Study On Acetylation Modification In Adipogenic Differentiation Of Mesenchymal Stem Cells C3H10T1/2

Objective: To investigate the effects and possible underying mechanism ofdeacetylase inhibitors suberoylanilide hydroxamic acid on the adipogenicdifferentiation of mesenchymal stem cells C3H10T1/2; analyse the changes ofintracellular protein acetylation levels and expression of11kinds deacetylase（Histone deacetylases, HDACs） during the process of adipogenic differentiation;analyse the role of HDAC11in the adipogenic differentiation of mesenchymal stemcell C3H10T1/2by RNA interference; To provide an experimental basis for furtherstudy of the molecular mechanism of acetylation modification on the adipogenicdifferentiation process of mesenchymal stem cells C3H10T1/2.Methods:1Establish an induction model of adipogenesis of mesenchymal stem cellsC3H10T1/2in vitro; verify the inhibition of deacetylase inhibitor SAHA on histonedeacetylase; and study impact of protein acetylation on C3H10T1/2cells proliferationand adipogenesis using SAHA.2Detected intracellular protein acetylation levels in the adipogenic differentiationprocess by Western blotting; Using Real time-PCR detected changes in the expressionof11HDACs in the process of adipogenic differentiation of C3H10T1/2cells;analyse the role of HDAC11in adipogenic differentiation of C3H10T1/2cells byRNA Interference and real time-PCR in order to explore the possible mechanisms ofacetylation modification involved in adipogenic differentiation regulation ofmesenchymal stem cell.Results:1The experiment successfully established the adipogenic differentiationmodel of C3H10T1/2cells in vitro; proved that SAHA can increase the intracellularprotein acetylation levels by inhibiting the effects of HDACs; C3H10T1/2cellstreated with SAHA not only changed their cell morphology to be flat, arrested theircell cycle at the G₀/G₁phase, also inhibited the adipogenic differentiation ofC3H10T1/2cells in a dose-dependent manner; SAHA also suppressed the expressionof key adipogenic transcription factor PPAR2γ，adipogenic markers Fabp4，perilipin and adipoq in a dose-dependent manner.2with the adipogenic differentiation of C3H10T1/2cells, the level of intracellularprotein acetylation was an up-regulated; the transcription levels of11kinds of HDACgenes showed great differences during the adipogenic differentiation of C3H10T1/2cells, the mRNA expression of HDAC3、 HDAC8and HDAC11increasedsignificantly, the mRNA expression of HDAC11had the most significant changeamong them; the mRNA expression of adipogenic differentiation key transcriptionfactors PPARγ2, adipogenic markers perilipin and adipoq decreased with the declineof HDAC11expression by RNA interference, but no significant change in Fabp4expression levels.Conclusion: Adipogenic differentiation process is accompanied by elevated levels ofintracellular protein acetylation, while the changes in the level of intracellular proteinacetylation can also affect the adipogenic differentiation;11kinds of HDACs havedifferent expression and roles during adipogenic differentiation, HDAC11maybe canaffect the adipogenic differentiation. These results provide an experimental basis forfurther study of the molecular mechanism of acetylation modification on theadipogenic differentiation process of mesenchymal stem cells.