Fig 2: CircRILPL1 improves mouse muscle regeneration in vivo.A Injection scheme for circRILPL1 into CTX-injured muscles. N = 3 mice for each group. B H&E staining of TA muscle at 0, 3, and 6 days after injection of CTX. Scale bars, 100 µm. C The expression of circRILPL1 in the above-injected muscles after injection of overexpression plasmid. The data were normalized to GAPDH mRNA. D The expression of mmu-miR-145a-5p in the above-injected muscles after injection of circRILPL1 plasmid. The data were normalized to U6. E After circRILPL1 expression plasmid injection, muscle H&E staining was performed on the 3rd and 6th day of muscle injury. The empty pCD2.1 vector was injected as a control. Mice in which CTX-muscle injury was not induced served as negative controls (NC). Scale bars, 100 µm. Fibers with intact structure (3 d) or centrally localized nuclei (CLN, 6 d) were quantified. F The above fibers were stained for eMyHC. Scale bars, 100 µm. The positively stained fibers were quantified. G, H The expression of IGF1R, PI3K, PDK1, and myogenic markers in the above-injected muscles. The data were normalized to GAPDH mRNA and represent the average of three independent experiments. Data are presented as means ± SEM. n = 3. *P < 0.05. **P < 0.01.

Fig 3: circRILPL1 serves as a miR-145 sponge.A CircRNAs with miR-145 absorption sites were screened from high-throughput sequencing data. B Luciferase activity of psiCheck2-miR-145 sensor in HEK293T cells co-transfected with miR-145 mimics and circRNAs, which are putatively binding to the miR-145 sequence. Luciferase activity was normalized by firefly luciferase activity. C Effect of circRILPL1 on the abundance of miRNAs. D Ago2-RIP assay for the amount of circRILPL1 and miR-145 in bovine myoblasts. E CircRNA pull-down assays were performed using a specific biotin-labeled circRILPL1 probe in myoblasts. Real-time qPCR was used to detect the expression levels of circRILPL1 and miR-145 in immunoprecipitates. n = 3. **P < 0.01. Compared with negative control (NC) probe. F Predicted circRILPL1 secondary structure and binding site to miR-145. G Luciferase reporter activity of circRILPL1-WT and circRILPL1-mut in HEK293T cells co-transfected with miR-145 mimics or NC mimics. H The miR-145 biosensor (psiCHECK-2-miR-145 2×) was transfected into HEK293T cells, together with mimics-NC, miR-145 mimics, pCD2.1-non, or pCD2.1-circRILPL1, and luciferase activities were measured after transfection. I Luciferase reporter activity of IGF1R-3’UTR in HEK293T cells with circRILPL1 knockdown or overexpression. J IGF1R expression in myocytes transfected with miR-145 mimics alone or co-transfected with circRILPL1. *P < 0.05. **P < 0.01.

Fig 4: circRILPL1 regulates IGF1R through miR-145.A CCK-8 assay for myocytes transfected with miR-145 mimics alone or co-transfected with circRILPL1. n = 6. B CCK-8 assay for myocytes with circRILPL1 overexpression and knockdown of IGF1R. n = 6. C, D EdU assay for myocytes transfected with miR-145 mimics alone or co-transfected with circRILPL1. Scale bars, 200 µm. E Cell proliferation and PI3K/AKT pathway-related protein analysis for myocytes transfected with circRILPL1 alone or co-transfected with si-IGF1R and miR-145 mimics. F The expression of MyoD, MyoG, and MyHC was detected by real-time qPCR. G Myocytes were transfected with circRILPL1 alone or co-transfected with si-IGF1R, immunofluorescence (MyHC) was used to analyze the level of muscle cell differentiation. Scale bars, 500 µm. H After 4 days of differentiation, the percentage of nuclei present in MyHC-positive myotubes with the indicated number of nuclei were quantified in cells. Data are presented as mean ± SEM of three independent experiments. I The protein expression of MyoD, MyoG, and MyHC was detected by western blots.