| Muscle satellite cells,as the basic unit in the process of skeletal muscle regeneration biology,directly affect the re-development and formation of skeletal muscle.According to the previous research,LncRNA(Long non-coding RNA)and miRNA(micro RNA)play an important regulatory role in the proliferation and differentiation of muscle satellite cells,affecting the formation of skeletal muscles.In the past time,the miR-133 a has been proved that it was specially expressed in the muscle.Moreover,it can interact with LncRNA,and thereby regulating muscle cell development.Even so,Studies on the regulation of the growth and development of bovine skeletal muscle satellite cells by miR-133 a and the interaction of LncRNA have not been reported.In this paper,The lnc RNA interacting with miR-133 a was obtained by screening data of RNA sequencing of fetal muscles of different developmental stages.Then we investigated the mechanism of miR-133 a and LncRNA interaction regulating the proliferation and differentiation of bovine muscle satellite cells.Our studies provided a reference for the study of the mechanism of miRNA and LncRNA regulating muscle cell growth and development.1..This study used the in vitro differentiation model of bovine primary skeletal muscle satellite cells established by the research group to explore the effect of miR-133 a on the development of bovine skeletal muscle satellite cells.The expression of miR-133 a in the differentiation process of muscle satellite cells showed an increasing trend by QRT-PCR.After overexpression or inhibition of miR-133 a,we detected the proliferation and differentiation of bovine skeletal muscle satellite cells using QRT-PCR,EDU staining,Western blot and immunofluorescence technique.The results showed that after overexpression of miR-133 a,the number of proliferation of muscle satellite cells was relatively decreased,while the expression of myocyte differentiation marker genes were significantly increased.The opposite result was obtained after knock-down of miR-133 a.The results of this study indicate that miR-133 a inhibits proliferation and promotes differentiation in the development of bovine skeletal muscle satellite cells.2.The potential interactions of three miR-133 a with LncRNAs were screened by analyzing the RNA sequencing data of the fetal muscles of different months old.The q RT-PCR detection was performed at the tissue and cell level to obtain a candidate interaction LncRNA(named LncRNA-133a)of Mi R-133.Following,the full length 1304 nt of LncRNA-133 a was first amplified by RACE technology.It was confirmed that LncRNA-133 a binds to miR-133 a by bioinformatics prediction and dual luciferase assay.In the proliferative phase of bovine skeletal muscle satellite cells,the expression of miR-133 a was decreased and the proliferation of muscle satellite cells was promoted after overexpression of LncRNA-133 a.After inhibition of LncRNA-133 a,the expression of miR-133 a was increased and the proliferation of muscle satellite cells was inhibited.In the differentiation stage of bovine skeletal muscle satellite cells,the expression of miR-133 a was not significantly changed and the differentiation of muscle satellite cells was promoted after overexpression of LncRNA-133 a.Inhibition of LncRNA-133 a,miR-133 a expression was not significantly altered and muscle satellite cell differentiation was inhibited.These results indicated that LncRNA-133 a was interacted with miR-133 a and regulated the proliferation and differentiation of bovine skeletal muscle satellite cells.3.In order to investigate the mechanism of interaction between LncRNA-133 a and miR-133 a,we identified that miR-133 a directly target LASP1 using bioinformatics prediction,RNA pull down and double luciferase assay.Overexpression of miR-133 a revealed a significant decrease in LASP1 m RNA and protein levels,while knockdown of miR-133 a increase the expression of LASP1.To investigate more explicit relationship,it was found that the proliferation of bovine skeletal muscle satellite cells was blocked and the differentiation process was promoted.When the LASP1 was interfered,the multiple treatment groups of double luciferase assay showed that LncRNA-133 a could compete with miR-133 a t increase the expression of target gene LASP1.In the proliferative phase,the expression of LASP1 m RNA and protein was significantly up-regulated after overexpression of LncRNA-133 a,while the inhibition of LncRNA-133 a resulted in the opposite result.In the differentiation phase,the expression of LASP1 m RNA and protein was significantly down-regulated after overexpression of LncRNA-133 a,while inhibiting LncRNA-133 a results in the opposite.LncRNA-133 a competitively binds to miR-133 a to increase expression of LASP1 by multiple treatment combinations of dual luciferase assays.In the proliferative phase,the expression of LASP1 m RNA and protein was significantly up-regulated after overexpression of LncRNA-133 a,while the inhibition of LncRNA-133 a resulted in the opposite result.In the differentiation phase,the expression of LASP1 m RNA and protein was significantly down-regulated after overexpression of LncRNA-133 a,while the opposite result was obtained by inhibiting the LncRNA-133 a.These results indicated that the target gene LASP1 of miR-133 a regulated the proliferation and differentiation of bovine skeletal muscle satellite cells,but the expression of LASP1 was regulated by LncRNA-133 a.Above all,a regulatory network of LncRNA-133a-miR-133a-LASP1 regulating the proliferation and differentiation of bovine skeletal muscle satellite cells was constructed,and the regulatory mechanism of miR-133 a and interaction LncRNA-133 a on the development of bovine skeletal muscle satellite cells was preliminarily elucidated.It provides important reference for further study of molecular mechanism of beef muscle growth and development.At the same time,it would be able to furnish important research target for meat production ability and improvement of meat quality. |
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