Survey on microRNAs Targeting MuRF1 and MuRF2 in Regenerating Skeletal Muscle

Skeletal muscle is a very adaptable tissue, mainly due to its regenerative capacity, which is fundamental importance for repairing large lesions and microlesions as well. Recently our research group observed that the E3 ligases MuRF1 and MuRF2, in addition to being implicated the atrophy process is also essential for muscle regeneration (data not published). In view of these results, in this study we investigated the molecular mechanisms involved in the increased expression of MuRFs during regeneration. Our hypothesis contemplates that certain microRNAs (miRNAs or miRs, regulators of gene expression at post‐transcriptional level) can regulate MuRFs expression during the regeneration process. Objective Search miRNAs that targets MuRFs, and investigate its impact on the skeletal muscle regeneration. Materials and Methods C57/BL6 mice (~8wk. 30±3g) were injected with cardiotoxin (CTX, 10μM) in the Tibialis Anterior (TA) muscle for 1, 3 and 10 days in order to induce muscle injury. After these periods the animals were euthanized and the TA muscles were removed for determination of RNA and protein expression levels. Plasmids pMIR‐3UTRMuRF1 and pMIR‐3UTRMuRF2 were produced out of the amplification of the 3′ UTR of each cDNA and cloned into the vector pMIR‐Report system. Vectors (300ng) were co‐transfected with the specified miRNA (miRNA mimics 40uM or mimic Control#1) in HeLa cells. Then, the luciferase assay was performed. Primary myoblast cultures were transfected with siRNA to MuRFs in pre differentiation and post differentiation phases. Procedures and experimental protocols were approved by the Ethics experimental Committee at University of Sao Paulo ‐ CEUA #118. Results The protein expression of MuRF1 rises after 3 days of injury (~100%; p<0.05 vs control), while its gene expression rises as soon as 1 day (~150%; p<0.05 vs control) after injury. MuRF2, gene expression and protein content, rises after 10 days of lesion (~ 50%; p <0.05 vs. control). Transcription factors of MuRFs, FOXO3a and NF‐Kb increase their protein expression after 3 days of injury (>100%; p<0.05 vs. control). The miRs 29c and 101a which target MuRF1 and miRs 133a and 133b which target MuRF2 (TargetScan6.2 analysis) are reduced by 1 and 3 days(~50–80%; p<0.05 vs. control). By using luciferase assays with co‐transfection pMIR‐3UTR‐MuRF1 or pMIR‐3UTR‐MuRF2 and their respective mimics, we showed that miR29c targets MuRF1 and miR133a and 133b target MuRF2. Interestingly, MuRF1 and MuRF2 siRNA knock down do not change the expected modulation of miRs 29c, 133a and 133b. Expression of miRs 133a and 133b are increased after the post differentiation period (~100%; p<0.05 vs pre‐differentiation) in cultured myoblasts. Conclusion With these partial results, we identify and validate the miRs 29c and 133a/b target MuRFs. Support or Funding Information Grant 2012/22488‐2, Sao Paulo Research Foundation (FAPESP)