Differences In The Effects Of Different Splice Isomers Of Bovine TUSC5 On Adipose Deposition And Their Mechanisms

Adipose deposition is an important factor in determining the economic value of beef cattle,but the mechanisms regulating adipose deposition are currently uncertain.TUSC5(tumor suppressor candidate 5)is an important key gene for fat deposition and there are two different alternative splice isoforms(TUSC5A and TUSC5B).However how the two different splice isomers of TUSC5 regulate fat deposition is uncertain.In this study,we used lentiviral technology to construct cell models stably expressing two different splice isoforms of the TUSC5 gene.By combining molecular biology techniques with high-throughput sequencing,we investigated the effects and differences of the two different splice isoforms of the TUSC5 gene on the regulation of lipid deposition,and analyzed the regulatory mechanisms of both in the process of lipid synthesis,thus refining the insight of the regulatory network of the TUSC5 gene of the different splice isoforms,The detailed results of the study are as follows:(1)Construction of TUSC5A and TUSC5B overexpression precursor adipocyte modelsThe lentiviral packaging system was constructed to overexpress TUSC5A and TUSC5B,and lentiviruses that can overexpress TUSC5A and TUSC5B were successfully packaged.Optimal titers for two separate lentiviral infestations of the precursor adipocyte line C3H10 using lentiviral gradient infestation analysis.The TUSC5A was 9.6 x 10~7TU/m L and the TUSC5B was 7.3 x 10~7 TU/m L.Using the lentivirus to infect precursor adipocytes and combined with puromycin for positive cell sorting,the purity of TUSC5A and TUSC5B precursor adipocyte lines reached more than 98.1%,and expressed 280 times more than the control group,successfully constructing the overexpression precursor adipocyte models of TUSC5A and TUSC5B.(2)Effect of overexpressing TUSC5A and TUSC5B on precursor adipocytesThe results of the growth curves of overexpressed TUSC5A and TUSC5B precursor adipocytes showed that the number of TUSC5A cells did not change significantly at 7 days compared to the control group,while the number of cells in the TUSC5B group was reduced by 0.32-fold and cell viability was diminished significantly(p<0.0001).Flow cytometric analysis revealed early and late apoptosis of 3.71%and 5.26%in TUSC5B precursor adipocytes(p<0.01).The q PCR results showed that the expression of proliferation-promoting gene PCNA was elevated significantly in TUSC5A cells(P<0.01).Apoptosis-promoting gene P53 was significantly elevated in TUSC5B cells(P<0.01).It indicated that TUSC5B may contribute to the apoptosis of precursor adipocytes via P53.q PCR and Western-blot of key genes for fat deposition revealed that ACC1,FASN,SCD1,LPL,PPARG,FABP4,GPDH and GLUT4 were significantly overregulated in TUSC5A precursor adipocytes and significantly repressed in TUSC5B precursor adipocytes.It indicates that TUSC5A and TUSC5B differentially regulate the expression of fat deposition genes ACC1,FASN,SCD1,LPL,PPARG,FABP4,GPDH and GLUT4.(3)Effect of overexpressing TUSC5A and TUSC5B on differentiation of adipocytesOil Red O staining of cells at 4,6,8 and 10 days during differentiation of TUSC5A and TUSC5B overexpressing cells revealed that TUSC5A promoted the accumulation of lipid droplets and TUSC5B inhibited the accumulation of lipid droplets.The results of triglyceride content on days 0,2,4,6,8 and 10 showed that TUSC5A promoted triglyceride accumulated during differentiation,while TUSC5B inhibited triglyceride accumulation.Further q PCR was used to detect the temporal expression of key genes for fat deposition,ACC1,FASN,SCD1,LPL,FABP4,PPARG,GPDH and GLUT4 in precursor adipocytes at days 0,2,4,6,8 and 10.The results revealed a promotive effect of TUSC5A for gene expression and a repressive effect of TUSC5B for gene expression.The results demonstrate at the phenotypic and molecular levels that TUSC5A has a promotive effect on fat deposition,while TUSC5B has an inhibitory effect on fat deposition.(4)Differences in the regulatory mechanisms of TUSC5A and TUSC5B on fat depositionTranscriptome sequencing was completed after 4 days of induced differentiation of TUSC5A and TUSC5B precursor adipocytes and normal precursor adipocytes(control group).Compared with the control group,there were 622 differentially expressed up-regulated genes and 312 differentially expressed down-regulated genes in the TUSC5A overexpression group;2541 differentially expressed up-regulated genes and 1497differentially down-regulated genes in the TUSC5B overexpression group(|log2FC|≥1,P≤0.05).Analysis of the expression of the key genes for fat deposition,FASN,SCD1,LPL,FABP4,PPARG,GPDH and GLUT4,showed that the transcriptome sequencing results were consistent with those obtained using the q PCR method in(3).The results of GO and KEGG analysis of differentially expressed genes were further validated against each other using a two-by-two comparison between the TUSC5A overexpression group,the TUSC5B overexpression group and the control group,showing that overexpression of TUSC5A and TUSC5B resulted in significant changes in the regulation of 12 pathways related to lipid synthesis and metabolism,including the PPAR signaling pathway and the adipocytokine signaling pathway.This means that TUSC5A and TUSC5B differ in the regulation of the above pathways,which in turn leads to differences in the ability to deposit fat.In this study,we constructed a precursor adipocyte model which overexpressing TUSC5A and TUSC5B and revealed that TUSC5A and TUSC5B have different effects on differentiation of precursor adipocytes through differential regulation of pathways including PPAR signaling pathway and adipocyte factor signaling TUSC5A.This study elucidates the contrasting roles and mechanisms played by different splice isoforms of TUSC5 in fat deposition,refining the insight into the TUSC5 gene to the level of different splice isoforms and providing a safe and rational implementation of molecular breeding in the future.