【作者】 彭永东；

【导师】 蒋思文；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 近年来,大量的研究表明miRNA在脂肪细胞分化和脂质代谢方面起着重要的调控作用,miRNA通过靶向脂肪生成的重要转录因子和脂质代谢关键基因调控脂肪细胞分化和脂质代谢。本论文基于5月龄大白猪和梅山猪的背部脂肪组织Solexa测序获得的差异表达的miRNAs,通过对miRNAs的序列分析、靶基因预测和功能分析等,靶定miR-224、miR-146b、miR-215和miR-135a。采用双荧素酶报告基因检测、Q-PCR、Western blotting等方法对4个miRNAs的作用和功能进行了研究,主要研究结果如下：1. miR-224调控脂肪细胞分化和脂肪酸代谢经UCSC网站比对发现,包括猪在内的多种哺乳动物的miR-224定位在X染色体上的相同的位置,即GABRE基因的内含子处；通过对miR-224的前体序列和成熟序列进行比对,它们在哺乳动物中的序列高度保守。通过多种靶基因预测软件预测发现,EGR2和ACSL4可能是miR-224的靶基因。双荧光素酶报告试验证实,与对照组相比, miR-224-5p明显抑制了EGR2-3’UTR和ACSL4-3’UTR双荧光素酶报告载体的荧光活性。在3T3-L1前脂肪细胞分化过程中miR-224-5p表达量先下降后上升。miR-224-5p转染组经诱导分化甘油三脂含量明显低于对照组,同时脂肪细胞分化标志基因PPARγ、aP2的表达量也明显下降。miR-224-5p在3T3-L1前脂肪细胞分化早期通过直接靶向EGR2,抑制其表达,进而抑制其下游的C/EBPβ基因的表达；抑制表达的EGR2基因至少是部分通过抑制C/EBPβ的表达抑制脂肪细胞分化标志基因PPARy, aP2的表达,导致甘油三脂积累受阻,从而抑制脂肪细胞分化。在脂肪分化晚期,miR-224-5p直接靶向ACSL4,抑制ACSL4的mRNA和蛋白质的表达,从而抑制脂肪酸p氧化过程中脂肪酸的活化,致使脂肪酸p氧化过程受阻,从而调控脂肪酸代谢。本研究首次证实了miR-224通过靶向EGR2和ACSL4在脂肪生成的不同时期发挥着不同的生物学功能。2. miR-146b调控脂肪细胞分化miR-146b序列比对发现,miR-146b的成熟序列有很高的保守性。通过多种靶基因预测软件预测发现,Runx1t1和Smad4可能是miR.146b的靶基因。双荧光素酶报告试验证实,与对照组相比,miR-146-5p明显抑制了Runx1t1-3’UTR和Smad4-3’UTR双荧光素酶报告载体的荧光活性。miR-146b-5p转染组经诱导分化后甘油三脂含量明显高于对照组,同时脂肪细胞分化标志基因C/EBPα、PPARγ、αP2的mRNA和蛋白表达量也明显上升。miR-146b-5p在3T3-L1前脂肪细胞分化早期靶向Runx1t1,抑制其表达,可能通过影响Runx1t1与C、EBPβ的相互作用从而调控C/EBPα启动子的活性促进脂肪细胞的分化；miR-146b-5p还能够靶向Smad4,抑制其表达,至少是部分通过抑制TGFβ通路下游基因CTGF的表达促进脂肪细胞的分化。3.miR-215调控脂肪细胞分化miR-215序列比对发现,miR-215的成熟序列有很高的保守性。通过多种靶基因预测软件预测发现,FNDC3B和CTNNBIP1可能是miR-215的靶基因。双荧光素酶报告试验证实,与对照组相比,miR-215-5p明显抑制了FNDC3B一3’UTR和CTNNBIP1-3’UTR双荧光素酶报告载体的荧光活性。miR-215-5p转染组经诱导分化甘油三脂含量明显低于对照组,同时脂肪细胞分化标志基因C/EBPα、PPARγ、αP2的mRNA和蛋白表达量也明显降低。miR-215-5p在3T3-Ll前脂肪细胞分化早期靶向FNDC3B,抑制其表达,从而抑制脂肪细胞的分化；miR-215-5p还能够靶向CTNNBIPl,抑制其表达,通过促进Wnt通路下游基因c-myc和CCNDl的表达抑制脂肪的分化。4. miR-135a调控脂肪酸碳链的延长miR-135a序列比对发现,miR-135a的成熟序列有很高的保守性。通过多种靶基因预测软件预测发现,ELOVL2和ElOVL6可能是miR-135a的靶基因。双荧光素酶报告试验证实,与对照组相比,miR-135a-5p明显抑制了ELOVL2和ELOVL6双荧光素酶报告载体的荧光活性。Q-PCR检测显示：在小鼠3T3-L1脂肪细胞中,miR-135a-5p抑制了ELOVL6的表达；而在小鼠Hepa1-6肝脏细胞中,ELOVL2和ELoVL6的表达都被miR-135a-5p的超表达所抑制。mir-135a可能参与调控脂肪酸碳链的延长。综上所述,本研究的结论是：①miR-224在脂肪细胞分化早期通过靶向EGR2抑制脂肪细胞的分化,在脂肪细胞分化晚期通过靶向ACSL4调控脂肪酸代谢；②miR-146b在脂肪细胞分化早期通过靶向Runx1t1和Smad4促进脂肪细胞分化；③miR-215在脂肪细胞分化早期通过靶向FNDC3B和CTNNBIP1抑制脂肪细胞分化；④miR-135a可能参与调控脂肪酸碳链的延长。更多还原

【Abstract】 In recent years, more and more studies indicate that miRNA plays an important role in adipocyte differentiation and lipid metabolism by targeting the key adipogenic transcription factors and the key genes of lipid metabolism. This study aimed to investigate the effect of miRNA on adipocyte differentiation and lipid metabolism based on the differentional expressions miRNAs of the backfat tissues of150-day-old Large White and Meishan pigs by Solexa sequencing. Then, these miRNAs with differentional expressions were subjected to sequence analysis, target prediction and function analysis and miR-224, miR-146b, miR-215and miR-135a became the object of our study. The four miRNAs were studied by using the dual luciferase reporter gene analysis, Q-PCR assay and Western blotting etc. The main results as follows:1. mR-224regulates adipocyte differentiation and fatty acid metabolismSsc-miR-224is located on Chromosome X and the same miRNA of the other mammals is also present in the same location, the intron of GABRE, using UCSC website. Furthermore, the pre-miRNAs and the mature miRNAs of miR-224are highly conserved in many mammals.Several bioinformatics target prediction softwares were used to predict targets of miR-224. The results showed that EGR2and ACSL4may be the targets of miR-224. miR-224-5p significantly inhibited the luciferase activity of EGR2and ACSL43’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.miR-224-5p abundance decreases first and then increases during adipogenesis of3T3-L1cells. The content of triglyceride was lower in miR-224-5p group, and meanwhile the expression levels of adipogenic maker genes (PPARy, aP2) were also decreased.The mRNA expression levels of EGR2was suppressed by miR-224-5p during early adipogenesis and the