顆粒蛋白前體在肌肉發育中抑制微核糖核酸-206表現以促進肌肉前驅細胞增生

Progranulin down-regulates miR-206 to promote myogenic progenitor cells proliferation in muscle development

許淳淼(Chum-Miao Hsu)

指導教授 ： 吳金洌

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01347

摘要

顆粒蛋白前體為分泌性的生長因子，已知參與在早期胚胎發育、細胞生長、傷口癒合、腫瘤形成。本實驗室先前的研究結果指出，在肌肉大量表現顆粒蛋白前體的轉基因斑馬魚上，顆粒蛋白前體具有促進肌肉生長的能力，但顆粒蛋白前體促進肌肉生長的確切機制至今仍是未知的，先前的研究結果也指出，使用反義核苷酸抑制斑馬魚顆粒蛋白前體基因表現，造成斑馬魚表現Pax7蛋白質的肌肉前驅細胞數量減少以及肌肉發育受損。然而減少的肌肉前驅細胞可以藉由恢復測試，額外注射MET (HGF受體)信息核糖核酸達到恢復的效果。根據這些結果，顆粒蛋白前體、MET以及肌肉前驅細胞之間的關係，可能在肌肉生長上扮演者關鍵的角色。另一方面，在一些研究中，肌肉專一性表現之微核糖核酸已被證實參與在肌肉生成及肌肉生長，MiR-206為肌肉專一性表現之微核糖核酸，具有負調控MET、Pax7表現以及抑制肌肉前驅細胞增生的能力，在初步的結果中，顆粒蛋白前體可能藉由MiR-206來調節MET、Pax7表現並促進肌肉前驅細胞增生。在本研究中，於C2C12肌肉芽母細胞大量表現顆粒蛋白前體，結果顯示顆粒蛋白前體促進了細胞的增生，且在基因表現層面，MET及Pax7的表現量上升，但miR-206的表現量卻下降。相反的，抑制C2C12肌肉芽母細胞的顆粒蛋白前體表現，MET及Pax7的表現量下降，而miR-206的表現量卻上升。進一步的結果指出，抑制顆粒蛋白前體表現所造成的MET及Pax7表現量降低，可以藉由抑制miR-206的表現以達到恢復的效果。而在斑馬魚系統中，轉基因魚(Tg(mlc2:grnA))的MiR-206表現受到抑制，尤其在斑馬魚受精後24及48小時，且受精後24小時，轉基因魚的肌肉前驅細胞數約為野生型斑馬魚的兩倍，且上升的細胞數是由細胞增生所造成的。在生長方面，3.5月時，轉基因魚的體中約為野生型斑馬魚的1.5倍。綜合以上結果，顆粒蛋白前體藉由調控miR-206的表現在肌肉生長扮演著重要的角色。未來將運用這些結果於更進一步的肌肉生長及水產養殖研究。

關鍵字

顆粒蛋白前體 ； 肌肉發育 ； 斑馬魚 ； 肌肉前驅細胞 ； MiR-206

並列摘要

Progranulin (PGRN) is a secreted growth factor that contribute to early embryogenesis, wound healing response and tumorigenesis. In our previous research, the transgenic zebrafish Tg(mlc2:grnA) overexpressing zebrafish progranulinA (GrnA) in musce showed that PGRN promotes muscle growth. But the mechanism how PGRN promotes muscle growth is still elusive. Previous research also showed that knockdown of GrnA resulted in impaired muscle growth and reduced quantity of Pax7+ myogenic progenitor cells (MPCs). Yet the reduced quantity of MPCs could be restored by ectopic expression of MET, the receptor for HGF. Hence, the relationship between PGRN, MET and Pax7+ MPCs may play a critical role in promoting muscle growth. On the other hand, some studies have identified that muscle specific microRNA (miRNA) participate in myogenesis and muscle growth. MiR-206 is one of muscle specific miRNA, that targets MET, Pax7 and inhibit MPCs proliferation. Additionally, premiere results revealed that miR-206 might be a downstream factor of PGRN. Here, we hypothesized that PGRN might down-regulate miR-206 to modulate MET, Pax7 expression and promote MPCs proliferation. Then the proliferation of MPCs provides more cells differentiating into myotube and promotes muscle growth. In this study, we demonstrated that overexpressing PGRN in C2C12 cells promotes cells proliferation. And the downstream factor, MET and Pax7 were up-regulated, but miR-206 was down-regulated. In contrast, knockdown of PGRN in C2C12 cells showed converse results in MET, Pax7 and miR-206 expression. And the reduced expression of MET and Pax7 caused by knocking down of PGRN can be rescued by inhibiting miR-206 expression. Moreover, in the zebrafish system, miR-206 was down-regulated in Tg(mlc2:grnA) at 24 and 48 hpf. Tg(mlc2:grnA) also showed increased MPCs at 24 hpf. And these increased MPCs resulted from enhanced proliferation of MPCs. As expected, the nucleus number in the somite was increased in Tg(mlc2:grnA) at 6 dpf. Additionally, Tg(mlc2:grnA) showed higher body weight compare to WT zebrafish. Based on these finding, PGRN/miR-206 axis plays an important role in muscle growth. And in the future, we will apply these findings to further muscle growth and aquaculture research.

並列關鍵字

PGRN ； Muscle development ； Zebrafish ； MPCs ； MiR-206

參考文獻

2. He, Z. & Bateman, A. Progranulin (granulin-epithelin precursor, PC-cell-derived growth factor, acrogranin) mediates tissue repair and tumorigenesis. J. Mol. Med. 81, 600–12 (2003).

3. He, Z., Ong, C. H., Halper, J. & Bateman, A. Progranulin is a mediator of the wound response. Nat. Med. 9, 225–9 (2003).

4. Bateman, A. & Bennett, H. P. The granulin gene family: from cancer to dementia. BioEssays. 31, 1245–54 (2009).

6. Chitramuthu, B. P., Baranowski, D. C., Kay, D. G., Bateman, A. & Bennett, H. P. Progranulin modulates zebrafish motoneuron development in vivo and rescues truncation defects associated with knockdown of Survival motor neuron 1. Neurodegeneration. 5, 41 (2010).

7. Daniel, R., Daniels, E., He, Z. & Bateman, A. Progranulin (acrogranin/PC cell-derived growth factor/granulin-epithelin precursor) is expressed in the placenta, epidermis, microvasculature, and brain during murine development. Dev. Dyn. 227, 593–9 (2003).