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目的:寻找人肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)中CD99转移相关选择性剪接异构体,探讨CD99选择性剪接异构体表达与ccRCC转移的关系。方法:通过选择性剪接数据库(alternative splicing database,ASD)对CD99基因可能存在的选择性剪接异构体进行预测。运用自行设计的5对特异性引物,采用RT-PCR技术,在转移组织、发生及未发生转移的癌组织、癌旁组织的各混合样本中检测6种预测异构体的表达,再对电泳所获条带进行克隆、测序。在各个组织样本中检测CD99Ⅰ、CD99Ⅱ和新型CD99选择性剪接异构体(CD99-Ⅲ)的表达。结果:从6种预测异构体中筛选出一种新型异构体(CD99-Ⅲ)。该异构体在转移组织中表达率(8/9)高于未发生转移的原发癌组织(10/21,P<0.05)。CD99Ⅰ型在样本中均表达,CD99Ⅱ型在发生转移的原位癌组织中表达率(6/9)远高于未转移的原发癌组织(3/21,P=0.008)。结论:CD99-Ⅲ为一种新型选择性剪接异构体,且CD99Ⅱ型、CD99-Ⅲ的表达可能与ccRCC的转移进展有关。
OBJECTIVE: To search alternative splicing isoforms of CD99 in clear cell renal cell carcinoma (ccRCC) and to investigate the relationship between the alternative splicing isoform of CD99 and the metastasis of ccRCC. Methods: The alternative splicing isoforms of CD99 gene were predicted by alternative splicing database (ASD). Five pairs of specific primers designed by ourselves were used to detect the expression of six predicted isoforms in mixed samples of metastasized and non-metastasized cancer tissues and adjacent tissues by RT-PCR. The resulting bands were cloned and sequenced. The expression of CD99I, CD99II and the novel CD99 alternative splice variant (CD99-III) was examined in various tissue samples. Results: A novel isoform (CD99-Ⅲ) was screened from six predicted isoforms. The expression of isoforms in metastatic tissues (8/9) was higher than that in primary cancerous tissues without metastasis (10/21, P <0.05). CD99 type Ⅰ was expressed in all the samples. The expression rate of CD99 Ⅱ was much higher in metastatic carcinoma tissues (6/9) than that in non-metastatic primary cancerous tissues (3/21, P = 0.008). CONCLUSION: CD99-Ⅲ is a novel isomer of alternative splicing, and the expression of CD99Ⅱ and CD99-Ⅲ may be related to the metastasis of ccRCC.