- 學位論文
SIRT1透過MURF3調節骨骼肌細胞分化
SIRT1 regulates differentiation of myoblasts through modulating MURF3 expression
摘要
肌肉老化的特徵是肌肉生成能力降低，進而導致肌肉質量下降。Sirtuin 1 (SIRT1) 屬於第三類組蛋白去乙醯酶，具有抑制細胞凋亡以及減緩細胞老化的作用。先前的研究指出白藜蘆醇活化SIRT1 有助於肌原母細胞的分化並減緩老化引起的肌肉疾病「肌少症」。因此，本篇研究的目的為探討SIRT1在骨骼肌的分化中所扮演的角色。我們利用小鼠肌原母細胞株C2C12進行實驗，發現以過氧化氫誘導細胞老化，細胞內的SIRT1表現量下降，並且伴隨著肌原母細胞的分化能力下降以及肌管 (Myotube) 形成減少。為了進一步釐清SIRT1與肌肉分化的相關訊息調控機轉，我們透過生物訊息分析軟體 (Ingenuity Pathway Analysis) 推測SIRT1可能透過MURF3影響肌肉的分化。然而，在MURF3的胺基酸序列中，我們未找到可能的去乙醯化位置，故推測SIRT1是間接透過轉錄因子RAR調控MURF3的基因表現。由染色質免疫沉澱實驗顯示，SIRT1藉由轉錄因子RARA結合於MURF3的啟動子而調控其基因的轉錄。綜合以上結果，本研究證實，在肌肉老化的過程中，SIRT1透過RARA/MURF3的訊息傳遞路徑影響肌肉細胞的分化能力。
並列摘要
Muscle senescence is characterized by decreasing of myogenic ability which may result in muscle mass loss. Sirtuin 1 (SIRT1), known as a histone deacetylase, has been reported to implicate in anti-apoptotic and anti-aging effects. In addition, SIRT1 activator (resveratrol) had been reported to promote myoblast differentiation and attenuate age-induced sacropenia. Accordingly, this study aimed to examine the role of SIRT1 in skeletal myoblasts differentiation. Our results demonstrated that SIRT1 levels were decreased in H2O2-induced senescent myoblast. Moreover, down-regulation of SIRT1 reduced myogenic capability and impaired myotube formation. To clarify this mechanism of SIRT1 and muscular differentiation, Ingenuity Pathway Analysis (IPA) was applied to predict muscle-specific RING finger protein 3 (MURF3) as a downstream molecule of SIRT1, however, none of potential acetylation site was identified in MURF3 peptide. Therefore, we hypothesized that SIRT1 might indirectly regulate MURF3 by interacting with a nuclear receptor RAR. MURF3 is an E3 ubiquitin ligase involved in the function of ubiquitin-proteasome system and is essential for microtubule maintenance and myoblast differentiation. Knockdown of endogenous SIRT1 reduced the expression and function of MURF3. By using chromatin immunoprecipitation assay, we further demonstrated that RARA was associated with MURF3 and regulated by SIRT1 activator. Taken together, our results suggest that SIRT1/RARA/MURF3 pathway plays an important role in myoblast differentiation during muscle senescence.