猪脂肪沉积关键基因的筛选及锌指蛋白KLF13的功能研究
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摘要
从生产角度,脂肪组织在不同部位的沉积对猪胴体性状和肉品质产生不同的影响,皮下脂肪沉积在皮下组织主要影响猪胴体性状,而肌内脂肪沉积在肌肉组织中主要影响猪肉品质。在组织发育特征上,皮下脂肪和肌内脂肪具有明显的时空特异性；进一步研究发现,在脂肪细胞生成过程中,来源于皮下和肌内的脂肪前体细胞呈现出不同的分化潜能。这些结果均暗示了在皮下和肌内脂肪前体脂肪成脂分化过程中可能存在不同的调控机制。因此,本研究的目的一方面通过比较猪皮下和肌内脂肪前体细胞在成脂分化过程中基因的表达水平,研究猪皮下和肌内脂肪前体细胞在分化过程中转录水平上的差异；另一方面通过筛选和分析猪皮下和肌内脂肪前体细胞成脂分化过程中的差异表达基因,鉴定出影响猪皮下和肌内脂肪分化的重要调控因子。
本研究的内容分为三部分：第一部分主要是利用RNA-Seq技术比较皮下和肌内脂肪前体细胞在成脂分化过程中基因的表达水平；并研究皮下和肌内脂肪前体细胞之间的差异表达基因在成脂分化过程中表达变化规律。第二部分主要是在猪皮下和肌内脂肪前体细胞成脂分化过程中分别研究基因表达的变化情况,并通过后续的生物信息学分析来靶定影响猪脂肪分化的重要候选调控因子。第三部分主要是通过分子生物学实验验证候选调控因子在猪脂肪分化过程中的作用,并对其调控机制进行研究。
第一部分,以猪的皮下脂肪和肌肉来源的血管基质(stromal vascular, SV)细胞为研究材料,利用高通量的RNA-Seq技术首先比较了皮下脂肪SV细胞(subcutaneous SV cell, ASVC)和肌内SV细胞(intramuscular SV cell, MSVC)在成脂分化第0d、2d和4d基因表达的差异；然后对ASVC和MSVC之间DEGs进行功能注释,最后研究ASVC和MSVC之间DEGs在成脂分化过程中的表达变化规律。主要的结果如下：
1、ASVC和MSVC之间的DEGs随着成脂分化时间的延长而逐渐减少。在成脂分化第0d,MSVC与ASVC相比有1509个DEGs;到成脂分化第2d,MSVC与ASVC相比DEGs减少到415个；最后在成脂分化第4d,MSVC与ASVC相比只有258个DEGs。
2、通过对ASVC和MSVC之间DEGs的功能注释发现,在分化前,ASVC中高表达的基因主要富集在细胞粘附、肌动蛋白的结合和粘附连接等功能单位中；MSVC中高表达的基因主要富集在调控细胞增殖、碳水化合物结合、胞外空间和基质等功能单位中。在成脂分化第2天,ASVC中高表达的基因主要富集在对激素刺激反应、肌动蛋白的结合和细胞骨架肌动蛋白等功能单位；MSVC中高表达的基因主要富集在外部刺激反应的调控、葡萄糖胺聚糖结合和胞外区域等功能单位。在成脂分化第4天,ASVC中高表达基因主要富集在蛋白刺激反应、多糖的结合和胞外区域等功能单位中,MSVC中高表达基因主要富集在外部刺激反应的调控和胞外区域等功能单位中。
3、针对ASVC与MSVC之间在成脂分化第0天的DEGs,利用STEM软件将它们在ASVC和MSVC成脂分化过程中表达模式(expression profile)进行聚类分析。结果显示,ASVC相比MSVC在成脂分化第0天高表达的基因在ASVC成脂分化过程中呈现下调表达的趋势,而ASVC相比MSVC下调表达的基因在ASVC成脂分化过程中呈现上调表达的趋势。另外,MSVC相比ASVC在成脂分化第0天高表达的基因在MSVC成脂分化过程中呈现下调表达的趋势,而MSVC相比ASVC下调表达的基因在MSVC成脂分化过程中呈现上调表达的趋势。
第二部分,在第一部分测序结果的基础上,分别筛选皮下和肌内SV细胞成脂分化过程(0d、2d和4d)中的差异表达基因(differentially expressed genes, DEGs);最后通过生物信息学分析靶定影响皮下脂肪和肌内SV细胞分化的重要候选调控因子。主要的结果如下：
