Celf1 Is Partially Responsible For Defective Myoblast Differentiation In Myotonic Dystrophy Imitated RNA Toxicity

Objective: Celf1 is partially responsible for defective myoblast differentiation in myotonic dystrophy RNA toxicity.Methods: 1. Cell culture and differentiation,Samples were collected on a serial of time points, including days 0(in propagating media), 1, 2, 4 and 6. 2. Vector construction and delivery, construct the GFP-CUG5 and GFPCUG200 plamisid, Maxipreps of these plasmids were transfected into C2C12 cells using Lipofectamine. p MSCV-Celf1Flag-Puro was constructed by inserting an Eco RI fragment containing Celf1 Flag from a donor plasmid pc DNA3-Celf1 Flag into p MSCV-Puro, Maxipreps of the resulting construct were used to prepare retroviral vectors using a packing cell line 293 T Ecopack,the retrovirus was next transduced into C2C12 cells. p LKO.1 based Celf1 sh RNA lentiviral production, 20 μg of sh RNA-encoding lentiviral vector was transfected along with 15 μg of packaging vector ps PAX2 and 10 μg of envelope vector p MD2.G into a 150 mm dish of 293 T cells and then transfected into C2C12. 3. Antibodies and immunoassays, for western blotting, membranes were incubated in phosphate-buffered saline, with the primary antibodies: mouse monoclonal antibody to Celf1, mouse antibody to Flag, and goat antibody to total actin.The membranes were next incubated with horseradish peroxidase-conjugated secondary. Bound antibodies were visualized with enhanced chemiluminescence reagents. 4. Realtime quantitative RT-PCR and Alternative splicing analyses by RT-PCR,use glyceraldehyde-3-phosphate dehydrogenase(GAPDH) as a constitutive control. Test the genes of Myo D(Myf3), Myogenin(Myo G), Mef2 c, Celf1 during myoblasts differentiation. Extracted total RNA, and then amplified using gene/exon specific primers, Zasp, MBNL-1, Z-TTN, M-TTN and BIN-1, PCR products were separated on 1% agarose gels and the bands were quantified using the Image J software. 5. Fluorescence activated cell sorting, FACS was used to determine the cell cycle distribution of propagating and early stage differentiated myoblasts. Briefly, cells were harvested by trypsinization and then washing、staining and FACS was performed on an Acuri C6 flow cytometer, and the data were analyzed using Flowjo software with the Dean–Jett–Fox model. 6. Quantification of staining and statistical analyzes,The fusion index of the cultures was calculated by dividing the total number of nuclei in myotubes(≥ 2 nuclei) by the total number of nuclei counted. The total myotube area was calculated as the percentage of the total image area covered by myotubes. At least 1000 nuclei were counted in each analysis, which covered at least 3 randomly selected culture areas. Analysis of data was performed by Student’s T tests in order to evaluate differences between groups. P<0.05 is considered significant.Result: 1. 3′-UTR CUG-expansion leads to accelerated cell cycling in myoblasts and impaired differentiation(1) Myotubes were evidently formed in GFP-CUG5-transfected cells, as visualized by alpha myosin heavy chain staining(MF20). In contrast, they were rarely detected in GFP-CUG200 cultures.(2) Quantification of three independent experiments showed that fusion indices in GFP-CUG5 cultures often reached 38.9 ± 8.7%, but they were only 2.2 ± 0.8% in GFP-CUG200 cultures. Similarly, myotube areas were 40.2 ± 12.0% in GFP-CUG5 cultures but only 3.3 ± 0.8% in GFP-CUG200 cultures.(3) RT-PCR showed CUG200 inhibited myo D expression in both proliferating and differentiating cells, as previously reported. Myo G and Mef2 c expression was inhibited by CUG200, consistent with defective differentiation.(4) Western blot indicated Celf1 protein level was elevated accordingly.(5) FACS results present that CUG200 led to significantly less cells in G1 phase, suggesting increased cell cycling. 2. Overexpression of Celf1 inhibits myoblast cell cycle exit and impairs differentiation(1) Western blot results showed, Flag-tagged Celf1 was only present in the p MSCV-Celf1 Flag transduced cells, which also showed an overall upregulation of Celf1 protein.(2) MF20 positive staining was sporadic in Celf1-overexpressing cultures.(3) Quantification of myotube formation by fusion indices(5.2±2.0% in Celf1 Flag vs. 34.9±11.7% in controls) and myotube areas(5.4±3.0% in Celf1 Flag vs. 29.8±12.7% in controls) suggests that overexpression of Celf1 severely impair terminal myocyte differentiation.(4) RT-PCR showed Celf1 overexpression led to an increase of Myo D expression in undifferentiated myoblasts during day1 to day6 compared with control group, and Myo G and Mef2 c were both inhibited.(5) FACS cell cycle analyses showed that Celf1 overexpression led to defective cell cycle exit during differentiation. 30.7% Celf1-overexpressing myoblasts were in G1 phase at undifferentiated state. 3. Knockdown of Celf1 promotes precocious myocyte differentiation(1) Effective reduction(~80%) of Celf1 was confirmed by western blot.(2) Immunostaining showed Celf1 knockdown resulted in significantly increased myotube formation over control cultures.(3) Fusion indices and myotube areas were 45.0±7.1% and 38.6±7.2%, respectively, vs. 22.0±1.9% and 16.1±2.6% in control cells.(4) By realtime RT-PCR, knockdown of Celf1 persisted throughout the gradual dropping course of its expression pattern. The expression of Myo D, Myo G and Mef2 c showed significant increases in Celf1 sh RNA cultures, supporting increased myocyte differentiation.(5) FACS cell cycle analyses showed,there were 16.0% of G1 cells in control group and 16.4% in Celf1 sh RNA cells during day0. 4. Knockdown of Celf1 partially rescues CUG-expansion induced myocyte differentiation defects(1) Western blot showed Celf1 protein was greatly reduced when compared to cells stably expressing a scrambled sh RNA.(2) MF20 staining showed, comparing to few multinucleate myotubes in GFP-CUG200 cells expressing a scrambled sh RNA, multinucleate myotubes were evident in cells expressing Celf1 sh RNA.(3) Fusion indices were improved from 2.3±1.4% to 17.6±1.1% by Celf1 sh RNA. Consistently, myotube areas were improved from 4.7 ± 3.1% to 12.7 ± 2.4%.(4) Realtime RT-PCR assays of myo D, Myo G and Mef2 c also support that Celf1 sh RNA partially rescued the differentiation defects in CUG- expansion cells.(5) FACS cell cycle analyses showed that Celf1 sh RNA corrected the aberrantly increased cell cycling in CUG200 cells. There were 43.3% of G1 cells in Celf1 sh RNA/ CUG200 cells, considerably greater than 31.5% G1 in scrambled sh RNA/ CUG200 cells.(6) Alternative splicing analyses showed, comparing to normal myoblast differentiation and in the presence of Celf1 sh RNA, the altered splicing patterns were not reversed.Conclusion: Celf1 plays a distinctive and negative role in terminal myocyte differentiation, which partially contribute to DM1 RNA toxicity. Targeting Celf1 may be a valid strategy in correcting DM1 muscle phenotypes, especially for congenital cases.