CAJ | 학술논문

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.
목적 관찰핵불균일핵당핵단백A2/B1(hnRNP A2/B1)재비소세포폐암(NSCLC)중적표체급기여DNA수복매O6-갑기조표령-DNA갑기전이매(MGMT)、8-간기조표령DNA당감매(OGG1)、양화환원인자1(Ref-1)、DNA의뢰성단백격매복합물DNA-PKcs화Ku mRNA지간적상호작용,병진일보탐토기재NSCLC발병궤제중적작용.방법 채용면역조화、Western blot급형광실시정량PCR방법,검측NSCLC환자암조직급정상폐조직hnRNP A2/B1적표체.채용면역공침정결합역전록취합매련반응(RT-PCR)방법,연구인폐린암세포주중hnRNP A2/B1단백시부여상술5충DNA수복매적mRNA직접결합,연후채용면역조화급형광실시정량PCR방법,검측NSCLC환자암조직급정상폐조직MGMT적표체정황.결과 면역조화염색현시,hnRNP A2/B1정위우세포핵,hnRNPA2/B1재NSCLC암조직중적표체양성솔(100%)화단백표체평분[(5.3±0.9)분]균현저고우정상폐조직[32%화(2.2±0.7)분,P＜0.01],재Ⅲ～Ⅳ기NSCLC조직중적표체략고우Ⅰ～Ⅱ기(P＜0.05),이여년령、성별、조직학류형급흡연상황무관(균P＞0.05).통과RT-PCR방법가이종인폐린암세포주면역공침정산물중확증출MGMT mRNA,제시hnRNP A2/B1여MGMT mRNA상결합.진일보적면역조화염색결과현시,재NSCLC조직중,MGMT적표체양성솔위32.0%,명현저우정상폐조직(78.0%),단백표체평분[(2.2±0.8)분]야현저저우정상폐조직[(4.1±1.2)분,P＜0.01].형광실시정량PCR결과현시,NSCLC조직중MGMT mRNA적표체량위1.8(0.6～3.1),명현저우정상폐조직[9.8(6.8～18.3),P＜0.01].결론 HnRNP A2/B1단백급mRNA재NSCLC조직중적표체균승고,hnRNP A2/B1여MGMT mRNA상결합,가능통과대MGMT mRNA적전록후조공삼여NSCLC적발생.
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.