1、分别对ASVC和MSVC成脂分化过程中DEGs筛选的结果显示,ASVC成脂分化过程中有985个DEGs, MSVC成脂分化过程中有1469个DEGs,其中409个DEGs在ASVC成脂过程中特异性差异表达,893个DEGs在MSVC成脂过程中特异性差异表达,576个DEGs在ASVC和MSVC成脂过程中共同差异表达。
2、利用STEM软件将ASVC成脂分化过程中的985个DEGs,以及MSVC成脂分化过程中的1469个DEGs进行表达模式聚类分析。STEM聚类分析显示各有4个表达模式分别显著性地富集于ASVC(profile1、4、5and14)和MSVC(profile1、4、11and14)成脂分化过程中(P<0.05)。
3、对ASVC和MSVC成脂分化过程中显著性富集表达模式中的DEGs进行功能注释。GO分析结果表明,ASVC和MSVC成脂分化过程中与脂肪分化和脂肪代谢相关的DEGs显著性富集在相同的表达模式(profile14)中。Pathway分析结果显示,ASVC的profile14中所包含的DEGs显著性富集在色氨酸代谢通路、PPAR信号通路、精氨酸和脯氨酸代谢通路,以及甘油酯代谢信号通路中(P<0.05)；MSVC的profile14和11中所包含的DEGs显著性富集在过氧物酶体通路、胞外基质受体互作通路、P13K-Akt信号通路、PPAR信号通路和脂肪酸代谢信号通路中(P<0.05)；其中PPAR信号通路在ASVC和MSVC成脂分化过程中共同显著性富集(P<0.05)。
4、在ASVC和MSVC成脂分化过程差异表达的基因中,筛选到114个转录因子基因；通过在线软件Genomatix中的MatInspector工具预测,这114个转录因子基因中有19个转录因子在猪源PPARγ启动子有结合位点。这个19个转录因子分别是STAT1、C/EBPβ、STAT2、En1、ETV1、KLF13、KLF15、BCL6、HES1、ZNF217、 VDR、PPARγ、ETS1、RUNX1、GATA3、HMGA1、GATA2、DLX2和Osr1。
第三部分,以第二部分筛选的转录因子KLF13为研究对象,首先通过KLF13的功能“获得”和“缺失”试验确定KLF13在猪脂肪分化过程中的作用,然后筛选KLF13发挥作用所介导的直接靶基因,随后通过启动子活性试验和染色质免疫共沉淀试验验证KLF13是否直接通过转录调控靶基因的活性来影响猪脂肪分化的,最后利用鼠源脂肪前体脂肪细胞系研究KLF13在鼠脂肪分化过程中的作用,来确定KLF13调控猪源和鼠源成脂分化是否存在种属特异性。
1、KLF13在猪脂肪SV细胞成脂分化早期被诱导表达,]mRNA水平在36h达到最高,随后表达量下降。用siRNA干涉KLF13的表达会明显降低脂肪SV细胞的成脂效率,并且极显著抑制脂肪SV细胞成脂分化第8天成脂关键基因PPARγ和aP2的表达(P<0.01)；反之,当超表达KLF13可以促进猪脂肪SV细胞的成脂分化,并且极显著地上调脂肪SV细胞成脂分化第8天PPARγ的表达(P<0.01),显著地提高aP2和Adiponectin的表达量(P<0.05)。
2、KLF13在猪肌肉SV细胞成脂分化早期被诱导表达,mRNA水平在36h达到最高,随后表达量下降。用siRNA干涉KLF13的表达会明显降低肌肉SV细胞的成脂效率,并且极显著抑制肌肉SV细胞成脂分化第8天PPARγ和Adiponectin的表达(P<0.01),以及显著抑制aP2的表达(P<0.05)。
3、KLF13在猪去分化的脂肪细胞(dedifferentiated fat cells, DFAT cells)成脂分化早期被诱导表达,mRNA水平在48h达到最高,随后表达量下降。用siRNA干涉KLF13的表达会明显降低DFAT细胞的成脂效率,并且siRNA处理能够极显著抑制DFAT细胞成脂分化第8天成脂关键基因PPARγ和Adiponectin的表达(P<0.01),以及显著抑制aP2的表达(P<0.05)。
