Browsing by Author "Passerin d'Entreves, Niccolo"
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- ItemThe Dose and Time Dependent Effects of HGF on Myf-5, MyoD and miR-31 Expression in Quiescent Primary Human Myoblasts(Stellenbosch : Stellenbosch University, 2017-03) Passerin d'Entreves, Niccolo; Myburgh, Kathryn H.; Van de Vyver, Mari; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Satellite cells are progenitor cells that persist in adult muscle in a quiescent state. Upon injury, growth or hypertrophy, satellite cells become activated, proliferate and differentiate into myoblasts. Hepatocyte growth factor (HGF) induces activation of quiescent satellite cells in vivo. Also, HGF binding to its receptor, c-Met, leads eventually to transcription of key myogenic genes namely Myf-5 and MyoD. Satellite cells’ responses to HGF are varied and time and concentration dependent effects on satellite cell fate have been reported, although published results are inconsistent. Previous studies have not standardised initial conditions prior to interventions, thus complicating comparison and conclusions. Finally, recent evidence implicates microRNAs (miRs) as active players maintaining quiescence in various cells, but HGF effects on miRNA expression are unknown in quiescent or activated satellite cells. The overarching aim was to determine the time and concentration dependent effects of HGF on quiescent satellite cells by analysing key myogenic gene and miRNA expression after first standardising myoblast characteristics. Primary human myoblasts (PHM) explanted from human muscle biopsies were cultured in a serum free media, supplemented with synthetic growth factor free serum (KOSR), for 10 days to induce quiescence. Multicolour flow cytometry and cell cycle analyses were performed to characterise the PHMs maintained in serum free culture with regard to undifferentiated and quiescent status. Intervention with rh-HGF included 4 conditions: 2 different concentrations and 2 different durations. The myogenic regulatory factors, Myf-5 and MyoD were analysed using Western blotting, while their mRNA expression, along with miR-31 expression, were analysed using qPCR. Effects of 10 d culture in quiescence media: PHMs maintained viability and undifferentiated morphology. Flow cytometry analyses of satellite cell markers indicated that 98% of PHMs remained positive for CD34 confirming their undifferentiated state. Activation and proliferation markers CD56 and Ki-67, decreased from 26% to 2% and from 85% to 46% respectively. While Myf-5 expression remained constant (~98%) for this period, MyoD expression dropped from 20% to 9%. Cell cycle progression was reduced: The percentage of G1-phase cells increased from 58% to 87%, while Sphase cells dropped from 42% to 10%. Effects of treating quiescent PHMs with rh-HGF: Treatment lead to significant (p<0.0001) time (24 h and 48 h) and concentration (2 ng/mL and 10 ng/mL) dependent increases in endogenous HGF protein. c-Met receptor content increased significantly (p<0.01) only with exposure to high dose HGF (10 ng/mL) within 24 h. Although MyoD mRNA expression decreased (~2 to 3-fold) with all HGF conditions, MyoD protein decreased only after 48 h in low HGF (2 ng/mL) compared to other treatment groups and untreated control. Myf-5 mRNA expression decreased moderately (up to ~0.5-fold) in all HGF conditions, but Myf-5 protein decreased only with high dose HGF (slightly). Finally, miR-31 expression decreased upon HGF treatment, although not consistently for all HGF conditions. This thesis confirmed time and concentration dependent effects of HGF on myoblasts. More specifically, diverse effects on MyoD and Myf-5 were not abolished here when satellite cells were first rendered quiescent. HGF affected miR-31, thus elucidating a new mechanism for influencing myoblasts.