The Effect And Mechanism Of MiR-324-5p On Pig Fat Deposition

Pork accounts for a large proportion in the meat consumption of Chinese residents.Pig fat deposition is regulated by many genes and non-coding RNAs(miRNA,Lnc RNA,and circ RNA).Micro RNA(miRNA)is a non-coding RNA with a length of 19-22 nucleotides(nt),which binds to target genes 3'UTR inhibits target gene translation.Numerous studies have shown that miRNAs can be involved in the deposition of subcutaneous and intramuscular fat in pigs.After analyzing the miRNAs sequencing results in pig subcutaneous and intramuscular adipocytes,it was found that the expression of miR-324-5p increased significantly after differentiation of subcutaneous and intramuscular adipocytes,and the seed sequence of miR-324-5p is highly conserved among pigs,rats,mice and other species.In addition to being deposited in adipocytes,lipid droplets can also be deposited in non-adipocytes such as skeletal muscle cells.The lipid droplets deposited in skeletal muscle cells form intramuscular adipocytes.Previous research in our laboratory found that oleate can induce fat deposition in skeletal muscle cells.Therefore,this study used pig subcutaneous adipocytes,mice and C2C12 myoblasts as research materials to explore the effects and regulatory mechanisms of miR-324-5p on pig subcutaneous adipocyte differentiation and C2C12 myoblast deposition lipid accumulation.In this experiment,miR-324-5p agomiR and antagomiR were transfected into porcine subcutaneous adipocytes to study the effect of miR-324-5p on porcine subcutaneous adipocyte differentiation;bioinformatics analysis predicted target gene of miR-324-5p,and the double-luciferase reporting experiment was used to verify the results;the target gene overexpression vector was constructed to study its role in adipoceyts differentiation and its targeting relationship with miR-324-5p.Using oleate to induce lipid accumulation in C2C12 myoblasts,overexpress or inhibit miR-324-5p,and explore the effect of miR-324-5p on oleate-induced intracellular lipid accumulation.Intraperitoneal injection of miR-324-5p agomiR in mice to verify the effect of miR-324-5p on fat deposition in mice at the in vivo level.The following main results were obtained: 1.MiR-324-5p bioinformatics analysisBy comparing the mature sequences of miR-324-5p between different species,it was found that miR-324-5p is highly conserved among species;The expression of miR-324-5p in different tissues of 7-day-old and 180-day-old Guanzhong black pigs was detected,and it was found that miR-324-5p had a higher expression level in adipose tissues of different-day-old of Guanzhong black pigs.In addition,the expression of miR-324-5p was increased in both subcutaneous and intramuscular adipose at the later stage of differentiation.GO analysis and KEGG analysis of miR-324-5p target genes revealed that miR-324-5p was associated with adipogenesis.2.miR-324-5p promotes differentiation of porcine subcutaneous adipocytesOil Red O and Bodipy staining results showed that overexpression of miR-324-5p can increase lipid droplet accumulation;real-time quantitative PCR and western blotting revealed that overexpression of miR-324-5p significantly increase the adipogenesis-related gene PPAR?(P < 0.05),ap2(P <0.05),and C/BP?(P < 0.05)m RNA and protein expression levels.But inhibition of miR-324-5p obtained the opposite result.3.MiR-324-5p can target KLF3Using miRNA target gene prediction software analysis showed that KLF3 could be a candidate target gene for miR-324-5p.Overexpression of miR-324-5p significantly inhibit the expression levels of KLF3 m RNA and protein(P <0.05),while inhibition of miR-324-5p significantly increase the expression of KLF3 m RNA(P <0.05).The results of the dual luciferase report experiment found that miR-324-5p significantly inhibit the activity of dual luciferase(P <0.05);overexpression of KLF3 significantly inhibited adipocyte differentiation(P <0.05),and at the same time overexpression of miR-324-5p could partially restore the inhibitory effect of KLF3 on genes related to lipid accumulation and adipogenesis.4.Overexpression of miR-324-5p promotes fat deposition in miceIntraperitoneal injection of miR-324-5p agomiR into obese mice induced by high-fat diet,it was found that overexpression of miR-324-5p significantly increase the weight of obese mice(P <0.05)without affecting their feed intake;body composition analysis found that overexpression of miR-324-5p significantly increase the weight of subcutaneous fat,visceral fat and brown adipose tissue(P <0.05)without affecting liver weight;H&E staining results found that overexpression of miR-324-5p can significantly increased the area of adipocytes in subcutaneous adipose tissue and visceral adipose tissue(P <0.05)without affecting the area of adipocytes in liver adipose.Real-time quantitative PCR and Western blotting revealed that overexpression of miR-324-5p significantly increase the expression levels of lipid synthesis-related genes FASN and SREBP1 c in adipose tissue(P <0.05),reduced the expression levels of lipolysis-related genes HSL and ATGL in subcutaneous adipose tissue and visceral adipose tissue(P <0.05),but did not affect liver fat and lipolysis;overexpression of miR-324-5p significantly reduced the expression of KLF3 in adipose tissue and liver.5.MiR-324-5p can promote oleate-induced lipid accumulation in myoblastsBodipy and oil red O staining found that overexpression of miR-324-5p promote oleate-induced lipid accumulation in myotubes and significantly increase long-chain fatty acid synthesis-related genes Evolv6(P <0.05),GPAM(P <0.05),and DGAT2(P <0.05)expression level,but inhibition the expression of ACC2(P <0.05);and inhibition of miR-324-5p obtained the opposite result.6.MiR-324-5p can improve mitochondrial function in C2C12 myoblasts induced by oleateMitochondrial and Bodipy staining revealed that overexpression of miR-324-5p can increase the number of lipid droplets and mitochondria,and significantly increase the number of mitochondrial copies(P <0.05);overexpression of miR-324-5p significantly increase mitochondrial metabolism and fatty acid ? oxidation-related genes Tfam,Cox7 a,CPT1,CD36 and Acadm expression levels(P <0.05);inhibition of miR-324-5p gave opposite results.The above results indicate that miR-324-5p promotes C2C12 lipid accumulation by promoting mitochondrial function.In summary,this study found that miR-324-5p can promote adipocyte differentiation and fat deposition by targeting KLF3 at the cellular and individual levels.In addition,it can also promote oleate-induced lipid accumulation in C2C12 myoblasts by enhancing mitochondrial function.This conclusion not only shows that miR-324-5p plays an important role in fat deposition,but also provides a theoretical basis for further exploring the molecular mechanism of fat deposition in livestock,especially pigs.