4、在脂肪SV细胞正常生长条件中直接干涉或超表达KLF13,检测成脂分化关键基因的表达变化情况,发现Ebf1、PPARγ和C/EBPα的表达会随着KLFl3表达的改变而发生同步变化。另外,在脂肪SV细胞成脂诱导条件中干涉或超表达KLF13,检测成脂分化关键基因的表达变化情况,发现KLF15、PPARγ和C/EBPα的表达会随着KLF13表达的改变而发生同步变化。综合这两部分的结果,我们将PPARγ和C/EBPα作为KLF13调控成脂分化所介导的直接候选靶基因。
5、通过启动子上转录因子结合位点预测发现,在猪C/EBPα启动子上没有KLF13的结合位点,而在猪PPARγ启动子存在一个KLF13的结合位点。通过启动子截短、突变和删除试验证明,KLF13可以直接作用于PPARγ启动子上-653～-636区域的"CTCCC"序列。染色质免疫共沉淀试验证实了KLF13能在猪脂肪SV细胞内结合PPARγ启动子。
6、在鼠前脂肪细胞系3T3-L1细胞成脂分化的前6天,KLF13的表达量没有出现明显的变化。用siRNA干涉KLF13的表达对3T3-L1前脂肪细胞的分化第8天的成脂表型,以及成脂相关基因PPARγ2、aP2和Adiponectin的表达都没有显著影响。在3T3-L1前脂肪细胞成脂诱导条件,分别干涉和超表达KLF13对PPARγ2的表达均没有显著影响。最后在鼠3T3-L1前脂肪细胞中,分别将猪PPARγ的启动子报告质粒和鼠PPARγ2的启动子报告质粒与KLF13的表达质粒共转染3T3-L1前脂肪细胞。研究结果发现KLF13可以极显著上调猪PPARγ的启动子活性(P<0.01),但对鼠PPARγ2的启动子活性没有显著性影响。
综上所述,本研究的结论是：
1、从猪皮下脂肪和肌肉分离的SV细胞在基因表达水平上存在较大差异,但这种差异会随着成脂分化时间的延长而逐渐减小。
2、在猪皮下和肌内SV细胞成脂分化过程中,与脂肪分化相关的基因呈现相同的表达模式,但调控皮下和肌内SV细胞成脂分化相关基因的信号通路存在较大差异。
3、KLF13是猪脂肪分化早期的一个正调控因子,其发挥作用是通过PPARγ介导的。KLF13可以直接转录激活猪PPARγ的表达来促进猪成脂分化,但对鼠PPARγ2的转录活性却没有影响。
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 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.
本研究的内容分为三部分：第一部分主要是利用RNA-Seq技术比较皮下和肌内脂肪前体细胞在成脂分化过程中基因的表达水平；并研究皮下和肌内脂肪前体细胞之间的差异表达基因在成脂分化过程中表达变化规律。第二部分主要是在猪皮下和肌内脂肪前体细胞成脂分化过程中分别研究基因表达的变化情况,并通过后续的生物信息学分析来靶定影响猪脂肪分化的重要候选调控因子。第三部分主要是通过分子生物学实验验证候选调控因子在猪脂肪分化过程中的作用,并对其调控机制进行研究。
第一部分,以猪的皮下脂肪和肌肉来源的血管基质(stromal vascular, SV)细胞为研究材料,利用高通量的RNA-Seq技术首先比较了皮下脂肪SV细胞(subcutaneous SV cell, ASVC)和肌内SV细胞(intramuscular SV cell, MSVC)在成脂分化第0d、2d和4d基因表达的差异；然后对ASVC和MSVC之间DEGs进行功能注释,最后研究ASVC和MSVC之间DEGs在成脂分化过程中的表达变化规律。主要的结果如下：
1、ASVC和MSVC之间的DEGs随着成脂分化时间的延长而逐渐减少。在成脂分化第0d,MSVC与ASVC相比有1509个DEGs;到成脂分化第2d,MSVC与ASVC相比DEGs减少到415个；最后在成脂分化第4d,MSVC与ASVC相比只有258个DEGs。
