| Intramuscular fat(IMF)content is one of the most important factors determining the quality of livestock and poultry meat.Rational and effective increase of IMF content is a key scientific problem to be solved in genetic improvement in livestock and poultry,and it is also one of the effective ways to fill the gap of consumers’ increasing demand for high-quality specialty livestock meat,and the "bottleneck" lies in finding out the main effector genes and acting mechanisms that specifically regulate IMF deposition.IMF deposition is a very complicated biological regulation process,including the proliferation and adipogenic differentiation of preadipocytes,fat acid synthesis and lipolysis,fatty acid metabolism and lipid transport.At the cellular level,it is mainly manifested by the increase in the number of preadipocytes(proliferation)and the increase in adipocytes size(adipogenesis).At the molecular level,it mainly depends on the complex and precise positive and negative regulation of lipid metabolism related genes,transcription factors,and epigenetic factors.Recent studies have shown that microRNA(miRNA),as an important class of epigenetic regulators,play an important regulatory role in the process of subcutaneous fat deposition in Meishan pigs.However,the regulating role and acting mechanism of miR-130b in the proliferation and adipogenic differentiation of intramuscular preadipocytes(IMA)in pigs and rats need to be further explored.This study is divided into four parts:1.Effect and mechanism of miR-130b on adipogenic differentiation of rat IMAIn this experiment,rat primary IMA were used as an in vitro model to investigate the effect and mechanism of miR-130b on adipogenic differentiation of IMA in late differentiation stage(7 d).Rat IMA were isolated aseptically and proliferated to 85%fusion,which were then randomly divided into four groups:(1)mNC group(miR-130b mimic NC transfected group);(2)mimic group(miR-130b mimic transfected group);(3)iNC group(miR-130b inhibitor NC transfected group);(4)inhibitor group(miR-130b inhibitor transfected group).IMA were induced differentiation for 7 days using the "cocktail" method after transfection.The results showed that the primary rat IMA were successfully obtained by different cell adhesion time.Compared with the mNC control group,miR-130b overexpression significantly inhibited the level of adipogenic differentiation,decreased the expression of 11β-hydroxysteroid dehydrogenase type 1(11β-HSD1),a key gene for lipogenic differentiation,and promoted the expression of lipoprotein lipase(LPL),a key gene for lipid hydrolysis.In addition,miR-130b overexpression significantly promoted the mRNA and protein expression of the apoptosis regulator Caspase-3.These results suggested that miR-130b was able to reduce the amount of lipid deposition in rat IMA by significantly inhibiting lipogenic differentiation,promoting lipolysis and inducing apoptosis of IMA.2.Effect and mechanism of miR-130b on the proliferation of rat IMAIn this experiment,we continued to use rat primary IMA as an in vitro model to further investigate the effect and mechanism of miR-130b on the proliferation and adipogenic differentiation of rat IMA at the early stages of adipogenic differentiation(24 h and 48 h).Rat IMA were isolated aseptically and proliferated to 85%fusion,which were randomly divided into four groups:(1)mNC group;(2)mimic group;(3)iNC group;(4)inhibitor group.All groups were induced differentiation for 24 h and 48 h using the "cocktail" method after transfection.The results showed that compared to the mNC group,miR-130b overexpression significantly inhibited the amount of lipid deposition in IMA after differentiation for 24 h and significantly suppressed IMA proliferation.In addition,miR-130b overexpression significantly inhibited the proliferation of rat IMA after differentiation for 48 h,leading to IMA cycle arrest,while induced the apoptosis level of rat IMA and significantly reduced the intracellular triglycerides(TG)content.Further assays of cell cycle and apoptosis-related genes expression showed that miR-130b overexpression significantly decreased the expression levels of the cell cycle protein gene cytokinin D1(Cyclind1),and the apoptosis suppressor gene B lymphocytoma-2 gene(Bcl-2)in rat IMA after differentiation for 48 h.Conversely,the expression level of intracellular Bcl-2 was significantly increased and apoptosis level was significantly decreased in the miR-130b inhibitor group compared with the iNC group.The results indicated that miR-130b significantly inhibited the proliferation of rat IMA and induced apoptosis at the early stage of adipogenic