Expression and promoter-alternation of osteoblastic insulin-like growth factors

以人软骨肉瘤软骨细胞系HCS-2/8为研究对象，建立了应用核糖核酸酶保护试验(RPA)检测胰岛素样生长因子基因表达和启动子变化的定量分析方法。IGF-2的启动子2P-1、2P-3和2P-4以及IGF-1的启动子1P-1和1P-2构建了RPA实验的探针，但由于HcS-2/8中转录产物很少，IGF-2的启动子2P-2未能构建。现在，我们继续利用它们的探针来确定RPA方法的检测限，并将在2P-2转录的RT-PCR产物积累后重建探针。在目前的发展研究中，我们发现：1.在HCS-2/8和人正常软骨细胞中，IGF-2的4个启动子同时被诱导表达；2.在HCS-2/8中，IGF-2的启动子2P-4转录产物中的4个碱基对在外显子6的3‘端缺失，位于翻译起始点9~6碱基的上游。3.与Hcs-2/8和人正常软骨细胞相比，胰岛素样生长因子-2和肿瘤抑制基因H19的表达呈平行相关。4.在Hcs-2/8中，IGF-2基因的限制性片段长度多态性表明，只有父亲的等位基因(S)在基因组水平上存在，而母亲的印记等位基因在软骨肉瘤中缺失。这可能与肿瘤的形成有关。

Using a chondrocytic cell line HCS-2/8 derived from human chondrosarcoma, the quantitative analytical method adapting the RNase protection Assay (RPA) was developped in order to examine the expression and promoter-alternation of insulin-like growth factor genes. The probe of the RPA experiments was constructed for the promoters 2P-1,2P-3 and 2P-4 of IGF-2 and the promoters 1P-1 and 1P-2 of IGF-1, but the promoter 2P-2 of IGF-2 failed to be constructed because the transcriptional product was very little in HCS-2/8. Now, we continue to investigate the determination of detectable limitation of the RPA method using their probes, and we will retry to construct the probe asfter the accumulation of RT-PCR product for 2P-2 transcript.During the present developmental investigations, we found some new facts as following :1.The simultaneous expression of four promoters of IGF-2 were induced in HCS-2/8 and human normal chondrocytes.2.A defect of four base pair in the promoter 2P-4 transcript of IGF-2 in HCS-2/8 was observed at the 3'-end of exon 6, at which the position is upstream of 9 to 6 base from the translation-starting point. This defect may be due to the alternative splicing specific to the chondrosarcoma.3.Comparing with HCS-2/8 and human normal chondrocytes, we found a parallel correlation between IGF-2 and H19 (tumor reppressor gene) expressions.4.The restriction fragment length polymorphism of IGF-2 gene in HCS-2/8 suggested that only the paternal allele (s) existed at the genomic level, but the maternal imprinting allele was absent in the chondrosarcoma. This perhaps correlate with the tumor formation.