downstream gene C/EBPβ was also suppressed. miR-224-5p could suppress EGR2expression by directly targeting it at least in part through indirect suppression of the C/EBPβ expression, decreasing the expressions of PPARy, aP2and accumulation of triglyceride, resulting in the inhibition of adipocyte differentiation. The mRNA and protein expression levels of ACSL4were both suppressed by miR-224-5p at terminal differentiation, inhibiting fatty acid activation of fatty acid β-oxidation and fatty acid β-oxidation, regulating fatty acid metabolism. We first found that miR-224plays different roles on different stages of adipogenesis by directly targeting EGR2and ACSL4.2. miR-146b regulates adipocyte differentiationThe mature miRNAs of miR-146b are highly conserved in many mammals through sequence alignment.Several bioinformatics target prediction softwares were used to predict targets of miR-146b. The results showed that Runxltl and Smad4may be the targets of miR-146b. miR-146b-5p significantly inhibited the luciferase activity of Runxltl and Smad43’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.The content of triglyceride was higher in miR-146b-5p group, and meanwhile the mRNA and protein expression levels of adipogenic maker genes(C/EBPa, PPARy, aP2) were also upregulated.miR-146b could suppress Runxltl expression by directly targeting it and may control the interaction of Runxltl and C/EBPβ, then prevent the transcriptional activation of the C/EBPa promoter and promote adipocyte differentiation. Furthermore, miR-146b could suppress Smad4expression by directly targeting it at least in part through suppression of the CTGF expression which is the downstream gene of TGFβ signaling pathway, resulting in promotion of adipocyte differentiation.3. miR-215regulates adipocyte differentiationThe mature miRNAs of miR-215are highly conserved in many mammals through sequence alignment. Several bioinformatics target prediction softwares were used to predict targets of miR-215. The results showed that FNDC3B and CTNNBIP1may be the targets of miR-215. miR-215-5p significantly inhibited the luciferase activity of FNDC3B and CTNNBIP13’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.The content of triglyceride was lower in miR-215-5p group, and meanwhile the mRNA and protein expression levels of adipogenic maker genes(C/EBPa, PPARy, aP2) were also decreased.miR-215-5p could suppress FNDC3B expression by directly targeting it and impair adipocyte differentiation. Furthermore, miR-215-5p could suppress CTNNBIP1expression by directly targeting it through promotion of the c-myc and CCND1expression which is the downstream gene of Wnt/β-catenin signaling pathway, resulting in inhibition of adipocyte differentiation. 4. miR-135a regulates fatty acid elongationThe mature miRNAs of miR-135a are highly conserved in many mammals through sequence alignment. Several bioinformatics target prediction softwares were used to predict targets of miR-135a. The results showed that ELOVL2and ELOVL6may be the targets of miR-135a. miR-135a-5p significantly inhibited the luciferase activity of ELOVL2and ELOVL63’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.miR-215-5p could suppress ELOVL6expression in mouse3T3-L1adipocytes and miR-215-5p also could suppress ELOVL2and ELOVL6in mouse Hepa1-6hepatocytes by using Q-PCR. miR-135a may regulate fatty acid elongation.In summary, the conclusions of this study are:①miR-224could suppress EGR2expression by directly targeting it and impair adipocyte differentiation during early adipogenesis; miR-224also could suppress ACSL4expression by directly targeting it and regulate fatty acid metabolism.②miR-146b could suppress Runxltl and Smad4expression by directly targeting them and promote adipocyte differentiation during early adipogenesis.③miR-215could suppress FNDC3B and CTNNBIP1expression by directly targeting them and impair adipocyte differentiation during early adipogenesis.④miR-135a may regulate fatty acid elongation.更多还原