudi、Pfeiffer细胞LITAF基因甲基化程度明显受抑制，基因表达得以恢复和增加。结论在B细胞淋巴瘤患者中普遍存在LITAF基因启动子区CpG岛高甲基化，由其所致的LITAF基因表达沉默或减少可能是B细胞淋巴瘤发生的一个重要因素，LITAF基因的高甲基化可望作为一个良好的候选分子诊断标记及可能的治疗靶点。

Objective To investigate promoter methylation status of LITAF gene in B-cell lymphoma and to explore transcription regulation of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on LITAF gene. Methods One hundred and five paraffin specimens including 54 cases of diffuse large B cell lymphoma （DLBCL）, 15 small lymphocytic lymphoma （SLL）, 8 mucosa-associated lymphoid tissue lymphoma （MALT） and 6 follicular lymphoma （FL） were included. Five reactive lymphoid hyperplasia samples were collected as control. Methylation status of CpG island in LITAF gene in the specimens and in Raji, Pfeiffer and Daudi cell lines were detected by methylation-specific PCR （MSP）. LITAF expression in Raji, Pfeiffer and Daudi cell lines with or without 5-Aza-CdR treatment was detected by Western blot and immunohistochemistry. The inhibitory ratio in the three cell lines was measured by MTT assay. Results The frequency of LITAF gene methylation in B-cell lymphoma was 89. 5% （94/105）. Among them, 3.8% （4/105） showed complete hypermethylation. In control group, however, there was no methylation in CpG island of LITAF gene promoter. The expression of LITAF was recovered or increased along with the ceU growth inhibition when the cells exposed to demethylating reagent. Conclusions LITAF gene silencing with aberrant CpG methylation is probably one of the critical events to the oncogenesis of B-cell lymphoma, whichmay have important implications as a candidate marker for diagnosis and target gene therapy.