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目的 观察核不均一核糖核蛋白A2/B1(hnRNP A2/B1)在非小细胞肺癌(NSCLC)中的表达及其与DNA修复酶O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、8-羟基鸟嘌呤DNA糖苷酶(OGG1)、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 采用免疫组化、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hnRNP A2/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hnRNP A2/B1蛋白是否与上述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,检测NSCLC患者癌组织及正常肺组织MGMT的表达情况.结果 免疫组化染色显示,hnRNP A2/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Ⅰ～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RT-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%,明显低于正常肺组织(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MGMT mRNA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 HnRNP A2/B1蛋白及mRNA在NSCLC组织中的表达均升高,hnRNP A2/B1与MGMT mRNA相结合,可能通过对MGMT mRNA的转录后调控参与NSCLC的发生.

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 O6-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.Coimmunoprecipitation(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 the same 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 Ⅲ-Ⅳ NSCLC,hnRNP A2/B1 expression was higher than that in stage Ⅰ-Ⅱ.There was no significant differences of hnRNP A2/B1 expression among patients of different age,sex,histological type,and smoking history.The results 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.