Transcriptional Regulation Analysis Of ROCK1 And ROCK2 Genes Of Pig

Due to the similarities between pig and human in anatomy, physiology, pathology, and genome, pig is increasingly used in medical research as an animal model to investigate human health problems, such as Parkinson’s Disease,coronary artery disease,obesity, and diabetes. Additionally, pork is one of the main sources of animal proteins for people all over the world, and the skeletal muscle is the most important tissue involved in the increase of body weight, indicating that it is necessary to research the genes associated with the development of muscle to better understand the underlying molecular mechanism.The ROCK family participates in various fundamental life activities. Meanwhile, our former results indicated that they were differentially expressed between Yorkshire and Meishan pigs at different development stages. Therefore, they were considered as condinate genes for further research. The main results are listed as follows:1. ROCK1 and ROCK2 expression profile in various tissues of 2 month old Yorkshire pig were detected via qRT-PCR.2. The 5’ progressive deletion plasmids of ROCK1 and ROCK2 promoter were constructed respectively, subsequently transfectd to PK and C2C12 cells. ROCK1(-744/-402 bp) and ROCK2(+37/+175bp) were determined as core promoter regions base on the relative luciferase activity.3. Site-directed mutagenesis of Sp1 binding sites in ROCK1(-744/-402 bp) can significantly reduce the relative luciferase activity, while overexpression of Sp1 can stimulate the relative luciferase activity of ROCK1-P5 in dose-dependent manner. EMSA, together with DNA pull down results showed the binding of Sp1 and ROCK1 promoter in vitro. The data of Ch IP assay indicate that Sp1, but not Sp3 directly bind with porcine ROCK1 promoter in vivo.4. Site-directed mutation of C/EBPα binding site in porcine ROCK2 core promoter and co-transfection of C/EBPα overexpression plasmid indicate the importance of C/EBPα binding sitelocated in ROCK2(+37/+175bp). EMSA assay confirmed the binding of C/EBPα and ROCK2 promoter in vitro.5. Overexpression and inhibiting results indicate that Sp1 can enhance the relative luciferase activity of ROCK1 promoter, and can promote the transcription and translation of ROCK1 gene. In addition, it can increase the expression of MYOD, myog, MyHC. The overexpression of C/EBPαshowed an similar ability towards the transcription and translation of ROCK2 gene.6. The luciferase activity of ROCK1-pmirGLO cotransfected with miR-33-5p, 376c-3p demonstrates each mi RNA can directly interact with 3’UTR of ROCK1 gene. They don’t significantly affect ROCK1 at transcription level, but significantly inhibit ROCK1 gene at the ptotein level.7. The expression of ROCK2 increased during the differentiation of C2C12 cells, while the expression of miR-142-3p decreased during the process.8. The luciferase activity of ROCK2-pmirGLO cotransfected with miR-142-3p demonstrates the mi RNA can directly interact with 3’UTR of ROCK2 gene. It significantly affects ROCK2 at both transcription and ptotein level.