Study On The Alternatively Spliced Variants Of CD99 In Clear Cell Renal Cell Carcinoma And Metastatic Disease

[Objective]To identify metastasis-associated splice variants of CD99 and investigate the mRNA expression of splice variants in clear cell renal cell carcinoma tissues and determine its relationship with the initiation and progression of ccRCC.[Method]cDNA microarray technique was used to detect the differentially expressed genes between the two ccRCC cell lines (RCC05-TXJ and RCC05-ZYJ). After screening with DNA microarray, CD99 gene was taken to be studied. Alternative Splicing Database was used to predict the splice variants of CD99.According to the prediction, the RT-PCR primers of CD99 gene were designed by using Primer Premier 5.0 software, Oligo 6.0 software and BLAST program. Using the specific primer sets,the splice variants were identified in metastatic tissues, primary ccRCCs and normal tissues adjacent to cance. Tissue specimens were obtained from the Departments of Urology and Orthopaedics, the 1st and 2nd Affiliated Hospitals of this University from Jan. 2004 to Sep. 2007, Specimens from 30 patients with ccRCC were collected immediately after surgical treatments. We obtained metastatic ccRCC specimens from 9 patients, including 2 metastasized to bone, 2 to lymph nodes near inferior vena and/or renal hilus and 5 cancer epistom in vein. All specimens were immediately frozen in liquid nitrogen and stored at -80°C. The variants were amplified by RT-PCR, and were cloned into pMD18-T vector, which were subsequently transformed into E.Coli DH5α.. After affirmation the existence of variants by gene sequencing, the mRNA expression of CD99 typeⅠ, CD99 typeⅡand CD99Ⅲwere detected in 9 metastatic tissues and its corresponding cancer tissues, 21 primary ccRCCs and normal tissues adjacent to cancer by RT-PCR.[Result]A new isoform of CD99 (CD99Ⅲ)was identified , which contained an insert about 100bp between exon 8 and 9 of the CD99. And this splice variant was detected in 8 of 9 metastatic tissues, significantly higher than that in primary ccRCCs without metastasis (P<0.05).The results revealed that the transcripts of CD99 typeⅠwas observed in all of the ccRCC specimens tested; CD99 typeⅡwas detected in 6 of 9 metastatic corresponding primary cancer tissues, higher than that in cancer tissues without metastasis( P =0.008) . [Conclution]CD99 typeⅡand CD99Ⅲ, the noval splice variant,may have association with the initiation and progression of ccRCC.