ANKRD Gene Family Function In The Skeletal Muscle Fiber Type Conversion

The mammalian skeletal fiber type conversions are very important for the meat quality, and are correlated with muscle proliferation, development, and differentiation and remolding. There are many researches focused on this issue, however, the mechanism of some key genes or regulators that directly and essentially regulate the muscle fiber conversions remain unclear. Many researches on skeletal muscle would hence be facilitated by decoding those key genes or regulators.This study was focused on the cardiac and skeletal muscle specially expressed ANKRD gene family. It is postulated that both the ANKRD 1 and ANKRD2 could modulate muscle fiber type conversions as they are not only correlated with cell proliferation, development, and differentiation, but they differentially express in different muscle types. Utilizing the Lentiviral Expression vectors and Stealth siRNA RNAi technologies, the hypothetical function that ANKRD gene family may modulate the skeletal fiber type conversions was somehow demonstrated. The results are as followed:1. The CDS sequences of mice and pig ANKRD 1, ANKRD2 were obtained based on the information in the GENEBANK database.2. Four lentiviral vectors:mice and pig ANKRD 1, ANKRD2, were successfully constructed by the TOPO cloning and Gateway sub-cloning.3. The mice ANKRD 1 and ANKRD2 lentiviral vectors were packaged into virus by the ViraPower HiPerform Lentiviral Expression Systems.4. The C2C12 myoblasts were infected by mouse ANKRD2 lentivirus then the mRNA expressions of the two ANKRD genes and the fiber type specific genes TNI was analyzed by real-time PCR. The results showed that the mRNA expression level of ANKRD2 was up-regulated for 405-fold while that of TN1 decreased for 3.5-fold.5. Six chemical synthetic stealth siRNAs and two negative controls were transfect into C2C12 respectively and two effective siRNAs of mouse ANKRD 1 and ANKRD2 were identified by semi RT-PCR.6. The C2C12 myoblasts were infected by mouse ANKRD 1 and ANKRD2 effective siRNAs then the mRNA expressions of the two ANKRD genes and two fiber type specific genes MCK and TNI were analyzed by real-time PCR. The results showed that:(1) the mRNA expression level of ANKRD 1 decreased for 5-fold while that of TN1 decreased for 7-fold; (2) the mRNA expression level of ANKRD2 decreased for 3-fold while that of TN1 increased for 60%.However, comparing with the negative control, the expression levels of MCK both in si-ANKRD1 and si-ANKRD2 have no obvious change.