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目的:检测胃癌中NR4A2(又称Nurr1)基因的表达,从NR4A2调控机制出发寻找相关选择性剪接异构体,探讨其与胃癌发生和转移的关系。方法:通过选择性剪接数据库(ASD)预测NR4A2基因可能存在的选择性剪接异构体,半定量PCR检测NR4A2及异构体的表达,基因测序技术鉴定新的剪接异构体;实时定量PCR检测41例样本中NR4A2及剪接异构体的表达水平;免疫组织化学方法检测28例样本中NR4A2蛋白表达。结果:NR4A2基因在胃癌原位、癌旁、转移组织中均有表达,确定了2种剪接异构体NR4A2-Ⅰ和NR4A2-Ⅱ。实时定量PCR检测41例样本中NR4A2及2种异构体在癌旁组织表达水平高于原位癌(P=0.017,P=0.007,P=0.004);在肝转移灶中表达水平低于原位癌(P=0.001,P=0.018,P=0.016),差异有统计学意义。免疫组化检测发现癌旁组织中NR4A2表达阳性率高于原位癌及转移灶,但差异无统计学意义(P=0.672)。结论:胃癌原位、癌旁和转移组织中至少存在NR4A2基因的2种剪接模式;NR4A2及异构体的表达变化与胃癌发生和转移相关。
OBJECTIVE: To detect the expression of NR4A2 (also known as Nurr1) gene in gastric cancer and search for the related alternative splicing isoforms from the mechanism of NR4A2 regulation to explore its relationship with the occurrence and metastasis of gastric cancer. Methods: The possible alternative splicing isoforms of NR4A2 were predicted by alternative splicing database (ASD). The expression of NR4A2 and its isoforms was detected by semi-quantitative PCR and the new splicing isoforms were identified by sequencing. Real-time PCR The expression of NR4A2 and splicing isoform in 41 samples was detected by immunohistochemistry. The NR4A2 protein expression in 28 samples was detected by immunohistochemistry. Results: NR4A2 gene was expressed in situ, adjacent tissues and metastatic tissues of gastric cancer. Two kinds of splicing isoforms NR4A2-Ⅰ and NR4A2-Ⅱ were identified. The expression of NR4A2 and its two isoforms in paracancer tissues was higher than that in carcinoma in situ (P = 0.017, P = 0.007, P = 0.004) (P = 0.001, P = 0.018, P = 0.016), the difference was statistically significant. Immunohistochemistry showed that the positive rate of NR4A2 in paracancer tissues was higher than that in situ carcinoma and metastasis, but the difference was not statistically significant (P = 0.672). CONCLUSION: There are at least two kinds of splicing patterns of NR4A2 gene in situ, paracancer and metastasis of gastric cancer. The expressions of NR4A2 and its isoform are related to the occurrence and metastasis of gastric cancer.