Study On The Regulation Mechanism Of Lnc133b,a MiR-133b Ralated LncRNA,in The Proliferation And Differentiation Process Of Bovine Skeletal Muscle Satellite Cells

Numerous studies show that long non-coding RNAs(lnc RNAs)play important roles in the processes of multiple regulation networks.lnc RNAs play significant roles in the development of skeletal muscle satellite cells.But there are very fewer reports of lnc RNAs in bovine skeletal muscle satellite cells.Based on the result of our previous high-throughput sequencing,we got a bovine skeletal muscle-relatived lnc RNA,named lnc133 b.Then,we identified the character and regulation mechanism of lnc133 b in the development of bovine skeletal muscle satellite cells.Combaining previous reports,we constructed the regulation pathway of lnc133 b in regulating the development of the bovine skeletal muscle satellite cells.Through performing high-throughput sequencing,we identified the lnc133 b which was completely complementary with the mature sequence of miR-133 b.The result of CPC(coding potential calculator)algorithm prediction showed that lnc133 b had a very low coding potential,similar to lnc RNA HOTAIR,which indicated that lnc133 b was a nc RNA.The luciferase report assay demonstrated that miR-133 b directely regulated the expression of lnc133 b and lnc133 b was a competitively “sponge” for miR-133 b.QRT-PCR was performed to detect the expression of lnc133 b,which found that the expression of lnc133 b gradually increased during the state of differentiation of bovine skeletal muscle satellite cells and came to the peak under DM2 conditions,indicating that lnc133 b affected the differentiation of skeletal muscle satellite cells.Cells fractionation followed by q RT-PCR showed that lnc133 b located in cytoplasm and nucleus.To detect the effect of lnc133 b on muscle cells development,the model of overexpression or downregulated lnc133 b in bovine skeletal muscle satellite cells were constructed.And the Ed U assay showed that over-expression or downregulate lnc133 b increased the ratio of positive cells of Ed U.The results of q RT-PCR and western blot showed that over-expression or downregulate lnc133 b significantly decreased the expression of Myo G and MHC,respectively.Our experiments demonstrated that over-expression or downregulate lnc133 b could promote the proliferation and decrease the differentiation of bovine skeletal muscle satellite cells.The bovine skeletal muscle satellite cells model of miR-133 b gain/loss-offunction was constructed to detect the interaction between lnc133 b and miR-133 b.The results showed that over-expression of lnc133 b significantly decreased the expression of miR-133 b,and downregulation of lnc133 b significantly increased the expression of miR-133b;Controry,over-expression of miR-133 b significantly decreased the expression of lnc133 b,and dowenregulation of miR-133 b significantly increased the expression of lnc133 b.The results indicated that lnc133 b negatively regulated the expression of miR-133 b and miR-133 b negatively regulated the expression of lnc133 b.We constructed lnc133b/miR-133b/IGF1 R axis.By the luciferase report assay,we demonstrated that miR-133 b directly regulated the IGF1 R.QRT-PCR and western blot were performed to detect the bovine skeletal muscle satellite cells model of miR-133 b gain/loss-of-function and lnc133 b gain/loss-of-function.The results showed that over-expression of miR-133 b significantly decreased the expression of IGF1 R in protein levels but not RNA levels,and downregulation of miR-133 b significantly increased the expression of IGF1 R in protein levels but not RNA levels,indicating that miR-133 b regulated the expression of IGF1 R in protein levels;Over-expression of lnc133 b significantly increased the expression of IGF1 R in protein levels but not RNA levels,and downregulation of lnc133 b significantly decreased the expression of IGF1 R in protein levels but not RNA levels,indicating that the expression of lnc133 b was positive correlation with the expression of IGF1 R,and further identified the ce RNAs mechanism between lnc133 b and miR-133 b.