Research On MicroRNA-127 Targeting ACO2 Gene And Its Action Pathway

Researchs about meat quality traits become one of the most important fields of animal nutrition in recent years. Porcine skeletal muscle fiber type and structural features is a key factor affecting the quality of meat.The difference of skeletal muscle fiber type of pigs was deemed as the main subject of this study.Based on the results of high-throughput sequencing, targeted gene of mi R-127 and its regulatory mechanism were researched in the study.In this study, 60 castrated hybrid male pigs(Duroc × Landrace × Large White) were divided into 6 stalls with 10 pigs in each stall. The experiment started at age 28 days and finished at age 161 days. On 63, 98, and 161 days, 6 pigs were slaughtered for sampling. Longissimus dorsi muscle were collected and made into paraffin sections to determin the proportion of muscle fiber types.We transfected mi R-127 mimics/inhibitor into skeletal muscle satellite cells which were collected from a 1-day-old piglet and then the expression of genes related to the development of muscle fiber types such as ACO2, SETD8, MEF2, MAPK1, MAPK3, MAPK4 and MYH7 were investigated with real-time fluorescence quantitative PCR(SYBR Green I). The expression of MYH7 protein was detected using western blot method. Finally, the normal sequence ACO2-Target N and mutant sequence ACO2-Target M were connected to a plasmid containing the luciferase reporter gene separately. Both of them were transfected into 293 A cells with mi R-127 mimics as the experimental group, and with NC as the control group.The activity of luciferase was tested to verify the targeting relationship between mi R-127 and ACO2 gene.The results were as follows:1. The proportion of type ? red muscle fibers increased during pig growth.The proportion at 63 d was significantlyhigher(P<0.05) than that at 98 d and 161 d, while there was no significant difference between the proportion at 98 d and 161 d.The proportion of type ?x and ?b white muscle fibers decreased during pig growth.The proportion at 63 d was significantly lower(P<0.05) than that at 98 d and 161 d, while there was no significant difference between the proportion at 98 d and 161 d.2. After skeletal muscle satellite cells transfected mi R-127 mimics, the expression of MYH7 protein, MYH7, ACO2, MEF2 and MAPK3 genes were significantly decreased(P<0.05), while the expression of SETD8, MAPK4 and MAPK1 did not change significantly(P>0.05). After skeletal muscle satellite cells transfected mi R-127 inhibitor, the expression of MYH7 protein, ACO2 and MEF2 genes were significantly increased(P<0.05), while the expression of SETD8, MYH7, MAPK4, MAPK3 and MAPK1 did not change significantly(P<0.05).3. Under conditions of lysine deficiency, MEF2 gene expression was significantly decreased(P<0.05) in the skeletal muscle satellite cells, and the MYH7 gene expression was intended to decrease(P>0.05).4.The mi R-127 mimics significantlydecreased(P<0.05) the firefly luciferase activity of pmir GLO-ACO2-Target N by 38.3%, but had no effect on pmir GLO- ACO2-Target M(P>0.05).In summary, mi R-127 can target ACO2 gene and decrease the expression of it.The content of intracellular lysine and the expression of MEF2 and MYH7 are also decreased. MYH7 gene is specifically expressed in slow muscle fibers.Thus, mi R-127 play a role in the regulation of muscle fiber types by this way.