The Investigation Of IGF-1 Anti-apoptosis Mechanism In Rat Cardiac Muscle Cells

【Aim】Age-related changes of the heart often cause a progressive decline in cardiac function that ultimately can lead to congestive heart failure, apoptosis is a key contributor to myocyte loss. IGF-1(insulin like growth factor 1, IGF-1) can prevent apoptosis and prolong cell survival, it is an attractive candidate for heart protection in clinical, but exactly how IGF-I modulates myocardial function remained obscure. The main purpose of this study is to identify a new IGF-1 protection cardiac muscle cells mechanism.【Methods】The high throughput gene expression profiling was observed by microarray. Differential expression of genes was calculated with three programs: Affymetrix, Genespring and Cyber T. The Microarry results were performed computational analysis on publicly available Gene expression Omnibus (GEO) datasets, and a new never reported apoptosis association gene was found.We investigated the regulation pathway from IGF-1 to BTEB and CYP1A1 by RT-PCR and Western-blot.Using BTEB and CYP1A1 specific siRNA, we down-regulated this two gene expression and observed apoptosis of rat cardiac muscle cell line H9C2 under serum free media by DNA ladder, Caspase 3 activity. The survival rate of cells was observed by typan blue exclusion. 【Results】We totally found 31 genes down-regulation by IGF-1 and got a new never reported apoptosis association gene-BTEB (Basic Transcription Element Binding Protein, BTEB). BTEB is a transcription factor and can regulate one of cytochrome c superfamily member CYP1A1.We investigated the regulation pathway from IGF-1 to BTEB and CYP1A1, and found IGF-1 can regulated CYP1A1 gene expression through BTEB.Using BTEB and CYP1A1 specific siRNA, we down-regulated this two gene expression and observed apoptosis of rat cardiac muscle cell line H9C2 under serum free media. Both of BTEB and CYP1A1 specific siRNA can protect H9C2 from apoptosis just like IGF-1.【Conclusion】Taken together, we found a new mechanism of IGF-1 protection cardiac muscle cell from apoptosis. CYP1A1 plays an important role in mitochondria apoptosis progress, our results set up a new connection between IGF-1 and mitochondria apoptosis pathway. This is not only enriching our knowledge for IGF-1 protection mechanism but also shed a new beacon light on IGF-1 clinical application.【Innovations】The innovations in this thesis are mainly showed as follows:1. Using DNA microarray we observed the gene expression profiling IGF-1 on rat cardiac muscle cells, we found a new IGF-1 protection cardiac muscle cells from apoptosis mechanism BTEB-CYP1A1. It is known that CYP1A1 play a very important role in mitochondria associated apoptosis, so our results make a connection between IGF-1 and mitochondria on apoptosis mechanism. Our founding not only enrich our reorganization of IGF-1 anti-apoptosis mechanism but also shed a beacon light on IGF-1 finally clinical application.2. To decrease the risk of obtaining false-positive data, 5 repetitive chips were usage in one group. Because of the high costs of microarray study, most investigators selected to limit the number of repetitive array experiments. It is possible that a limited number of repetitions may lead to false-positive results. Our data were obtained from 5 repetitive experiments that increased the specificity of DNA microarray study and excluded minimally expressed genes.3. The selection of different analysis program will obtain different DNA microarray results. For most clinical investigators, they don't have the background knowledge for statistic. How to select suitable statistic software to estimate DNA microarray data is a major blockage in the field. We analyzed our microarray data by three independent softwares: Affymetrix, Genespring and Cyber T, our results showed that the last two softwares could acquire more accurate gene expression profiling. It would provide a clue for others clinical investigators.