Microrna-128 Targets Myostatin At Coding Domain Sequence To Regulate The Proliferation And Differentiation Of Myoblasts

MicroRNAs(miRNAs or miRs)play a critical role in skeletalmuscle development.In a previous study,we observed that miR-128 was highly expressed in skeletal muscle.However,its function in regulating skeletal muscle development is not clear.We found that miR-128 targeted mRNA of myostatin(MSTN),a critical inhibitor of skeletal myogenesis,at coding domain sequence(CDS)region by bioinformatic analysis.To detected the effect of miR-128 on MSTN and its role in C2C12 myoblast proliferation and differentiation,we applied EdU(5-ethynyl-2'-deoxyuridine)proliferation assay,cell cycle flow cytometry,quantitative RT-PCR,immunofluorescence analysis and western blot analysis after transfected with miR-128 inhibitor,NC,miR-128mimics or pre-miR-128.We also detected the expression of miR-128 and MSTN in murine muscle.The results are as follows:1.The Stem-loop RT-PCR results showed that miR-128 expression in murine muscle increased as animal age increased,in addition,miR-128 expression showed an increasing trend during C2C12 cell differentiation,which suggests that miR-128 is involved in the regulation of the proliferation.2.Overexpression of miR-128 inhibited proliferation of mouse C2C12 myoblast cells but promoted myotube formation.MSTN protein expression was reduced after miR-128 overexpression,and MSTN protein expression was increased after transfection of the miR-128 inhibitor,but MSTN mRNA abundance did not change,which suggests that miR-128 effect the expression of MSTN protein,inhibiting mRNA translation rather than degradation is the main method of miR-128 to regulate MSTN gene expression.3.The dual luciferase reporter plasmid pMSTN and miR-128 mimics were co-transfected into C2C12 myoblasts and HEK293T cells.The result of dual luciferase reporter assay showed the luciferase activity was lower after being co-transfected with plasmid pMSTN and miR-128 mimics both in the C2C12 cells and in HEK293T cells,this suggests that miR-128 could target MSTN in skeletal muscle cells.4.The RT-PCR results showed that the expression of myogenic factor 5(Myf5),myogenin(MyoG),paired box(Pax)3,and 7 were significantly increased in the miR-128 overexpression group than NC group at both 48 and 72 h post transfection;The mRNA expression of Myf5,MyoG and Pax3 were less for the miR-128 inhibitor group than the NC group at 72 h.These results indicate that ectopic expression of miR-128 effected proliferation and differentiation of myoblasts.5.The proliferation of C2C12 cells was measured by the 5-ethynyl-2'-deoxyuridine(EdU)proliferation assay and propidium iodine flow cytometry,The results showed that overexpression of miR-128(mimics and pre-miR-128)repressed proliferation of C2C12 cells and increased the percentage of C2C12 cells in the G1 and G2 phases,decreased the percentage of cells in the S phase.Overexpression of miR-128 increased the inhibition of G1 phase of C2C12 cells.6.The mmu-miR-128 precursor expression vector and miR-128 mimics were transfected to C2C12 cells to study the effects of miR-128 overexpression on the differentiation of C2C12 cells.At 12 h after transfection,cells were induced to differentiate in DM.MyoG expression showed an notable increasing trend during C2C12 cell differentiation analyzed by immunofluorescence,and the mRNA expression of MyHC and MyoG were greater analyzed by RT-PCR.MSTN protein expression was less but MyHC protein expression was greater for the miR-128 analyzed by western blot.These results suggest that overexpression of miR-128 promoted C2C12 myoblast differentiation.The expression of MyHC and MyoG decreased notably when miR-128 was inhibited,demonstrated the effects of miR-128 inhibition on the differentiation of myoblast.In summary,our experimental results demonstrated that miR-128 involves in the regulation of the proliferation and differentiation of skeletal myoblasts by targeting MSTN CDS region.