摘要
目的观察核不均一核糖核蛋白A2／B1（hnRNPA2／B1）住非小细胞肺癌（NSCLC）中的表达及其与DNA修复酶O^6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）、8-羟基吗嘌呤DNA糖甘酶（OGGI）、氧化还原因子1（Ref-1）、DNA依赖性蛋白激酶复合物DNA—PKcs和Ku mRNA之间的相互作用，并进一步探讨其在NSCLC发病机制中的作用。方法采刚免疫绀化、Western blot及荧光实时定量PCR方法，检测NSCLC患者癌组织及正常肺组织hnRNPA2／B1的表达。采用免疫共沉淀结合逆转录聚合酶链反应（RT—PCR）方法，研究人肺鳞癌细胞株中hnRNPA2／B1蛋向是否与上述5种DNA修复酶的mRNA直接结合，然后采用免疫组化及荧光实时定量PCR方法，榆测NSCLC患者痛绀织及正常肺组织MGMT的表达情况。结果免疫组化染色显示，hnRNPA2／B1定位于细胞核，hnRNPA2／B1在NSCLC癌组织中的表达阳性率（100％）和蛋白表达评分[（5．3±0．9）分]均显著高于正常肺组织[32％和（2．2±0．7）分，P〈0．01]，在Ⅲ～Ⅳ期NSCLC组织中的表达略高于J～Ⅱ期（P〈0．05），而与年龄、性别、组织学类型及吸烟状况无关（均P〉0．05）。通过tiT—PCR方法可以从人肺鳞癌细胞株免疫共沉淀产物中扩增出MGMT mRNA，提示hnRNP A2／B1与MGMT mRNA相结合。进一步的免疫组化染色结果显示，在NSCLC组织中，MGMT的表达阳性率为32．0％，明娃低于止常肺组织（78．0％），蛋白表达评分[（2．2±0．8）分]也显著低于正常肺组织[（4．1±1．2）分，P〈0．01]。荧光实时定量PCR结果显示，NSCLC组织中MGMTmRNA的表达量为1．8（0．6～3．1），明显低于正常肺组织[9．8（6．8～18．3），P〈0．01]。结论HnRNPA2／BI蛋白及mRNA在NSCI．C组织中的表达均升高，hnRNPA2／B1与MGMTmRNA相结合，可能通过对MGMTmRNA的转录后调控参与NSCLC的发牛。
Objective To study the expression of heterogeneous nuclear ribonucleoprotein A2/B1 （hnRNP A2/B1） in non-small cell lung cancer （NSCLC）, and the interaction between hnRNP A2/B1 protein and mRNA of DNA repair enzymes O^6-methylguanine DNA-methyltransferase （MGMT）, 8-oxoguanine DNA glycosylase （ OGG1 ）, redox factor 1 （ Ref-1 ） , DNA-dependent protein kinase （ including DNA-PKcs and ku）. Methods The expression and distribution of hnRNP A2/B1 were detected by immunohistochemistry and Western blot on 50 NSCLC samples from patients who underwent resection in Zhongshan Hospital. The hnRNP A2/B1 mRNA expression was tested by real-time PCR. Co- immunoprecipitation （co-IP） combined RT-PCR was used to investigate whether hnRNP A2/B1 could be bound with the mRNA of the above mentioned 5 DNA repair enzymes in human lung cancer cell line （ HTB- 182）. Then immunohistochemistry and real-time PCR were used to detect the expression of MGMT in thesame group of patients. Results HnRNP A2/B1 protein and mRNA expressions were increased in the NSCLC tissues than that in the corresponding normal lung tissues. HnRNP A2/B1 was expressed predominantly in the nuclei of tumor cells. The positive rate and immunohistochemistry score of hnRNP A2/B1 in tumor tissue were significantly higher than that in normal tissue （P 〈 0.01 ）. In stage m-IV NSCLC, hnRNP A2/B1 expression was higher than that in stage I-Ⅱ. There was no significant differences of hnRNP A2/BI expression among patients of different age, sex, histological type, and smoking history. The resuhs of co-IP combined RT-PCR suggested that hnRNP A2/B1 is bound with MGMT mRNA, and MGMT expression is decreased in tumor tissue of NSCLC. Conclusions The results of this study show that hnRNP A2/B1 protein and mRNA are highly expressed in NSCLC, and hnRNP A2/B1 is bound with MGMT mRNA, which indicate that it might be one of the mechanisms of hnRNP A2/B1 participating in the pathogenesis of NSCLC.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2011年第2期110-114,共5页
Chinese Journal of Oncology
基金
上海市复旦大学青年科学基金（154）
上海市卫生局科研课题（2009020）
