Suppression of WWP1 Gene Via RNAi Induced the Reduction of Proliferation Rate of C2C12 Myoblasts

Abstract

The WW domain containing E3 ubiquitin protein ligase 1 (WWP1), which plays an important role in ubiquitin-proteasome pathway to degrade unneeded or damaged proteins, was recently identified as the responsible for chicken muscular dystrophy. Despite intensive studies on oncogenic characters, the role of WWP1 to muscular diseases has not yet been fully understood. Previously, we transfected either of wild and mutated types of WWP1 gene into C2C12 mouse myoblasts to monitor the expression pattern of muscle-differentiation markers, so that excessive WWP1 expression enhanced the expression of the myosin heavy chain (MyHC) Ia gene but lowered the expression of the MyHC IIb gene, while mutated WWP1 gene transfected into myoblasts was distinct from these cases in that the MyHC gene or genes expression inhibited the normal myoblast differentiation. However, the mechanism for the mutation to inhibit muscle differentiation remains elucidated. The current study attempted to suppress the WWP1 expression by RNAi technique and to observe its effect on C2C12 cells. The effect of WWP1 suppression was clearly different from that of the R441Q missense mutation in the WWP1 gene. The WWP1 suppression reduced the proliferation rate of C2C12 myoblasts, while clear difference was not observed in the proliferation rate by the transfection of the mutated WWP1 gene into the cells. Our RT-PCR analysis indicated that the reduction of the WWP1 expression is the specific effect mediated by RNAi and that the reduction of proliferation rate observed in this study is largely attributed to the suppression of the WWP1 expression. These data indicated that the mutation responsible for chicken muscular dystrophy does not eliminate the enzymatic activity and provides some new function for the gene.

Keywords

• c2c12
• chicken
• muscular dystrophy
• rnai
• wwp1