非小细胞肺癌中ASPPl和ASPP2基因启动子甲基化的意义

Promoter methylation of ASPP1 and ASPP2 genes in non-small cell lung cancers

目的检测非小细胞肺癌（NSCLC）中ASPPl和ASPP2基因启动子CpG岛的甲基化状态和相应的蛋白表达，及癌细胞的凋亡水平和p53蛋白的表达，探讨ASPP基因在NSCLC中的作用及其临床意义。方法采用甲基化特异性PCR（MSP）检测90例NSCLC组织、25例癌旁肺组织中ASPPl、ASPP2基因启动子的甲基化水平，并测序证实其甲基化位点；免疫组织化学EnVision法检测NSCLC组织中ASPPl、ASPP2和p53蛋白的表达；原位末端转移酶标记检测法（TUNEL）检测癌细胞凋亡，计算其凋亡指数（AI）。结果ASPPl基因启动子甲基化率在NSCLC中为42．2％（38／90），癌旁组织中为16．0％（4／25），二者比较差异有统计学意义（P=0．019）；ASPPl基因启动子甲基化与患者年龄、性别、组织类型和分化程度无相关性（均P〉0．05），但与患者的TNM分期和淋巴结转移存在相关性（P=0．031，P=0．030）；90例肺癌组织中ASPPl甲基化者其蛋白表达率明显低于ASPPl未甲基化者（P=0．002）；ASPPl蛋白阳性组的AI值高于阴性组（P=0．022）。90例NSCLC和25例癌旁组织中均未检测到ASPP2基因启动子甲基化；ASPP2蛋白表达阳性组和阴性组之间AI值差异也无统计学意义（P=0．282）。ASPPl和p53表达呈负相关，（r=-0．259，P〈0．01），ASPP2和p53的表达状态无关（r=-0．119，P〉0．05）。结论NSCLC中ASPPl基因启动子甲基化可能是ASPPl蛋白表达下调的原因，高甲基化可能与NSCLC的恶性进展有关．ASPPl蛋白的表达能促进细胞凋亡。

Objective To investigate the methylation status of CpG islands in the promoter region and the protein expression of ASPP1 and ASPP2 genes in non-small-cell lung cancer （ NSCLC ） and their relationship with cellular apoptosis and p53 gene expression. Methods The 5＇ CpG island methylation patterns of ASPP1 and ASPP2 were evaluated by methylation specific polymerase chain reaction （ MSP ） followed by confirmation of sequencing. Immunohistochemistry was used to detect the expression of ASPPI, ASPP2 and p53 in lung carcinoma tissue samples （n = 90） and adjacent non-neoplastic lung tissue samples （n = 25 ） . TUNEL assay was used to detect the apoptotic activity. Results The presence of ASPP1 methylation was significantly higher in NSCLC than that in the adjacent non-neoplastic lung tissue [42. 2% （38/90） vs. 16. 0% （4/25）, P =0. 019]. ASPP1 promoter methylation had a close relationship with TNM stage and lymph node metastasis（P =0. 031, P =0. 030） , but was not related to the age, sex, histological types and the grades of tumor differentiation （P =0. 389, P =0. 278, P =0. 570, and P =0. 103）. Tumors with ASPP1 promoter methylation demonstrated a lower expression of ASPP1 as compared with those without the methylation （P = 0. 002 ）. ASPP1 expression was associated with a higher apoptotic index （AI） （ P = 0. 022） and a decreased lo53 expression（r = -0. 259, P 〈 0. 01 ）. Methylation in the promoter region of ASPP2 gene was not detected in lung cancer （n = 90 ） or adjacent non-neoplastic lung tissue （n = 25 ）. Expression of ASPP2 protein did not correlate with AI （P = 0. 282） and p53 status in NSCLC. Conclusions High methylation of ASPP1 gene promoter regions is one of the important mechanisms that down-regulate its protein expression in NSCLC. ASPP1 promoter methylation may be associated with the malignant progressionof the tumor, and ASPP1 expression promotes cellular apoptosis.