| LncRNA is an important transcriptional regulator that can participate in various physiological activities and metabolic processes through epigenetic regulation,transcriptional regulation,and post-transcriptional regulation.At present,a large number of functional LncRNAs have been identified,but their specific functions and mechanisms need to be further studied.Based on the RNA-seq results of muscle-mixed samples from three different developmental stages of full-sibs Mongolia black beef cattle,the key LncRNAs related to muscle development have were screened and their function and mechanism in the development of bovine skeletal muscle satellite cells.The function and mechanism of action have been studied in our study.The main findings are as follows:1.In the previous study,full-sibs Mongolia black beef cattle samples were obtained by superovulation and embryo transfer techniques,and muscle mixed samples(leg gluteal muscle,scapular muscle,dorsal lumbar muscle and intercostal muscle)of 3 month old fetus,6month old fetus and neonatal calf were collected for high-throughput RNA sequencing.In this study,the sequencing results were analyzed in depth,and 12632 annotated m RNAs and212,851 new unannotated LncRNAs were obtained.A total of 9913 LncRNAs with differential expression at different stages and 90 LncRNAs closely related to muscle development were screened out.Through variance analysis,bioinformatics analysis and fluorescence quantitative detection analysis,a LncRNA with high expression in bovine skeletal muscle satellite cells and muscle tissue was obtained and named as Lnc403 as the main research object.2.The structure and function analysis of Lnc403 revealed that the full length of Lnc403 was 2689bp,which was located in the upstream of Myf6 gene of bovine chromosome 5.The expression of Lnc403 in myoblasts and myotubes was detected by nuclear separation and q-PCR.And it was found that Lnc403 was to be mainly located in the nucleus.3.The transfection technique was used to transfect si Lnc403 RNA and Lnc403 recombinant vector to bovine skeletal muscle satellite cells,and the Lnc403 function loss and gain model were successfully constructed.Based on the above cell model,the effects of Lnc403 on the proliferation and differentiation of bovine skeletal muscle satellite cells were detected by q-PCR,EDU,Western blot and other techniques.The results showed that the change of Lnc403 expression had no significant effect on the proliferation of skeletal muscle cells,but it has obvious inhibitory effect on the differentiation of myoblasts,both interference and overexpression of Lnc403 could hinder the differentiation process of bovine myoblasts.4.This study further screened and analyzed the target factors that Lnc403 may regulate during bovine myogenic differentiation.The results showed that the expression of the adjacent gene Myf6 was opposite to Lnc403 expression,confirming the negative regulation between Lnc403 and Myf6.At the same time,436 target proteins directly binding to Lnc403 were obtained by RNA-pull down technology.Bioinformatics analysis showed that these proteins were mainly enriched in biological processes and regulatory pathway including cell proliferation,cell morphology regulation,transcriptional regulation,m RNA splicing,ribosome biosynthesis,RNA degradation and transport,regulation of the actin cytoskeleton et al.Five target proteins related to muscle development were screened for PTK2,THRB,BRK1,PDGFD and KRAS.And the targeted regulation relationship between Lnc403 and KRAS,which closely related to the proliferation and differentiation of myocytes,was analyzed.The results showed that expression of KRAS m RNA and protein decreased when Lnc403 was down-regulated.Meanwhile Lnc403 expression was up-regulated,KRAS m RNA expression was still down-regulated,but protein expression did not change significantly.In addition,when the expression level of Lnc403 was changed,the expression of Raf gene in the RAS-RAF-MEK-ERK cascade signaling pathway involved in KRAS gene was also significantly decreased.In summary,this study screened a functional long-chain non-coding RNA Lnc403,and analyzed its structure,function and potential targets.The inhibitory effect of Lnc403 on myocyte differentiation was confirmed.It is speculated that Lnc403 may participate in muscle development through a network regulation mode.This study Provide new evidence for the mechanism of how LncRNA plays a regulatory role in the process of bovine muscle formation. |
