Further analysis reveals that MuRF-1 dampens proliferation of skeletal muscle cells through inhibiting autophagy, and provides a potential approach for atrophy therapy

Further analysis reveals that MuRF-1 dampens proliferation of skeletal muscle cells through inhibiting autophagy, and provides a potential approach for atrophy therapy. Autophagy has been shown to be involved in multiple biological events of both muscle cells and muscle stem cells [13,16]. up with a new mechanism of MuRF for muscle atrophy, which potentially invoking a novel therapeutic approach for the disease. value less than 0.05 was considered as statistically significant. Results Association between MuRF expression and autophagy In order DM1-Sme to understand the possible association between MuRF-1 expression and autophagy, we prepared total RNA and protein lysates of skeletal muscles from muscle atrophy patients or healthy controls. We performed real time PCR and western blot for MuRF, and western blot for LC3, a commonly used marker for autophagy, and Atg5, a key regulator for autophagy. Our results showed that MuRF was downregulated in atrophy muscles, both in mRNA and protein level (Physique 1A and ?and1B),1B), while autophagy was inhibited in atrophy muscles based on LC3 (Physique 1B). Further analysis revealed a strong association between MuRF expression (protein) and autophagy (based on LC3 II) (Physique 1C). Thus, our data shows a potential association between MuRF-1 and autophagy in muscle. Open in a separate windows Physique 1 MuRF expression and autophagy in atrophy muscle. Muscle from patients or health control were homogenized, followed by preparation of total RNA with Trizol, or preparation of protein lysates with RIPA. (A) Total RNA was reverse-transcribed and then real time PCR was performed using specific primer pairs. Data are DM1-Sme mean SEM of three impartial experiments. Protein lysates were analyzed by immunoblotting with specific antibodies for the expression of MuRF, LC3. Densitometry quantitation of MuRF bands over -actin or LC3 II over -actin using ImageJ (B). DM1-Sme (C) Correlation analysis between expression of MuRF and LC3 II. Rabbit Polyclonal to RBM34 Data are mean SEM of three impartial experiments and analyzed with Students test, * 0.05 was considered as statistically significant. Overexpression of MuRF inhibits autophagy To explore whether MuRF regulates autophagy in muscle cells, we decided to overexpress MuRF in myoblast cell C2C12, which display a low or no expression of MuRF (Physique 2A). We constructed a MuRF-expressing plasmid and transfected C2C12 cells, which considerably upregulated the expression of MuRF in C2C12 cells (Physique 2A). Starvation strongly induces autophagy in C2C12 cells, while overexpression of MuRF obviates the induction of autophagy by starvation, roughly the same as treatment with autophagy inhibitor, 3-MA (Physique 2A). As autophagy regulates cellular proliferation, which plays essential role in muscular atrophy, so we would like detect whether overexpression of MuRF affects C2C12 proliferation. As shown in Physique 2B, autophagy inhibition with 3-MA inhibits C2C12 proliferation, and overexpression of MuRF showed a similar effect, strongly suggesting essential role of autophagy in MuRF-mediated proliferation. So our results support the idea that MuRF restrains proliferation of skeletal muscle cells through inhibiting autophagy. Open in a separate window Physique 2 MuRF overexpression inhibits autophagy in C2C12 cells. A. C2C12 cells were transiently transfected with MuRF plasmid. 48 h later, the cells were subjected to starvation for 4 hours and lysed, followed by SDS-PAGE and transfer to nitrocellulose membrane. After blotting with LC3 antibody, the membrane was incubated with HRP-conjugated secondary antibody, and visualized with an ECL chemiluminescence kit. B. C2C12 cells with or without MuRF overexpression were plated in 24-well plates, and MTT assay was used to determine the proliferation DM1-Sme of the cells. Data are mean SEM of three impartial experiments and analyzed with Students test, * 0.05 was considered as statistically significant. Knockdown of MuRF promote autophagy To confirm the effect of MuRF on autophagy, we further knocked down MuRF with siRNA specifically targeting on MuRF in skeletal muscle cell line C2C12 overexpressed MuRF, and our western blot results confirmed the efficiency of the knockdown on MuRF (Physique 3A). Next we induced autophagy by starvation in C2C12 cells with MuRF knockdown; we observed knockdown of MuRF reversed autophagy in C2C12 cells (Physique 3A). Similarly, knockdown of MuRF also restored the proliferation of C2C12 cells (Physique 3B). Collectively, our study confirmed that MuRF regulates skeletal muscle cells proliferation via dampening autophagy. Open in a separate window Physique 3 MuRF knockdown reverses autophagy in C2C12 cells. A. C2C12 cells overexpressed MuRF were transiently transfected with MuRF specific siRNA. 48 h later, the cells were subjected to starvation for 4 hours and lysed, followed by SDS-PAGE and transfer to nitrocellulose membrane. After blotting with MuRF antibody,.