| Skeletal muscle is the largest tissue in the mammalian body.The loss of its functional or regenerative properties can lead to skeletal muscle diseases in human.On the other hand,increasing muscle yield and improving meat quality are issues that have been explored in animal production.Understanding the mechanism of skeletal muscle formation not only helps to reveal the molecular basis of skeletal muscle diseases and find therapeutic targets for the treatment of skeletal muscle-related diseases,but also provides new strategies for increasing muscle yield and improving meat quality.Therefore,elucidating the mechanism of skeletal muscle development and regeneration will be of great significance to both human medicine and animal husbandry.Long non-coding RNAs（lncRNAs）are a class of RNAs with a fragment length greater than 200 bp that do not have coding functions.However,reports in recent years have found that some lncRNAs have small open reading frames,which have coding potential and can encode micropeptides.With the application of transcriptome sequencing,a large number of novel lncRNAs were detected in skeletal muscle.However,the mechanisms of only a small number of lncRNAs have been reported,and the regulatory mechanisms of a large number of lncRNAs are still unknown.In this study,mouse C2C12 cell line and C57BL/6 mouse were used as research models.Two key lncRNAs（2310043L19Rik and Mir22hg）were screened and identified by analyzing and integrating a large number of differential lncRNAs obtained from published transcriptome sequencing data,and elucidated their roles in regulating skeletal muscle cell proliferation and differentiation,muscle regeneration.This study was divided into two parts.The main research contents and results are as follows:Experiment 1 LncRNA 2310043L19Rik inhibits differentiation and promotes proliferation of myoblast by sponging miR-125a-5pAccording to previous RNA-seq results,lncRNA 2310043L19Rik（lnc-231）was identified and was significantly elevated during mouse myoblast differentiation and highly expressed in skeletal muscle.Furthermore,the expression levels of muscle atrophy markers Atrogin-1 and lnc-231 were negatively correlated during myogenesis.Intracellular overexpression of lnc-231 inhibited the formation of myoblasts into multinucleated myotubes,thus restraining their differentiation in vitro.In addition,knockdown of lnc-231using RNA interference（si RNA）technology was found to promote the differentiation of myoblasts.In flow cytometry assays,overexpression of lnc-231 significantly increased the S-phase cell ratio.Conversely,knockdown of lnc-231 caused a reduction in the proportion of cells in S phase.The results showed that lnc-231 could positively regulate the transition from G0/G1to S in myoblasts.Furthermore,overexpression of lnc-231 increased the ratio of Ed U-positive cells,whereas knockdown of lnc-231 caused a decrease in the ratio of Ed U-positive cells.These results suggested that lnc-231 may promote myoblasts proliferation.In addition,we examined the expression of the proliferation marker gene,Ki67 and cyclin-dependent kinases（CDK2,CDK4,and CDK6）.q RT-PCR results showed that the m RNA expression levels of Ki67,CDK2,CDK4 and CDK6 were increased after overexpressing lnc-231.Conversely,the m RNA levels of Ki67,CDK2,and CDK6 were decreased after knockdown of lnc-231.Western blot results showed that over expression of lnc-231 increased the protein levels of CDK2 and CDK6,but knockdown of lnc-231inhibited the expression of CDK2 and CDK6.Nucleocytoplasmic separation assays found that lnc-231 was highly expressed in the cytoplasm,and it was predicted by biological information that it could bind to the bound miR-125-5p.The targeting relationship between lnc-231 and miR-125a-5p was confirmed by dual-luciferase reporter vector experiments.E2F3 is a target gene of miR-125-5p.Overexpression of miR-125a-5p suppressed E2F3protein levels,and miR-125a-5p co-transfection with lnc-231 rescued the decrease in E2F3protein levels compared with transfection of miR-125a-5p alone.Likewise,co-transfection of lnc-231 and miR-125a-5p increased the ratio of Ed U-positive cells,the ratio of S-phase cells,and improved cell viability compared with transfection of miR-125a-5p alone.These results indicated that lnc-231 sponge miR-125a-5p through a competitive endogenous RNA（ce RNA）mechanism to relieve the inhibition of its target gene E2F3,thereby promoting the proliferation of myoblasts.Experiment 2 Long non-coding RNA Mir22hg-derived miR-22-3p promotes skeletal muscle differentiation and regeneration by inhibiting HDAC474 lncRNAs annotated in the UCSC database were overlapped by analyzing the published data and these lncRNAs were consistently up-regulated during C2C12differentiation.Previous Gene Ontology（GO）functional analysis indicated that Mir22hg may be involved in muscle contraction in myotubes and may affect skeletal muscle myogenesis,but the exact mechanism remains unclear.Our results have shown that Mir22hg is significantly elevated during C2C12 differentiation and is highly expressed in skeletal muscle.Moreover,Mir22hg first increased and then decreased during regeneration after skeletal muscle injury,which suggested that Mir22hg may play an important role in myogenesis and skeletal muscle regeneration.Overexpression of Mir22hg promoted myotube formation in vitro.Furthermore,knockdown of Mir22hg by si RNA inhibited myotube formation.These results suggest