| Intramuscular fat(IMF),one of the most important forms of fat deposition in animals,is a key index to evaluate muscle tenderness and flavor of animals.With the pursuit of high-quality pork,how to improve the IMF content has become one of the hot topics in pork research.Laiwu pig is one of the typical fatty breeds in China with high IMF content,however,the IMF content varies greatly among individuals in pork production which might be due to the different expression of key genes.In the current study,we found that mi R-34a/LEF1 was involved in the IMF deposition based on the results from whole transcriptome sequencing.Thus,the effects and mechanisms of mi R-34a/LEF1 on IMF accumulation were investigated in this study.1 Study on the differential deposition of IMF in Laiwu pig individualsThe study was performed to screen the samples for RNA-seq by investigating the IMF contents of Laiwu pig individuals as well as their genetic relationship.The samples were divided into two groups including H group(IMF > 12%)and L group(IMF < 5%),which were used for whole transcriptome sequencing.The results were presented as following:(1)The IMF content in the longissimus dorsi muscle of 29 Laiwu pigs was detected.And the results showed that the highest IMF content was 13.93% and the lowest was 2.17%.(2)According to IMF content and genetic relationship,eight samples were selected for RNA-seq.The detailed information of these samples was as follows: the IMF content in H group was 12.45%,13.93%,13.27% and 13.38%,respectively;the IMF content in L group was 4.23%,4.29%,4.31% and3.23%,respectively.These pigs are half-sibling relationships,respectively.t test was performed to determine the difference of IMF content in H and L,and it was found that the IMF content in group H was significantly higher than that in L group(P < 0.01).Oil red O staining showed that the lipid droplets area of the liver and the longissimus dorsi muscle cross-section in group H were larger than that in group L.(3)Fatty acid content in H and L was detected,and it was found that myristic acid methyl ester(C14:0)(P < 0.05)and arachidonic acid methyl ester(C20:4n6)(P < 0.01)in group H were significantly higher than that in group L.2 The gene expression profiling associated with IMF deposition in Laiwu pigs through whole transcriptome sequencingThe selected samples with different IMF contents of Laiwu pigs in group H and L were used for RNA sequencing.The threshold was set as |fold change| > 1 ? Padj < 0.05 for the identification of candidate genes related to IMF accumulation.And then the regulatory network between noncoding RNAs(nc RNAs)and m RNAs was constructed to investigate the potential mechanisms for IMF deposition.(1)A total of 17 differentially expressed lnc RNAs were obtained by RNA sequencing(the number of upregulated and downregulated lnc RNAs were 11 and 6,respectively).GO enrichment analysis showed that these differentially expressed lnc RNAs were mainly enriched in fatty acid biosynthetic process,fatty acid metabolic process,regulation of fat cell differentiation and fatty acid binding molecular functions.And KEGG enrichment analysis showed that these lnc RNAs were significantly enriched in fatty acid elongation(P = 0.015)and arachidonic acid metabolism(P = 0.019)signaling pathways.Furthermore,a total of 14 differentially expressed circ RNAs were identified,and the functional analysis of these circ RNAs were not enriched in the biological processes related to IMF deposition.(2)According to the analysis of RNA sequencing,a total of 180 differentially expressed m RNAs were obtained,including 112 upregulated and 68 downregulated m RNAs.Functional analysis of the differentially expressed m RNAs showed that these m RNAs were significantly enriched in the GO terms,such as cellular response to fatty acids,insulin receptor binding,and glycerol kinase activity.The KEGG enrichment analysis demonstrated that these m RNAs were enriched in the signaling pathways related to fat deposition and lipid metabolism,such as m TOR,TGF-?,AMPK,Wnt,PI3K-Akt,MAPK and insulin regulation signaling pathways.(3)Eight upregulated mi RNAs and 16 downregulated mi RNAs were obtained in group H compared with group L.Functional analysis showed that these mi RNAs were mainly enriched in the processes of lipid metabolism,synthesis and cellular response to lipid.Meanwhile,these mi RNAs were primary involved in Wnt,Hedgehog,m TOR,AMPK,MAPK,PI3K-Akt,PPAR and insulin signaling pathways.(4)Association analysis of differentially expressed nc RNAs and m RNAs showed that a total of seven genes were identified by intersection analysis of lnc RNAs-m RNAs,including two downregulated genes and five upregulated genes.Three groups of association relationship were obtained between mi RNAs and m RNAs,including mi R-532-HMCES/PLD3,mi R-34a-LEF1 and mi R-6782-3PDDX54/ARHGEF7/JARIC2.Go analysis showed that these three groups were enriched in 11 GO items(P < 0.05)related to muscle development and fat deposition.And LEF1 was included in all of these items.Meanwhile,the Wnt signaling pathway(involving LEF1)related to IMF deposition was enriched according to KEGG analysis.Therefore,in the current study,mi R-34a/LEF1 network and Wnt signaling pathway were taken as the main targets for exploring the mechanisms of IMF accumulation.3 The functions and mechanisms of mi R-34a/LEF1 in the regulation of IMF deposition3T3-L1 and C2C12 cells were used in this study.The target relationship between mi R-34 a and LEF1,expression of adipogenic makers(PPAR?,C/EBP?,FABP4 and Perilipin1)and ?