肌肉生成轉錄因子MRFs基因之表現及功能在正常及病態生理中的調控

All trunk and limb skeletal muscle (SKM) cells in vertebrates are derived from myogenic stem cells (MSC) residing in the paraxial mesoderm tissue called somites during embryogenesis. In response to local myogenic signals, such as FGFs and Wnts, MSC express the stem cell markers Pax3 and Pax7 and later the expression of Myf5 or MyoD to drive the MSC Px3+/Pax7+ to become myoblasts. Upon the stimulation of differentiation signals, myoblasts (Myf5+ or MyoD+) start expressing Myogenin and Mef2c that cooperatively drive the terminal myogenic differentiation process. Later stage of muscle development, including alignment and maturation of myotubes, is regulated by Mrf4, which is also the dominant myogenic regulatory factor (MRF) in postnatal muscles. The expression of MyoD, Myf5, Myogenin, and Mrf4 is highly SKM-specific and they are collectively called as myogenic regulatory factors (MRFs). MRFs recognize a CANNTG cis-element with their basic helix-loop-helix (bHLH) DNA-binding domain, and they control the expression of most, if not all, SKM-specific genes. Besides, they are also important regulators of SKM metabolism and regeneration in adulthood. Cancer is the 2nd leading cause of death globally and the metastasis of cancer cells invading various vital organs is the major cause of cancer–related death. Besides, cancer cells can release soluble factors to affect not invaded organs, such as skeletal and heart muscles, and they induce serious fat and muscle wasting, resulting in a syndrome called cachexia that are associated with about 80% of cancer patients at advanced stages. These cancer cells derived soluble factors thus act as strong cachectic signals to SKM. It is estimated that about 20-30% cancer death is directly linked to cachexia. Currently, there is no effective treatment for alleviating this syndrome. As MRFs play critical role in SKM regeneration and metabolism, we would like to analyze the crosstalk between the SKM-promoting myogenic signals and SKM-reducing cachectic signals on their expression and functions. The myogenic signals that will be tested include RA, T3/4, Wnt3a, Shh, FGF8, and bFGF as they have been involved in MRFs expression. Cachectic signals will be provided in the form of C26 colon cancer (C26) or Lewis lung cancer cells (LLC) conditioned medium. The effects of myogenic and cachectic factors will be tested on cultured myoblasts to pinpoint down the cis-elements and transcription factors that mediate the activating or repressing effects on MRF genes. The roles of these mediators will be further verified in the syngeneic mice carrying C26 or LLC tumor. As MRFs play critical roles in myogenesis, elucidation of their regulatory mechanisms by myogenic and cachectic factors will certainly pave the way to the developing of drugs antagonizing SKM cachexia induced by cancer or other chronic diseases.Therefore, the specific aims of this study can be summarized below:1.Examining the effects of myogenic and cachectic signals on MRFs expressionand functions.2.Identifying MRFs cis-elements mediating the effect of myogenic and cachecticsignals.3.Examining the crosstalk between myogenic and cachectic signals on MRFs ciselements.4.Rescuing the expression and functions of MRFs in cachectic SKM