pig7基因在急性白血病细胞中的表达及其临床意义

Expression of pig7 in acute leukemia and its clinical significance

目的检测 pig7基因在急性白血病(AL)细胞中的表达水平及其临床意义,在基因甲基化调控方而探讨 pig7基因表达异常的可能机制。方法应用实时定量逆转录 PCR(RT-PCR)方法对138例 AL 患者、21名正常人骨髓标本以及6个白血病细胞株进行了 pig7转录本的检测,并在全反式维甲酸(ATRA)作用下观察 NB4细胞分化效应及 pig7基因表达情况。进行限制性内切酶分析以鉴定白血病细胞中存在的 pig7转录剪接体。通过甲基化特异性 PCR(MSP)检测白血病细胞 pig7基因启动子区域是否存在过度甲基化。结果 AL 进展期(包括初治、复发/难治)患者骨髓细胞 pig7 mRNA 相对表达水平与正常骨髓细胞相比明显降低(RQ 中位数分别为0.62和18.30,P<0.01),其中急性髓系白血病(AML)与急性淋巴细胞白血病(ALL)差异无统计学意义,而初治组与复发/难治组比较 pig7mRNA 水平差异有统计学意义,前者明显高于后者(RQ 中位数分别为1.43和0.16,P<0.05)。pig7低表达患者完全缓解率也较低(P<0.05)。NB4细胞经 ATRA 诱导分化后 pig7表达水平由1.61±0.72增至44.75±3.93(P<0.01)。酶切结果提示白血病细胞中仅存在 SIMPLE 剪接体。在 K562、HL-60及 Nalm-6细胞 pig7启动子区域存在过度甲基化,而 U937、NB4和 kasumi-1细胞中该区域非甲基化状态占优势。结论 pig7基因在 AL 的表达下调为白血病发病机制的探讨和疗效预测提供了新的思路。

Objective To investigate pig7 expression level in acute leukemia （AL） and its clinical significance and explore the possible mechanisms for pig7 silence in terms of methylation control. Methods Expression levels of pig7 mRNA in bone marrow samples from 138 patients with de novo AL and 21 normal controls and in 6 leukemic cell lines were detected by quantitative real-time reverse transcription PCR （ RTPCR）. Differentiation induction effect by all-trans retinoic acid （ATRA） and concomitant change in pig7 expression were also monitored in NB4 cells. Endonuclease analysis was employed to determined the identity of pig7 transcript present in AL samples. Methylation specific PCR （MSP） was used to elucidate if hypermethylation was responsible for pig7 silence in AL. Results Compared with that in normal control, pig7 expression was markedly decreased （0.62 vs 18.30, median, P 〈 0.01 ） in AL patients on progression （at diagnosis, relapse or refractory）. No significant difference was observed between acute myeloid leukemia （AML） and acute lymphoblastic leukemia （ALL）. AL at diagnosis had a higher pig7 level than those with relapsed or refractory disease （ 1.43 vs 0. 16, median, P 〈 0.05 ）. The complete remission （CR） rate after chemotherapy was found to be significantly correlated with pig7 expression levels （ P 〈 0.05 ）. Differentiated NB4 cells showed an increased level of pig7 expression （ from 1.61 ± 0.72 to 44.75 ± 3.93, P 〈 0.01 ）. Only one form of pig7 transcripts i. e. , Small integral membrane protein of late endosome （ SIMPLE ） , was detected in AL patients. Hypermethylation of pig7 promoter was identified in K562 and HL-60 cells, in contrast to non-methylation predominant in U937 cells. Condusion Aberrant clown-regulation of pig7 provides novel insights into leukemogenesis and therapy response prediction in AL.