that Mir22hg is a key positive regulator of myoblast differentiation.Mir22hg is also highly conserved in many species,such as human and mouse.And the seed sequence of miR-22-3p is particularly highly conserved.In addition,q RT-PCR results showed that the expression of miR-22-3p was significantly positively correlated with the expression of Mir22hg during differentiation（r=0.732,P=0.0019）.The results of tissue expression profiling showed that miR-22-3p was also highly expressed in skeletal muscle and heart,and overexpression of Mir22hg also promoted the expression of miR-22-3p.After mutating the miR-22-3p seed sequence in the Mir22hg sequence,the miR-22-3p in the overexpressing Mir22hg-mut group was not significantly elevated.The above results suggest that Mir22hg may be processed to produce mature miR-22-3p through post-transcriptional processing.Nucleocytoplasmic separation assays and RNA-FISH confirmed that the expression of Mir22hg in the cytoplasm was higher than that in the nucleus.Mir22hg may play a role in regulating its target genes by producing miR-22-3p.Seven target genes of miR-22-3p（ERBB3,HDAC4,MECP2,DDIT4,KLF6,VSNL1,and SCAMP1）were predicted by using three online tools（miRTar Base,Target Scan and RNA22）.The dual-luciferase reporter vector assays confirmed that HDAC4 was directly target of miR-22-3p.Immunofluorescence and western blotting results both showed that overexpression of miR-22-3p significantly inhibited the expression of HDAC4,and we confirmed that Mir22hg inhibited HDAC4 by producing miR-22-3p,which indicated that HDAC4 is a direct target of miR-22-3p.Mir22hg inhibits the expression of HDAC4 by producing miR-22-3p.Knockdown of HDAC4 significantly increased the expression of MEF2C,a transcription factor downstream of HDAC4.Furthermore,immunofluorescence results showed that knockdown of HDAC4 significantly increased the ratio of My HC and Myo G positive cells.These results suggested that HDAC4 was a negative regulator of myoblast differentiation.Both co-transfection in vitro with miR-22-3p inhibitor assays and miR-22-3p seed sequence mutation in Mir22hg confirmed that Mir22hg inhibited HDAC4by the production of miR-22-3p and promoted the expression of MEF2C,thus promoting myoblast differentiation in vitro.The above studies confirm that Mir22hg can promote the differentiation of myoblasts,we speculate that Mir22hg may regulate muscle fiber regeneration after skeletal muscle injury.C57BL/6 mice were used as a research model and injected lentivirus into the tibialis anterior（TA）muscle to knock down Mir22hg.And skeletal muscle injury model（injection of 1.2%Ba Cl2into the TA muscle）was conducted.H&E staining showed that the repair rate of skeletal muscle in the Mir22hg knockdown group was lower than that in the control group.Many of the nuclei in the Mir22hg knockdown group were still located in the center of the myofiber in day 9.While in the control group,most of the myofiber nuclei had migrated to the edge of the myofiber with characteristics of mature fibers.And the TA muscle weight in the Mir22hg knockdown group was significantly lower than that in the control group on day 12.q RT-PCR results showed that during the repair of injured TA muscle,the m RNA expression of My HC and skeletal muscleα-Actin in the Mir22hg knockdown group was significantly lower than that in the control group on day 1 and day 3.In the Mir22hg knockdown group,My HC and Myo G protein levels were also significantly lower than those in the control group on days 3,5,7 and 9.In contrast,the HDAC4 protein of the Mir22hg knockdown group showed higher expression than the control group on day7 and day 9.What’s more,Mir22hg expression was significantly positively correlated with the ratio of TA muscle to body weight（r=0.5055,P=0.0084）.H&E staining showed that in the Mir22hg knockdown group,the cross-sectional area of muscle fibers in the TA muscle was significantly lower than that in the control group.These results suggested that Mir22hg knockdown in skeletal muscle weaken the regenerative capacity of skeletal muscle fibers,thereby delaying the repair of injured muscle,ultimately leading to a reduction in skeletal muscle weight and cross-sectional area.In conclusion,we identified two long non-coding RNAs 2310043L19Rik（lnc-231）and Mir22hg that were highly expressed in skeletal muscle.Lnc-231 inhibits myoblast differentiation and adsorbs miR-125a-5p through a ce RNA mechanism,weakening its inhibition of the target gene E2F3,and promoting myoblast proliferation.Mir22hg was significantly increased during myoblast differentiation and was highly expressed in skeletal muscle.Mir22hg,as a miR-22-3p precursor,can be processed to generate mature miR-22-3p,which inhibits its target gene HDAC4,thereby increasing the expression of downstream MEF2C and ultimately promoting myoblast differentiation.Knockdown of Mir22hg in skeletal muscle in vivo can delay repair and regeneration after skeletal muscle injury,resulting in reduced skeletal muscle cross-sectional area and weight loss.Mir22hg is highly conserved not only in mice and primates（humans,rhesus,baboons,etc.）,but also in some livestock animals（such as cattle,sheep）.Therefore,Mir22hg not only serves as a new potential therapeutic target for the treatment of skeletal muscle injury,skeletal muscle atrophy and other muscle diseases,but also may provide a new way to increase the meat production and quality of cattle and sheep. |
PDF Full Text Request