| With the widespread application of high-throughput sequencing technology,a large number of circRNAs are identified in muscle tissues.And more and more research unveiled that circRNAs including circ-FUT10,circ-WDR77 and civc-ZNF609 closely relate to the growth and development of muscles.As a widely investigated circular RNA,CDRlas regulates the expression of target genes IGF1R and Spl through competitively binding miR-7.According to our data,CDR1as promotes the differentiation of goat skeletal muscle satellite cells(SMSCs).Meanwhile,MyoD is a marker of muscle satellite cells activation and promotes myogenic differentiation by regulating the expression of muscle-specific genes,and MyoD has a wide range of regulatory functions within the whole genome.So we speculate that MyoD may directly or indirectly regulate the expression of muscle-related circRNAs during myogenic differentiation,though it has not been reported so far.Here,we investigated whether MyoD regulated CDR1as gene expression and its underpinning mechanism.The results obtained are as follows:1.We utilized RT-qPCR to explore the expression patterns of CDR1as and MyoD in different muscle tissues from goats at embryonic 120 days and the differentiating SMSCs.CDR1as predominantly expressed in the gastrocnemius,followed by the longissimus dorsi and the lowest in the semitendinosus.Then,the mRNA levels of MyoD,MyHC and MyoG were similar to CDR1as.Moreover,the expression levels of CDR1as and MyoD were increased and then decreased during the SMSCs differentiation.These results suggest that CDR1as may be co-expressed with MyoD in muscle tissues and SMSCs.2.Compared with the control group,overexpression of MyoD promoted myotubes formation and promoted the expression of differentiation-related genes MyoG,MyHC and Myomaker(P<0.01),while the CDR1as expression was also significantly increased(P<0.01)in goat SMSCs.Moreover,the proliferation-related gene PCNA was also significantly up-regulated(P<0.01).Conversely,disrupted MyoD inhibited the SMSCs differentiation and down-regulated the expression of muscle development-related genes,and markedly reduction of CDR1as expression was also presented(P<0.05).These findings indicate that MyoD regulates the expression of CDR1as.3.Bioinformatics analysis discovered that the approximately 1.9 kb fragment of CDR1as 5’-flanking region had a 60%similarity to the human CDR1as promoter.Moreover,this region contains the eukaryotic promoter regulatory elements including CAAT-box and TATA-box,which contains muscle-related transcription factor binding sites like MyoD.Meanwhile,the predicted core promoter sequence was within the interval 1294 to 1344 of this fragment,embeding the transcription initiation site.4.We further divided the CDR1as promoter region(1.9 kb)into six fragments and constructed six vectors(CP1-CP6).C2C12 cells were transfected with each deletion vector individually,and cells were induced differention for 48 h and dual luciferase assay was performed.The results showed that the promoter activity of CP5 containing a MyoD binding site was the highest compared to other constructs.We co-transfected CP5 wild-type vector(CP5-WT)with pEGFP-MyoD or si-MyoD into C2C12 cells.The transcriptional activity of CP5 was significantly increased with pEGFP-MyoD(P<0.01),while knockdown of MyoD significantly reduced the CP5 transcriptional activity(P<0.05).And coincided results exhibited in HeLa cells.Expectedly,the transcriptional activity of CP5-Mut(mutated MyoD binding site)was unaffected by MyoD in both C2C12 and HeLa cells.The binding ability of MyoD to the CDR1as promoter was examined using ChIP-PCR.The results showed that MyoD protein binded to the CDR1as promoter region and overexpression of MyoD enhanced the binding.These findings establish that CDR1as is initiated and activated by MyoD.5.We treated SMSCs with actinomycin-D,and found that the half-life of MyoD mRNA was increased by forced-expressing CDR1as.In other word,CDR1as could enhance the stability of MyoD mRNA in the cytoplasm.And potential of CDR1as directly binding to MyoD mRNA was ditected using online software.Further CDR1as and MyoD were almost completely co-localized in cytoplasm of SMSCs during proliferation and differentiation,unveiled by fluorescence in situ hybridization(FISH).In summary,we hypothesized that CDR1as might affect MyoD mRNA stability by binding to it in the cytoplasm.In conclusion,CDRlas was co-expressed with MyoD in goat muscle tissues and SMSCs.MyoD could bind the promoter region of CDR1as gene to promote the expression of CDR1as.And in turn,CDR1as might enhance MyoD mRNA stability by directly binding to it in the cytoplasm.Here we report the mutual regulation between CDR1as and MyoD,which will provide an important reference for further study the function of circRNAs in skeletal muscle. |
