Explore The Reversal Effect Of MiR-322/503 On DM1 Imitated Inhibition Of Myoblasts Differentiation

Objective:Explore the reversal effect of miR-322/503 on DM1 imitated inhibition of myoblasts differentiation 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.pMSCV-Celf1Flag-Puro was constructed by inserting an EcoRI fragment containing Celf1 Flag from a donor plasmid pcDNA3-Celf1 Flag into pMSCV-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.donor plasmid pMSCV-miR322/503-puro was provided by Dr.Liu Yu,packaging vector with 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,use glyceraldehyde-3-phosphate dehdrogenase(GAPDH)as a constitutive control.test the genes of MyoD(Myf3),Myogenin(MyoG),Mef2 c,Celf1 during myoblasts differentiation.5.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(35.4±4.1)%,but they were only(2.6±1.1)% in GFP-CUG200 cultures.Similarly,myotube areas were(35.6±2.2)% in GFP-CUG5 cultures but only(2.7 ± 0.8)% in GFP-CUG200 cultures.(3)RT-PCR showed CUG200 inhibited myoD expression in both proliferating and differentiating cells,as previously reported.MyoG and Mef2 c expression was inhibited by CUG200,consistent with defective differentiation.(4)Western blot indicated Celf1 protein level was elevated accordingly.2.Overexpression of Celf1 inhibits myoblast differentiation(1)Western blot results showed,Flag-tagged Celf1 was only present in the pMSCV-Celf1 Flag transduced cells,which also showed an overall upregulation of Celf1 protein.(2)MF20 positive staining was sporadic in Celf1-overexpressing cultures.3.miR-322/503 reverse myoblast differentiation inhibition induced by CTG trinucleotide repeat abnormal amplification(1)Immunostaining showed miR-322/503 overexpression resulted in signifi-cantly increased myotube formation over control cultures.(2)Fusion indices and myotube areas were(26.6 ± 5.5)% and(7.9 ± 2.5)%,respectively,vs.(6.4 ±1.0)% and(2.7± 1.0)% in control cells.(3)By realtime RT-PCR,overexpression of miR-322/503 increased the expression of MyoD,MyoG and Mef2 c significantly,supporting increased myocyte differentiation.(4)western blot showed miR-322/503 reduced the expression of Celf1.4.miR-322/503 reverse myoblast differentiation inhibition induced by RNA toxicity(1)MF20 staining showed,comparing to few multinucleate myotubes in control cultures,multinucleate myotubes were evident in cells expressing miR-322/503.(2)Fusion indices were improved from(6.2 ± 1.5)% to(36.1 ± 2.1)% by miR-322/503 overexpression.Consistently,myotube areas were improved from(5.6 ± 1.0)% to(16.2 ± 1.5)%.(3)Realtime RT-PCR assays of myoD,MyoG and Mef2 c also support that miR-322/503 rescued the differentiation defects in CUG-expansion cells.(4)Western blot showed Celf1 protein was decreased when compared to control group.Conclusion:miR-322/503 reverse DM1 imitated inhibition of myoblasts differentiation...