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

成骨细胞胰岛素样生长因子的表达和启动子改变

基本信息

批准号：
06671854
负责人：
TAKAHASHI Kojiro
金额：
$ 1.41万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06671854/
关键词：
Insulin-like growth factor II Insulin-like growth factor I Osteoblastic cell Chondrosarcoma Chondrocytes Transcriptional promoter RT-PCR RNase Protection Assay Insulin-like growth factor-II Insulim-like growth factor-I 軟骨様細胞転写調節 I

项目摘要

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.

以人软骨肉瘤细胞系HCS-2/8为材料，建立了一种定量分析胰岛素样生长因子基因表达和启动子改变的RNA酶保护试验（RPA）方法。针对IGF-2的启动子2 P-1、2 P-3和2 P-4以及IGF-1的启动子1 P-1和1 P-2构建了RPA实验的探针，但IGF-2的启动子2 P-2由于在HCS-2/8中转录产物很少而未能构建。目前，我们继续研究使用它们的探针确定RPA方法的检测限，并将在2 P-2转录本的RT-PCR产物积累后尝试构建探针。1.在HCS-2/8和人正常软骨细胞中同时诱导了IGF-2的4个启动子的表达; 2.在HCS-2/8中观察到IGF-2的启动子2 P-4转录本在外显子6的3 '端存在4个碱基对的缺陷，在该位置处，该位置是从分离起始点开始的9至6个碱基的上游。通过与HCS-2/8和正常人软骨细胞的比较，发现IGF-2和H19之间存在平行相关性4. IGF-2基因在HCS-2/8中的限制性片段长度多态性表明，在基因组水平上仅存在父本等位基因，但软骨肉瘤中不存在母亲印记等位基因。这可能与肿瘤的形成有关。