-catenin(a marker of Wnt signaling pathway),and lipid droplets accumulation,were detected in3T3-L1 cells after transfected with mi R-34 a and/or LEF1.Then,the effects of mi R-34 a on lipid droplets deposition induced by oleate in C2C12 cells were detected in this study.Besides,through transfection with mi R-34 a,we detected the effects of mi R-34 a on LEF1 expression and IMF deposition in the co-culture of 3T3-L1 and C2C12 cells.Finally,3T3-L1 cells were transfected with mi R-34 a mimics,and the supernatant of these cells during differentiation was collected and used as the conditioned medium to culture C2C12 cells.And the lipid droplets accumulation in C2C12 cells were detected.The results were as follows:(1)Tissue expression profiling analysis showed that mi R-34 a and LEF1 were predominantly expressed in subcutaneous fat and muscle.The LEF1 protein expression of H and L groups were also detected using Western blot.Compared with L group,LEF1 protein expression in H was significantly decreased(P < 0.05).With the differentiation of 3T3-L1 cells,mi R-34 a expression was significantly increased(P < 0.05).While,LEF1 was significantly decreased at the early stage of differentiation(d 0-d 8)(P < 0.05),and then it began to be upregulated from the low point after induction for 8 days.RNA fluorescence in situ hybridization and immunofluorescence staining showed that mi R-34 a and LEF1 in 3T3-L1 cells were expressed and located in cytoplasm.Interestingly,LEF1 transferred into the nucleus after 3T3-L1 cells differentiation.(2)According to the websites of Target Scan and Starbase,7 nt base in LEF13'UTR could be complementary to the seed sequence of mi R-34 a,which indicated the targeting relationship between mi R-34 a and LEF1.Dual luciferase assay was used to verify the targeting relationship between mi R-34 a and LEF1.And the results showed that mi R-34 a overexpression effectively reduced luciferase activity of pmir GLO-LEF1-WT reporter(P < 0.001),which demonstrated that LEF1 was the target of mi R-34 a.(3)The functions of mi R-34 a in the fat deposition in 3T3-L1 cells.A.After mi R-34 a was overexpressed,3T3-L1 cells were induced to differentiation(d 0?d 8).mi R-34 a mimics significantly increased the expression of PPAR?,C/EBP?,FABP4 and Perilipin1(P < 0.05),and decreased LEF1 expression(P < 0.05).Western blot showed that LEF1 and ?-catenin(nucleus)expression was significantly decreased,while PPAR? and C/EBP? levels were increased(P < 0.05).Bodipy and oil red O staining demonstrated that mi R-34 a overexpression promoted the accumulation of lipid droplets.On the contrary,mi R-34 a inhibitor decreased adipogenic markers expression(m RNA and protein levels)and promoted LEF1 and ?-catenin protein levels(P < 0.05).Furthermore,it also decreased the lipid droplets deposition.These results suggested that mi R-34 a promoted fat accumulation in 3T3-L1 cells.B.The functions of LEF1 in fat deposition of 3T3-L1 cells.The results showed that LEF1 overexpression in 3T3-L1 cells significantly decreased the m RNA expression of PPAR?,C/EBP?,FABP4 and Perilipin1(P < 0.05).While these adipogenesis markers were markedly elevated after LEF1 inhibited.These results suggested that LEF1 inhibited fat deposition in 3T3-L1 cells.C.The verification of the targeting relationship between mi R-34 a and LEF1 by rescue assay.To further explore the relationship between mi R-34 a and LEF1,mi R-34 a mimic NC + pc DNA3.1,mi R-34 a mimics + pc DNA3.1-LEF1,mi R-34 a inhibitor NC + si NC and mi R-34 a inhibitor + si LEF1 were co-transfected into 3T3-L1 cells.It was found that the expression of lipid markers has no difference,which indicated that mi R-34 a regulated lipid deposition in 3T3-L1 cells by targeting LEF1.(4)The functions of mi R-34 a in the fat deposition of C2C12 cells.mi R-34 a overexpressed in C2C12 cells significantly upregulated the expression of PPAR?,C/EBP?,and FABP4(P < 0.05).The formation and accumulation of lipid droplets were promoted(P < 0.05)while the expression of LEF1 was inhibited(P < 0.05).However,inhibition of mi R-34 a showed opposite results.These results suggested that mi R-34 a promoted lipid accumulation in myoblasts by targeting LEF1.(5)The effects of mi R-34 a on fat deposition in the co-culture of C2C12 and 3T3-L1 cells.It was found that mi R-34 a mimics significantly elevated the gene expression of PPAR?,C/EBP?,and FABP4,and promoted the fat deposition,but reduced LEF1 and My OD expression(P < 0.05).While mi R-34 a inhibitor decreased the expression of adipogentic markers and fat deposition,and increased LEF1 and My HC levels(P < 0.05).These results indicated that mi R-34a/LEF1 was involved in the regulation of fat deposition in the co-culture of C2C12 and 3T3-L1 cells.(6)The effects of conditioned culture mediums derived from 3T3-L1 cells(mi R-34 a overexpressed)differentiation on the accumulation of lipid droplets in C2C12 cells.Firstly,the 3T3-L1 cells were transfected with mi R-34 a mimics and the conditioned culture mediums during differentiation(d 0-d 8)of these preadipocytes were collected.Then,the medium was used for the incubation of C2C12 cells.The results showed that with the differentiation of 3T3-L1 cells,the conditioned medium significantly promoted the expression of adipogenic markers and the accumulation of lipid droplets in myoblasts(P < 0.05),but decreased the LEF1 expression(P< 0.05).These results indicated that mi R-34a/LEF1 might regulate IMF deposition by the secreta of adipocytes during differentiation.In conclusion,mi R-34 a promoted lipid deposition in adipocytes and myoblast cells by targeting LEF1 to regulate IMF deposition,which might result in the difference of IMF content among laiwu pig individuals. |
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