The Dose and Time Dependent Effects of HGF on Myf-5, MyoD and miR-31 Expression in Quiescent Primary Human Myoblasts

Passerin d'Entreves, Niccolo (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OPSOMMING: Satellietselle is voorvader stamselle wat in ‘n onaktiewe toestand in volwasse spiere gevind word. Met besering, groei of hipertrofie, word satellietselle geaktiveer, waarna hulle vermenigvuldig en differensiëer na mioblaste. Hepatosiet groeifaktor (HGF) ontlok aktivering van onaktiewe satellietselle in vivo. Ook sal HGF binding aan sy reseptor, c-Met, transkripsie van belangrike miogeniese gene, naamlik Myf-5 en MyoD, bemiddel. Satellietselle toon egter verskillende reaksies teenoor HGF, wat tyd- en konsentrasie-afhanklik is, maar resultate in verskeie gepubliseerde studies toon dat satellietsel noodlot nie noodwendig konsekwent is nie. Aangesien eksperimentale kondisies egter nie gestandardiseer was voor supplementering nie, is inter-studie vergelykings gekompliseerd. MikroRNSe (miRs) speel ook ‘n aktiewe rol in handhawing van verskeie selsoorte as onaktief, maar dit is nog onbekend of HGF miR uitdrukking onaktiewe of onlangs geaktiveerde satellietselle beïnvloed. Die hoofdoel was om die tyd- en konsentrasie-afhanklike gevolge van HGF op gevestigde onaktiewe satellietselle te bestudeer deur die ontleding van die belangrikste miogeniese gene en moontlike gepaardgaande miR- uitdrukking. Primêre menslike mioblaste (PHM) afkomstig ban spierbiopsies is vir 10 dae gekweek in serum-vrye medium, aangevul met sintetiese groeifaktor-vrye serum (KOSR), om onaktiwiteit teweeg te bring. Multikleurvloeisitometrie en selsiklusanalise is uitgevoer om karakteristieke van PHM te ontleed m.b.t. ongedifferensiëerdheid en die toestand van onaktiwiteit. Supplementering van PHM met rh-HGF het onder 4 kondisies plaasgevind: twee verskillende konsentrasies en twee verskillende tydsperiodes. Die miogeniese regulerende faktore Myf-5 en MyoD is daarna ontleed met behulp van die immunoklad-tegniek, terwyl hul mRNS en miR-31 uitdrukking, met kwantitatiewe PKR ontleed is. Effek van 10 dae in serum-vrye kultuur: lewensvatbaarheid en ‘n ongedifferensiëerde morfologie is gehandhaaf in PHM, selsiklus progressie is terselfdertyd onderdruk. Vloeisitometriese analise het bewys dat 98% van PHM deurgaans positief was vir CD34 wat ongedifferensiëerdheid kwantitatief bevestig. Merkers van aktivering en vermeerdering, CD56 en Ki-67, het gedaal van 26% tot 2%, en van 85% tot 45% respektiewelik. Alhoewel Myf-5 uitdrukking konstant gebly het oor die tydperk (~98%), het MyoD uitdrukking gedaal van 20% tot 9%. Selsiklus: die persentasie G1-fase selle het toegeneem van 58% na 87%, terwyl die persentasie S-fase selle gedaal het van 42% tot 10%. Effek van rh-HGF toediening: HGF het gelei tot beduidende (p<0.0001) stygings in endogene HGF proteine wat beide tyd (24 uur en 48 uur) en konsentrasie (2 ng/mL of 10 ng/mL) afhanklik was. Reseptorvlakke van c-Met het aansienlik toegeneem (p<0.01) na 24 uur behandeling met hoë dosis HGF. Alhoewel MyoD mRNS twee-tot drievoudig verminder het onder alle HGF kondisies, het MyoD protein slegs ná 48 uur behandeling met die lae HGF dosis gedaal. Dalings in Myf-5 mRNS was matig (tot ~0.5-voudig) met lae en hoë HGF, maar Myf-5 protein het slegs met hoë dosis HGF toediening gedaal. Laastens het miR-31 uitdrukking afgeneem met HGF toediening, ofskoon hierdie bevinding in party HGF kondiesies egter onbeslis was. Hierdie tesis beam tyd- en konsentrasie-afhanklike uitwerkings van HGF op mioblaste. Meer spesifiek, diwerse uitwerkings op MyoD en Myf-5 is nie deur eersgaande sinkronisasie van die onaktiewe staat verwyder nie. HGF beinvloed miR-31, dus is ‘n nuwe meganisme vir effekte op mioblaste onthul.

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