Studies On Molecular Biological Function Of Porcine STMN1Gene In The Muscle Growth And Development

Stathmin (STMN1) is a microtubule-destabilizing protein that is able to regulate its own activity to change the dynamic balance of the microtubule system depending on its phosphorylation level and then be involved in regulating cell proliferation and differentiation. In many reports, STMN1was highly expressed in rapid proliferation and differentiation cells, such as tumor cell and porcine muscle cells in early embryonic stage. However, it was expressed little in normal tissue cells. In this research, the STMN1gene structure was predicted. The present study aimed to elucidate the effect of STMN1on C2C12cell proliferation and differentiation by infecting C2C12cells with eukaryotic expression plasmid encoding STMN1gene and small molecule siRNA-STMN1. The results of this study were shown as follows:1. From bioinformatics analysis results, the porcine STMN1gene promoter region was located at+817bp to+1066bp. And some transcription factor binding sites of MyoD, GATA-1and HSF2genes, and a CpG island about1000bp were found nearby the transcriptional start site. The STMN1protein was highly conservative in different species by multiple sequence alignment.2. The promoter regions and3’ UTR of STMN1gene in Meishan pig, TC pig, Qingping pig, Tunchang pig, Duroc, Yorkshire were amplified, and found SNPs after sequencing. The result suggested that the consistency of STMN1gene sequences in the promoter region in different pig varieties was very high. There were T/C, A/G, A/G, A/G, C/T, C/T, C/G, C/T, C/T transition mutations in319bp,853bp,993bp,1161bp,1593bp,1859bp,1942bp,2154bp,2400bp of the promoter region and also14transition mutations in3’UTR region. These mutations were defined as potential SNPs.3. The eukaryotic expression plasmid pcDNA3.1-STMN1was successfully constructed, also the small molecule siRNA-STMN1was designed and synthesized, and then were respectively transfected into C2C12cells to raise/lower STMN1gene expression in C2C12cells. 4. After the STMN1gene expression was raised/lowered, the fluorescent quantitative PCR method was used to detect the expression of the genes associated with muscle development in C2C12cells, such as MyoG, Myf5, MyHC, MyoD. The results said that the expression of the muscle development related genes in C2C12cells was up-regulated while the STMN1gene expression was raised, and the expression was down-regulated while the STMN1gene expression was lowered. The gene expression level was analyzed by statistical software SPSS.And it was found that the effect of changing STMN1gene expression for the expression of the muscle development related genes in C2C12cells was not significant (P>0.05). It demonstrated that STMN1gene was involved in the muscle differentiation, and further studies are needed to determine whether STMN1gene plays an important role in the muscle differentiation.5. The Roche ARCE instrument was implemented for the real-time detection of C2C12cells after raised/lowered the STMN1gene expression. The measured data was analyzed by statistical software SPSS, and then found that the STMN1gene was significantly affected the proliferation of C2C12cells (P<0.01). At the same time the method of Western blot was used to detect the expression of the cell proliferation mark gene Ki67. The results were in accordance with the results of the Roche ARCE method. It was concluded that STMN1gene played an important role in cell proliferation.