| MicroRNA is known to be an important regulator of muscle growth and development.The regulation of micro RNA on the skeletal muscle phenotype of animals is mainly achieved by regulating the proliferation and differentiation of muscle cells.Chicken is a very good animal in the process of regulating muscle development.The micro RNA regulation mechanism that analyzes muscle growth and development is one of the breakthroughs in chicken molecular breeding improvement.In this experiment,nine tissues including the heart,liver,spleen,lung,kidney,ovary,brain,leg muscle and chest muscle of Tibetan chicken were collected for miRNA sequencing,differentially expressed miRNAs in muscle tissue were screened,and chicken myoblasts were cultured in primary culture.Based on this,two of the miRNAs were verified at the cellular level.The main results are as follows:(1)We have selected 9 differentially expressed miRNAs in muscle tissue including gga-miR-133a-3p,gga-miR-133a-5p,gga-miR-133 b,gga-miR-301b-3p,gga-miR-1a-3p,gga-miR-1b-3p,gga-miR-22-3p,gga-miR-454-3p,and gga-miR-30a-3p.(2)The expression levels of gga-miRNA-301 b and gga-miRNA-454 increased first and then decreased during the proliferation and differentiation of chicken myoblasts,and gga-miRNA-301 b showed a significant increase at 48 h and 96h(P<0.01),it showed a significant decrease(P<0.01)at 96 h and 144 h,and reached a maximum at about 96 h adherence.gga-miRNA-454 showed a significant increase(P<0.01)at 144 h after adherence for 48 h to 144 h.There was a significant decrease(P<0.05)within 144 h to 192 h.,and t he maximum value was reached at 144h(3)Transfection of miRNA mimic significantly up-regulated the expression of miR-454 and miR-301 b in cells(P<0.01),while miRNA inhibitor significantly inhibited the expression of endogenous miR-454 and miR-301b(P<0.01)and miRNA mimic and miRNA inhibitor can be stably maintained in the cell for 24-96 h.(4)Through EDU and CCK8 cell proliferation assay,it was found that overexpression or inhibition of gga-miR-301b-3p expression had no significant effect on myoblast proliferation,overexpression or inhibition of gga-miR-454-3p expression.There was no significant effect on the proliferation of myoblasts.(5)gga-miR-301b-3p belongs to an intron miRNA,and its overexpression or inhibition does not promote or inhibit the expression of its host gene SKA2,and there is no negative feedback regulation mechanism with the host gene SKA2 gene.(6)Overexpression of miRNA-301 b significantly inhibited myoblast differentiation,and My HC fluorescently labeled protein area was significantly reduced(P<0.001).The m RNA levels of differentiation-related genes My HC,Myo G and Myo D were significantly decreased at 24 h and 48h(P<0.05).Inhibition of miR-301 b expression promoted the differentiation of myoblasts,and the area of myhc fluorescently labeled protein increased significantly(P<0.001).The m RNA levels of differentiation-related genes My HC,Myo G and Myo D increased significantly at 24 h and 48h(P<0.05).(7)Cell Giemsa staining and cellular immunofluorescence labeling of skeletal muscle myosin and real-time quantitative results showed that overexpression of miRNA-454 significantly inhibited myoblast differentiation,and myhc fluorescently labeled protein area was significantly reduced(P<0.001).The m RNA levels of differentiation-related genes My HC and Myo G decreased significantly at 24 h and 48 h after differentiation(P<0.05).Inhibition of miR-454 expression promoted the differentiation of myoblasts,and the area of myhc fluorescently labeled protein increased significantly(P<0.001).The m RNA levels of My HCand Myo G showed significant increase in differentiation at 24 h and 48h(P<0.05).(8)The predicted result of the target gene revealed that the miR-301 b has a binding site with the 3’-UTR of the TBL1XR1 gene,and the miR-454 has a binding site with the 3’-UTR of the SBF2 gene.By constructing a dual luciferase reporter detection system,it was found that TBL1XR1 and SBF2 are target genes of gga-miR-301 b and gga-miR-454,respectively.The above results indicated that 9 differentially expressed miRNAs in muscle tissue were screened by high-throughput sequencing of transcriptomes from nine different tissues of chickens.There is no negative feedback regulation of miR-301 b and host gene SKA2 during the proliferation and differentiation of chicken primary myoblasts.miR-301 b inhibits cell differentiation by targeting TBL1XR1 and down-regulating the expression of differentiation-related genes.miR-454 inhibits cell differentiation by targeting SBF2 and down-regulating the expression of differentiation-related genes,further highlighting the proliferation of miRNAs in chicken primary myoblasts.The importance of the process of differentiation. |
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