differentiation,thereby suppressing the lipid deposition in rat IMA.3.Effect and mechanism of miR-130b on proliferation and adipogenic differentiation of porcine IMAIn this part,the primary IMA derived from the longest muscle of Erhualian piglets was used as an in vitro model to further explore the effect and mechanism of miR-130b on the proliferation and adipogenic differentiation of porcine IMA.Primary IMA derived from the longest muscle of Erhualian piglets were isolated aseptically and cultured until 85%fusion,and then were randomly divided into four groups:(1)mNC group;(2)mimic group;(3)iNC group;(4)inhibitor group.All groups were induced adipogenic differentiation for 48 h(early stage of differentiation)and 7 d(late stage of differentiation)with the "cocktail" method.The results showed that miR-130b overexpression significantly upregulated the expression of proliferating cell nuclear antigen(PCNA),Cyclinb1,and decreased the expression of cell cycle-dependent kinase inhibitor P27 at the early differentiation stage compared with mNC group.At the late differentiation stage,overexpression of miR-130b significantly inhibited the mRNA and protein expression of PPAR-γ and SREBP1,while the expression of the PPAR-γdownstream genes FAS,GR,PPAR-α.FTO,Perilipin and SCD1,were all significantly reduced.However,compared with the iNC group,the mRNA expression of FAS,GR,PPAR-γ,FTO,Perilipin,and 11β-HSD1 were significantly increased in miR-130b inhibitor group.These above results indicated that miR-130b could significantly promote IMA proliferation at the early stage of differentiation,while at the late stage of differentiation,miR-130b significantly inhibited adipogenic differentiation in porcine IMA,mainly by suppressing the expression of key adipogenic genes,which in turn inhibited lipogenic differentiation of porcine IMA.4.Effect of miR-130b on the gene expression profile of porcine IMA at the late stage of differentiationIn order to further investigate the mechanism of miR-130b inhibiting lipid deposition in porcine IMA at the late stage of differentiation,in this part of study,primary IMA derived from the longest muscle of Erhualian piglets were still used as an in vitro model,which were randomly divided into three groups:(1)NC group;(2)mimic group;(3)inhibitor group.All groups were induced adipogenic differentiation for 7 d.The results showed that there were several differentially expressed mRNAs(DE mRNAs)in miR-130b mimic group and miR130b inhibitor group,respectively.Comparing with the NC control group,these DE mRNAs were enriched in several signaling pathways.Among them,DE mRNAs of miR-130b mimic group were enriched in biological functions like apoptotic and protein kinase binding,and mainly enriched in signaling pathways such as AMPK,mitophagy,protein processing in endoplasmic reticulum,autophagy and mTOR.In contrast,DE mRNAs of miR-130b inhibitor group were enriched in cellular components such as nuclear outer membrane-endoplasmic reticulum membrane network,endoplasmic reticulum membrane,endoplasmic reticulum subcompartment,cytoplasmic dynein complex and collagen trimer.In addition,the DE mRNAs were mainly enriched in the PPAR signaling pathway.Besides,miR-130b mimic significantly inhibited Caspase-3 mRNA and protein expression in porcine IMA and reduced apoptosis in porcine IMA compared with NC group,while the miR-130b inhibitor significantly promoted the mRNA and protein expression of Caspase-3 in porcine IMA compared with NC group.These results showed that the inhibition of lipid deposition in porcine IMA at the late stage of differentiation by miR-130b may be related to the inhibition of apoptosis signal pathway.In summary,miR-130b was able to inhibit intracellular lipid deposition at early stage of differentiation by inhibiting proliferation and inducing apoptosis in rat IMA.While in late stage of differentiation,miR-130b reduced intracellular lipid deposition mainly by significantly inhibiting adipogenic differentiation,promoting lipolysis and inducing apoptosis in rat IMA.In addition,miR-130b significantly promoted the proliferation of porcine IMA at the early stage of differentiation,but was able to reduce the lipid deposition in porcine IMA by inhibiting adipogenic differentiation at the late stage of differentiation.These above results indicate that miR-130b plays a key role in the regulation of IMF deposition and lipid metabolism,and can be a potential candidate gene related to IMF deposition,providing a theoretical basis for further improvement of meat quality and molecular breeding in livestock and poultry in the future. |
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