2、通过对ASVC和MSVC之间DEGs的功能注释发现,在分化前,ASVC中高表达的基因主要富集在细胞粘附、肌动蛋白的结合和粘附连接等功能单位中；MSVC中高表达的基因主要富集在调控细胞增殖、碳水化合物结合、胞外空间和基质等功能单位中。在成脂分化第2天,ASVC中高表达的基因主要富集在对激素刺激反应、肌动蛋白的结合和细胞骨架肌动蛋白等功能单位；MSVC中高表达的基因主要富集在外部刺激反应的调控、葡萄糖胺聚糖结合和胞外区域等功能单位。在成脂分化第4天,ASVC中高表达基因主要富集在蛋白刺激反应、多糖的结合和胞外区域等功能单位中,MSVC中高表达基因主要富集在外部刺激反应的调控和胞外区域等功能单位中。
3、针对ASVC与MSVC之间在成脂分化第0天的DEGs,利用STEM软件将它们在ASVC和MSVC成脂分化过程中表达模式(expression profile)进行聚类分析。结果显示,ASVC相比MSVC在成脂分化第0天高表达的基因在ASVC成脂分化过程中呈现下调表达的趋势,而ASVC相比MSVC下调表达的基因在ASVC成脂分化过程中呈现上调表达的趋势。另外,MSVC相比ASVC在成脂分化第0天高表达的基因在MSVC成脂分化过程中呈现下调表达的趋势,而MSVC相比ASVC下调表达的基因在MSVC成脂分化过程中呈现上调表达的趋势。
第二部分,在第一部分测序结果的基础上,分别筛选皮下和肌内SV细胞成脂分化过程(0d、2d和4d)中的差异表达基因(differentially expressed genes, DEGs);最后通过生物信息学分析靶定影响皮下脂肪和肌内SV细胞分化的重要候选调控因子。主要的结果如下：
1、分别对ASVC和MSVC成脂分化过程中DEGs筛选的结果显示,ASVC成脂分化过程中有985个DEGs, MSVC成脂分化过程中有1469个DEGs,其中409个DEGs在ASVC成脂过程中特异性差异表达,893个DEGs在MSVC成脂过程中特异性差异表达,576个DEGs在ASVC和MSVC成脂过程中共同差异表达。
2、利用STEM软件将ASVC成脂分化过程中的985个DEGs,以及MSVC成脂分化过程中的1469个DEGs进行表达模式聚类分析。STEM聚类分析显示各有4个表达模式分别显著性地富集于ASVC(profile1、4、5and14)和MSVC(profile1、4、11and14)成脂分化过程中(P<0.05)。
3、对ASVC和MSVC成脂分化过程中显著性富集表达模式中的DEGs进行功能注释。GO分析结果表明,ASVC和MSVC成脂分化过程中与脂肪分化和脂肪代谢相关的DEGs显著性富集在相同的表达模式(profile14)中。Pathway分析结果显示,ASVC的profile14中所包含的DEGs显著性富集在色氨酸代谢通路、PPAR信号通路、精氨酸和脯氨酸代谢通路,以及甘油酯代谢信号通路中(P<0.05)；MSVC的profile14和11中所包含的DEGs显著性富集在过氧物酶体通路、胞外基质受体互作通路、P13K-Akt信号通路、PPAR信号通路和脂肪酸代谢信号通路中(P<0.05)；其中PPAR信号通路在ASVC和MSVC成脂分化过程中共同显著性富集(P<0.05)。
4、在ASVC和MSVC成脂分化过程差异表达的基因中,筛选到114个转录因子基因；通过在线软件Genomatix中的MatInspector工具预测,这114个转录因子基因中有19个转录因子在猪源PPARγ启动子有结合位点。这个19个转录因子分别是STAT1、C/EBPβ、STAT2、En1、ETV1、KLF13、KLF15、BCL6、HES1、ZNF217、 VDR、PPARγ、ETS1、RUNX1、GATA3、HMGA1、GATA2、DLX2和Osr1。
第三部分,以第二部分筛选的转录因子KLF13为研究对象,首先通过KLF13的功能“获得”和“缺失”试验确定KLF13在猪脂肪分化过程中的作用,然后筛选KLF13发挥作用所介导的直接靶基因,随后通过启动子活性试验和染色质免疫共沉淀试验验证KLF13是否直接通过转录调控靶基因的活性来影响猪脂肪分化的,最后利用鼠源脂肪前体脂肪细胞系研究KLF13在鼠脂肪分化过程中的作用,来确定KLF13调控猪源和鼠源成脂分化是否存在种属特异性。
