Screening Of Key Genes Related To Porcine Fat Deposition And Functional Study Of Zinc Finger Protein KLF13

Subcutaneous fat and intramuscular fat are different fat depots. From the perspective of pig production, adipose tissue from different fat depots contributes differentially to carcass traits and meat quality. Subcutaneous fat is the layer of subcutaneous tissue that mainly affects pig carcass quality. Intramuscular fat is deposited within muscle, which is an important factor affecting pork quality. Subcutaneous fat develops earlier than intramuscular fat. Preadipcoytes originating from subcutaneous and intramuscular fat have distinct adipogenic potential. These results indicated that the regulation of adipogenesis of subcutaneous and intramuscular adipocytes were different. Hence, in the present study, RNA-Seq technology was used to compare the differences of gene expression levels between subcutaneous SV cell (ASVC) and intramuscular SV cell (MSVC) differentiation on days0,2, and4, and the differentially expressed genes (DEGs) were screened during ASVC and MSVC adipogenic differentiation, respectively. The main objective was to identify and verify the regulatory factors during porcine adipogenic differentiation.The contents of the research were mainly composed of three parts: the first part was to investigate the diffeneces of genes expression between ASVC and MSVC during adipogenic differentiation, and reveal the expression pattern of DEGs between ASVC and MSVC during adipogenic differentiation. The second part was to investigate the change of genes during adipogenic differentiation of ASVC and MSVC, and use bioinformatics analysis to identify candidate regualatory factors affecting porcine adipogenic differentiation. The third part was to investigate the effect of KLF13on porcine adipogenic differentiation, and to elucidate the regulation mechanism of KLF13affecting porcine adipogenic differentiation.The first part: SV cells were collected from postnatal porcine longissimus dorsi muscle and subcutaneous adipose tissue, and then induced to differentiate into adipocytes in vitro. RNA-Seq was used to screen DEGs between ASVC and MSVC adipogenic differentiation on days0,2, and4, respectively. Then, GO and pathway analysis were preformed to further understand the biological functions of DEGs between ASVC and MSVC. Finally, the expression pattern of DEGs between ASVC and MSVC were determined by STEM platform during adipogenic differentiation. The main results are as follows:1. The number of DEGs between ASVC and MSVC was gradually diminished as the adipogenic differentiation.1509DEGs were detected between ASVC and MSVC on day0;415genes were differentially expressed between ASVC and MSVC on day2and258genes were differentially expressed between ASVC and MSVC on day4.2. GO analysis was preformed to further understand the biological functions of DEGs between ASVC and MSVC. Results showed that genes up-regulated on ASVC day0of adipogenic differentiation were mainly enriched in regulation of cell adhesion, antin binding, adherens junction and so on. The genes up-regulated on MSVC day0of adipogenic differentiation were mainly enriched in regulation of cell proliferation, carbohydrate binding, extracellular region and so on. The genes up-regulated on ASVC day2of adipogenic differentiation were mainly enriched in response to hormone stimulus, actin binding, actin cytoskeleton and so on. The genes up-regulated on MSVC day2of adipogenic differentiation were mainly enriched in regulation of response to external stimulus, glycosaminoglycan binding, extracellular region part and so on. The genes up-regulated on ASVC day4of adipogenic differentiation were mainly enriched in response to protein stimulus, polysaccharide binding, extracellular region and so on. The genes up-regulated on MSVC day4of adipogenic differentiation were mainly enriched in regulation of response to external stimulus, extracellular region and so on.3. The expression profiles of DEGs between ASVC and MSVC on day0were determined by cluster analysis based on the STEM platform. The results indicated that the up-regulated genes on ASVC differentiation day0compared with MSVC were inhibited during ASVC adipogenic differentiation. The down-regulated genes on ASVC differentiation day0compared with MSVC were inhibited during ASVC adipogenic differentiation. Additionally, the up-regulated genes on MSVC differentiation day0compared with ASVC were inhibited during MSVC adipogenic differentiation. The down-regulated genes on MSVC differentiation day0compared with ASVC were inhibited during MSVC adipogenic differentiation.The second part:based on the results of RNA-Seq derived from the first part, DEGs were respectively screened during ASVC and MSVC differentiation. Then, bio informatics analysis was used to identify candidate regualatory factors affecting porcine adipogenic differentiation. The main results are as follows:1. The DEGs were screened during ASVC and MSVC adipogenic differentiation, respectively. The results indicated that985DEGs were expressed in ASVC differentiation and1469DEGs were expressed in MSVC differentiation. Among these DEGs,576DEGs were co-expressed in ASVC and MSVC differentiation,409DEGs were specific for ASVC differentiation, and893DEGs were specific for MSVC differentiation.2. The expression profiles of DEGs during ASVC and MSVC adipogenic differentiation were determined by cluster analysis based on the STEM platform. The results showed that4expression profiles (profile1,4,5, and14) were significantly enriched in ASVC differentiation (.P<0.05), and4expression profiles (profile1,4,11, and14) were significantly enriched in MSVC differentiation (P<0.05).3. GO analysis was preformed to further understand the biological functions of the genes within significant gene expression profiles. Results showed that DEGs related to adipocyte differentiation and fatty metabolism were significantly enriched in both adipose and muscle gene profile14(P<0.05). The genes in adipose profile14or muscle profiles11and14were mapped to terms in the KEGG database. The results showed that genes within adipose profile14were significantly enriched in tryptophan metabolism, PPAR signaling pathway, arginine and proline metabolism, as well as glycerolipid metabolism (P<0.05). Genes within muscle profiles11and14were significantly enriched in peroxisome, ECM-receptor interaction, PI3K-Akt signaling pathway, PPAR signaling pathway, as well as fatty acid metabolism (P<0.05).4.114transcription factor genes of the DEGs during ASVC and MSVC differentiation were identified. The tool of Genmatix Matlnspector was used to scan transcription factor binding sites within the promoter of PPARγ. Among the114 transcription factors, the binding sites of19transcription factors were identified within PPARy promoter. The19transcription factors were STAT1, C/EBPβ, STAT2, Enl, ETV1, KLF13, KLF15, BCL6, HES1, ZNF217, VDR, PPARy, ETS1, RUNX1, GATA3, HMGA1, GATA2, DLX2, and Osrl.In the third part, we used "gain-of-function" and "loss-of-function" experiments to investigate the role of candidate transcription factor KLF13in porcine adipogenic differentiation, and then the molecular mechanism of KLF13regualting porcine adipogenic differentiation were investigated through promoter reporter system and ChIP trial. Finally, we evaluated the effect of KLF13on mouse adipogenic differentiation to confirm whether exist the species-specific differences of KLF13regulating adipognic differentiation in porcine and mouse. The main results are as follows:1. The expression of KLF13was markly upregualted during the early stage of ASVC adipogenic differentiation, reaching a maximum at36h. Inhibition of KLF13by siRNA suppressed ASVC adipogenic differentiation, and significantly inhibited expression of PPARy and aP2(P<O.01). Conversly, overexpression of KLF13stimulated ASVC adipogenic differentiation, and significantly up-regualted expression of PPARy (P0.01), aP2and Adiponectin (P<0.05).2. The expression of KLF13was markly upregualted during the early stage of MSVC adipogenic differentiation, reaching a maximum at36h. Inhibition of KLF13by siRNA suppressed ASVC adipogenic differentiation, and significantly inhibited expression of PPARy and Adiponectin (P<0.01), and aP2(P<0.05).3. The expression of KLF13was markly upregualted during the early stage of porcine DFAT (dedifferentiated fat) cells adipogenic differentiation, reaching a maximum at48h. Inhibition of KLF13by siRNA suppressed ASVC adipogenic differentiation, and significantly inhibited expression of PPARy and Adiponectin (P<0.01), and aP2(P<0.05).4. After2days transfection of KLF13siRNA or pcDNA3.1-KLF13, adipose SV cells were harvested. Real-time PCR was used to determine the mRNA expression of adipocyte differentiation-related genes. The results indicated that Ebfl, PPARy and C/EBPa showed coordinate regulation between KLF13overexpression and knockdown. After1day transfection of KLF13siRNA or pcDNA3.1-KLF13, adipose SV cells were stimulated in adipogenic induction medium for2days. Real-time PCR was used to determine the mRNA expression of adipocyte differentiation-related genes. The results indicated that KLF15, PPARγ and C/EBPα showed coordinate regulation between KLF13overexpression and knockdown. We overlapped the over results, and PPARγ and C/EBPα were identified as candidate target genes of KLF13.5. Bio informatics analysis predicted that there was no KLF13binding site in porcine C/EBPα promoter, but there was a KLF13binding site in porcine PPARγ promoter. The results of promoter truncation, mutation and deletion indicated that KLF13could interact with the-653～-636sequence located in porcine PPARγ promoter. ChIP assay indicated that KLF13could bind to porcine PPARγ promoter in cells.6. The expression of KLF13was not significantly changed during the first6days of3T3-L1adipogenic differentiation. Inhibition of KLF13by siRNA did not influence ASVC adipogenic differentiation, and expression of PPARγ, Adiponectin and aP2on3T3-L1differentiation day8. After1day transfection of KLF13siRNA or pcDNA3.1-KLF13, adipose SV cells were stimulated in adipogenic induction medium for2days. Real-time PCR was used to determine the mRNA expression of adipocyte differentiation-related genes. The results indicated that the expression of PPARγ was not regulated by KLF13overexpression and knockdown. Finally, porcine PPARγ and mouse PPARγ2promoter reporter were respectively co-transfected with pcDNA3.1-KLF13into3T3-L1cells. The results showed that KLF13could significantly promote the activity of porcine PPARγ promoter (P<0.01), but not mouse PPARγ2promoter.In summary, the conclusions of this study are:1. Great difference in gene expression exists between ASVC and MSVC, but the difference will gradually diminish as the adipogenic differentiation.2. DEGs related to adipocyte differentiation and fatty metabolism during ASVC and MSVC adipogenic differentiation were significantly enriched in the same expression profile, and the signaling pathways that regulate DEGs related to adipocyte differentiation and fatty metabolism were different between ASVC and MSVC adipogenic differentiation.3. KLF13s a key pro-adipogenic transcription factor through regulating PPARy transactivation at the early stage of porcine adipogenic differentiation, but KLF13do not influence mouse PPARy2transactivation.