Effect Of STAT3Proteins In IGF-i Mediated Proliferation Process Of L6Myoblasts

Objective:To investigate suitable concentration of IGF-Ⅰ contributed to the L6myoblasts proliferation in vitro, validate the effect of STAT3proteins (signal transducers and activators of transcription3) in IGF-Ⅰ mediated myoblasts proliferation.Methods:Applied10ng/ml,50ng/ml,100ng/ml,200ng/ml,300ng/ml IGF-Ⅰ to L6myoblasts for respectively0h,12h,24h,48h, with2%FBS as control group. The effect of IGF-Ⅰ on myoblast proliferation were measured by MTT assay. Applied100ng/ml IGF-Ⅰ to act on L6myoblast for Oh,12h,24h,48h, extracted the RNA and protein of each experimental group, test the genetic transcription level of STAT3,protein expression level of STAT3and p-STAT3with RT-PCR technique and Western Blotting immune imprinting technology; Applied JAK-STAT3pathway inhibitor AG490to process myoblasts in advance, and then applied100ng/ml IGF-Ⅰ to L6myoblasts for24h, tested the genetic transcription level of STAT3,protein expression level of STAT3and p-STAT3with RT-PCR technique and Western Blotting immune imprinting technology.Results:Myoblast proliferation ability got enhanced statistically differently with the increase of IGF-Ⅰ concentration (P<0.05), when the concentration of IGF-Ⅰ was less than100ng/ml,. When the concentration of IGF-Ⅰ rised up to100ng/ml or above, cell proliferation ability also showed some increase but without any statistical difference along with the increase of IGF-Ⅰ concentration (P>0.05); In0-24hour period, cell proliferation ability increased gradually, peaking at the24hour time point, and differences between time-point of groups showed significantly statistically important (P<0.05). Cell proliferation ability slowed down but still at relatively high level; also, differences between time-point of groups showed significantly statistically important (P<0.05).The expression of STAT3protein of the experimental groups induced by100ng/ml IGF-Ⅰ was higher than of the control group either in the genetic transcription level or in the protein expression level, and it showed significantly statistically different between test and control groups. However, no obvious difference was observed of total STAT3protein expression level between experimental groups at each time point. In0-24hour time period, p-STAT3protein expression level kept increasing, which peaked at the24-hour time point, while there was a decrease of p-STAT3expression level noticed after the24-hour time point. It has been figured out that there were significantly statistical differences of p-STAT expression between every two-time points. Cells of AG490treatment group showed obviously less proliferation activity than cells of untreated group, where it showed significantly statistical difference (P<0.05).No significant difference has been found of total STAT3protein expression levels between the AG490pretreatment group and untreated group. However, the expression level of p-STAT3in AG490pretreatment group was much lower than in the group without AG490processing; what is more, significantly statistical difference has been calculated between the expression levels of two groups.Conclution:IGF-Ⅰ can promote the proliferation of myoblasts to some extent. STAT3protein is activated in IGF-Ⅰ mediated myoblast proliferation, the cell proliferation ability was significantly reduced when STAT3protein phosphorylation was partly blocked, we can infer that the STAT3proteins play an important role in IGF-Ⅰ mediated myoblast proliferation.