1、KLF13在猪脂肪SV细胞成脂分化早期被诱导表达,]mRNA水平在36h达到最高,随后表达量下降。用siRNA干涉KLF13的表达会明显降低脂肪SV细胞的成脂效率,并且极显著抑制脂肪SV细胞成脂分化第8天成脂关键基因PPARγ和aP2的表达(P<0.01)；反之,当超表达KLF13可以促进猪脂肪SV细胞的成脂分化,并且极显著地上调脂肪SV细胞成脂分化第8天PPARγ的表达(P<0.01),显著地提高aP2和Adiponectin的表达量(P<0.05)。
2、KLF13在猪肌肉SV细胞成脂分化早期被诱导表达,mRNA水平在36h达到最高,随后表达量下降。用siRNA干涉KLF13的表达会明显降低肌肉SV细胞的成脂效率,并且极显著抑制肌肉SV细胞成脂分化第8天PPARγ和Adiponectin的表达(P<0.01),以及显著抑制aP2的表达(P<0.05)。
3、KLF13在猪去分化的脂肪细胞(dedifferentiated fat cells, DFAT cells)成脂分化早期被诱导表达,mRNA水平在48h达到最高,随后表达量下降。用siRNA干涉KLF13的表达会明显降低DFAT细胞的成脂效率,并且siRNA处理能够极显著抑制DFAT细胞成脂分化第8天成脂关键基因PPARγ和Adiponectin的表达(P<0.01),以及显著抑制aP2的表达(P<0.05)。
4、在脂肪SV细胞正常生长条件中直接干涉或超表达KLF13,检测成脂分化关键基因的表达变化情况,发现Ebf1、PPARγ和C/EBPα的表达会随着KLFl3表达的改变而发生同步变化。另外,在脂肪SV细胞成脂诱导条件中干涉或超表达KLF13,检测成脂分化关键基因的表达变化情况,发现KLF15、PPARγ和C/EBPα的表达会随着KLF13表达的改变而发生同步变化。综合这两部分的结果,我们将PPARγ和C/EBPα作为KLF13调控成脂分化所介导的直接候选靶基因。
5、通过启动子上转录因子结合位点预测发现,在猪C/EBPα启动子上没有KLF13的结合位点,而在猪PPARγ启动子存在一个KLF13的结合位点。通过启动子截短、突变和删除试验证明,KLF13可以直接作用于PPARγ启动子上-653～-636区域的"CTCCC"序列。染色质免疫共沉淀试验证实了KLF13能在猪脂肪SV细胞内结合PPARγ启动子。
6、在鼠前脂肪细胞系3T3-L1细胞成脂分化的前6天,KLF13的表达量没有出现明显的变化。用siRNA干涉KLF13的表达对3T3-L1前脂肪细胞的分化第8天的成脂表型,以及成脂相关基因PPARγ2、aP2和Adiponectin的表达都没有显著影响。在3T3-L1前脂肪细胞成脂诱导条件,分别干涉和超表达KLF13对PPARγ2的表达均没有显著影响。最后在鼠3T3-L1前脂肪细胞中,分别将猪PPARγ的启动子报告质粒和鼠PPARγ2的启动子报告质粒与KLF13的表达质粒共转染3T3-L1前脂肪细胞。研究结果发现KLF13可以极显著上调猪PPARγ的启动子活性(P<0.01),但对鼠PPARγ2的启动子活性没有显著性影响。
综上所述,本研究的结论是：
1、从猪皮下脂肪和肌肉分离的SV细胞在基因表达水平上存在较大差异,但这种差异会随着成脂分化时间的延长而逐渐减小。
2、在猪皮下和肌内SV细胞成脂分化过程中,与脂肪分化相关的基因呈现相同的表达模式,但调控皮下和肌内SV细胞成脂分化相关基因的信号通路存在较大差异。
3、KLF13是猪脂肪分化早期的一个正调控因子,其发挥作用是通过PPARγ介导的。KLF13可以直接转录激活猪PPARγ的表达来促进猪成脂分化,但对鼠PPARγ2的转录活性却没有影响。
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
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.
引文
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