Cloning of promoter of CA125 gene and tissue specific gene therapy for ovarian cancer

从EMBL SP 6/T7噬菌体文库中克隆CA 125基因5'端500 bp的基因组DNA，测序CA 125基因组DNA外显子1上游5 kb的序列，用SMART PCR cDNA Synthesis试剂盒确定转录起始位点。外显子1分为外显子1A和外显子1B，正常淋巴细胞的转录起始位点为外显子1A，而卵巢癌细胞的转录起始位点为外显子1B。在启动子区5 ′上游每隔250-500 bp设置一条正义引物。通过双荧光素酶测定试剂盒（Promega）测定荧光素酶测定。在转录起始位点上游500 bp处有明显的转录活性。在转录起始位点上游1825 bp处有增强子活性。用卵巢癌HEY细胞系、宫颈癌SKGIIIa细胞系和正常人角质形成细胞K42细胞系检测CA 125基因转录活性的组织特异性。CA 125基因在转录起始位点上游1825 bp处转录活性最高。CA 125基因在HEY细胞中的转录活性是SV 40基因的7倍，在SKGIIIa细胞中是SV 40基因的1.9倍，在K42细胞中是SV 40基因的1.8倍。这些结果表明CA 125基因在卵巢癌细胞中具有组织特异性活性。然后将CA 125基因插入腺病毒E1 A启动子区，制备条件复制型腺病毒。该载体在卵巢癌细胞中显示出高的组织特异性抗增殖活性，因为该载体的IC 50在HEY细胞中为0.01 MOI，在SKGIIIa细胞中为100 MOI。因此，利用CA 125基因启动子进行卵巢癌的基因治疗可能具有组织特异性，在卵巢癌基因治疗的临床试验中有希望。

Genomic DNA was cloned from EMBL SP6/T7 phage library using 500 bp of 5' end of CA125 gene and sequenced 5 kb upstream of exon 1 of CA125 genomic DNA.Transcriptional start site was determined by SMART PCR cDNA Synthesis kit. Exon 1 is devided into exon 1A and exon 1B.It is reported that transcriptional start site of normal lymphocite is exon 1A.However, this study demonstrated that transcriptional start site of ovarian cancer cell line was exon 1B.Deletion mutant was constructed by using PCR, in which antisense primer was contructed including transcriptional start site of exon 1B.Sense primer was setted in every 250-500 bp size 5' upstream of promoter region. Luciferase assay was determined by dual luciferase assay kit (Promega). Significant transcriptional activity was demonstrated in 500 bp upstream of transctiprional start site. Enhancer activity was shown in 1825 bp uptream of transctiprional start site. Ovarian carcinoma HEY cell line, cervical carcinoma SKGIIIa cell lilne and normal human keratinocyte K42 cell line were used to determine the tissue specificity in the transcriptional activity of CA125 gene. The highest transcriptional activity of CA125 gene was shown in 1825 bp upstream of transcriptional start site. The transcriptional activity of CA125 gene was 7 times that of SV40 gene in HEY cells, 1.9 times that of SV40 gene in SKGIIIa and 1.8 times that of SV40 gene in K42 cells. These results shows the tissue specific activity of CA125 gene in ovarian carcinoma cells. Then we inserted the 1875 bp of CA125 gene into E1A promoter region of adenovirus to make a conditioned replicative adenovirus. This vector showed the high tissue specific anti-proliferative activity in ovarican carcinoma cells, because IC50 of this vector was 0.01 MOI in HEY cells and 100 MOI in SKGIIIa cells. From these results, gene therapy by using CA125 gene promoter may be tissue specific for ovarian carcinoma and promissing in the clinical trial of gene therapy of ovarian cancer.