Study On The SCD1 Gene Expression By MiR-200c And MiR-429 In Ovine Subcutaneous Adipocytes

Stearoyl-CoA desaturase 1(SCD1)is a lipogenic enzyme and is a potential target for regulating adipose metabolism.SCD1 can desaturate from palmitic and stearic acids in the de novo synthesis of fatty acids to palmitoleic and oleic acids,respectively,and thus participate in fat metabolism by regulating the content of unsaturated fatty acids.According to the previous reports,SCD1 gene can be regulated by multiple miRNAs at the same time.However,these studies have focused on species such as humans,rats,fish and pigs,and are rare in sheep.This study aimed to predict and validate key miRNAs with a target relationship with SCD1,and to explore the regulation of miRNAs on SCD1 in ovine subcutaneous adipocytes.The results may provide some scientific bases to improve meat quality by increasing the content of unsaturated fatty acids in carcass.Four bioinformatics softwares,i.e.Target Scan,mir DB,Star Base and miRanda,were used to predict miRNAs with a target relationship to SCD1.The dual luciferase assay was used to verify the target relationship between miRNAs and SCD1.The regulation of miRNAs on SCD1 was explored at the transcriptional and protein levels by using both qPCR and Western-blotting.(1)The number of miRNAs that had a target relationship with SCD1 predicted by mir DB,Starbase,Miranda and Targetscan was 113,41,39 and 8,respectively.The intersection of Wayne diagrams showed that three miRNAs,i.e.miR-200 c,miR-429 and miR-216 a,were the key miRNAs.After preliminary screening,miR-200 c and miR-429 were selected as the research objects of this experiment.The seed region sequences of miR-200 c and miR-429 were completely complementary to the 1395~1401 bases of the SCD1 3’ UTR.In other words,they had the same binding site.(2)The dual luciferase assay showed that,the fluorescence activity of the overexpressing group(co-transfected with pmir-SCD1 and miR-200 c mimic or miR-429 mimic)was significantly lower(P < 0.05)than that of the negative control.This result indicated that both miR-200 c and miR-429 have a target relationship with SCD1.(3)The qPCR and Western-blotting results showed that the overexpression group(transfected with miR-200 c mimic or miR-429 mimic)down-regulated the expression of SCD1 significantly at both transcription level(P < 0.05)and protein level(P < 0.01).The inhibiting-expression group(transfected with miR-200 c inhibitor or miR-429 inhibitor)up-regulated the expression of SCD1 significantly at both transcription level(P < 0.05)and protein level(P < 0.01).These results indicated that both miR-200 c and miR-429 inhibited the expression of SCD1 in ovine subcutaneous adipocytes.Co-transfection of miR-200 c mimic and miR-429 inhibitor group and NC group were highly significant(P < 0.01),and co-transfection of miR-200 c inhibitor and miR-429 mimic group was not significantly different from the NC group(P > 0.05).The last contrast result indicated that miR-200 c was more effective than miR-429.(4)Observation under microscope revealed that the number of lipid droplets in the overexpression group(transfected with miR-200 c mimic or miR-429 mimic)was less than that in the negative control,and less than the corresponding inhibitory expression groups(transfecting miR-200 c or miR-429 inhibitor).It showed that both miR-200 c and miR-429 inhibited the formation of subcutaneous lipid droplet.This study demonstrated that miR-200 c and miR-429 had target relationships with SCD1,and had the same binding site.It is cleared that both miR-200 c and miR-429 targeted SCD1 and negatively regulated the expression of SCD1 in ovine subcutaneous adipocytes,and the regulation of miR-200 c was more effective.These provide scientific bases for regulating the content of unsaturated fatty acids and improving meat quality.