选择性多聚腺苷酸化在牛肌内前脂肪细胞分化中的功能研究

The mRNA alternative polyadenylation (APA) in eukaryotes not only generates proteomic and functional diversity, but also plays important roles in regulating gene expression. In the past few years, exciting results have further highlighted the crucial role of APA in the control of global physiological events such as proliferation, differentiation, transformation, and development programs. But the APA expression profile and its functions during the differentiation of bovine intramuscular preadipocytes is still unclear. In the research, the entire transcriptome differential expression profiles of APA during preadipocytes differentiation were analyses by SAPAS (sequence of APA site) technology. Candidate genes with APA involved in fat metabolism pathways were screened by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes). The mechanisms APA regulating the gene expression were investigated by molecular biology technology. At the same time, the recombinant siRNA Lentivirus vector targeted candidate gene and recombinant Lentivirus vector carrying siRNA-resistant candidate genes with different 3'-UTR isoforms were constructed. The bovine preadipocytes were transfected or co-transfected using Lipofectamine2000 with siRNA Lentivirus vector and/or siRNA-resistant Lentivirus vector to overexpress one isoform with certain 3'-UTR or down regulate the expression of endogenous candidate gene and rescue expression of one isoform carrying mutation type candidate gene at the same time. The effect of different isoforms of candidate gene on differentiation of bovine preadipocytes were further observed and detected by cytochemical staining, qRT-PCR (real time quantitative PCR), northern blot and western blot. The results of this study should make a foundation for the regulation mechanisms of bovine intramuscular fat deposition and the discovery of new functional candidate genes related to fat deposition.

选择性多聚腺苷酸化(APA)在基因表达调控中起着重要作用。大量研究表明APA对细胞的增殖、分化和发育等也有关键的调控作用。但APA在脂肪细胞分化过程中的表达模式及功能研究尚属空白。本研究采用SAPAS高通量测序技术检测牛肌内前脂肪细胞分化过程中APA表达谱的差异；利用GO和KEGG方法筛选出参与代谢通路的具有APA的候选基因，利用分子生物学技术研究APA调控候选基因表达的机制；构建靶向候选基因的siRNA慢病毒表达载体和抗siRNA的候选基因APA变异体的表达载体，二者分别或共转染牛肌内前脂肪细胞，过表达某种变异体或在抑制内源性侯选基因表达的同时回复表达某种变异体，应用细胞化学染色、qRT-PCR、Northern blot、Western blot等方法分析该变异体对牛肌内前脂肪细胞分化的影响。研究结果为进一步揭示牛肌内脂肪沉积的调控机制，挖掘牛肌内脂肪沉积相关的新功能基因奠定了基础。

选择性多聚腺苷酸化(APA)在基因表达调控中起着重要作用，而且APA对细胞的增殖、分化和发育等也有关键的调控作用中起着重要作用。采用RNA-Seq与SAPAS高通量测序技术相结合的分析方法，并利用相关的分子生物学技术研究了脂肪细胞分化过程中APA变化模式及其功能。 主要研究结果或发现有：（1）RNA-Seq共鉴定了3T3-L1前脂肪细胞与其分化的脂肪细之间的差异表达基因4443个，其中上调和下调基因分别为2013、2430个；SAPAS测序共获得RPKM>5的基因6954个，其中2027个有2个以上的tandem APA位点。结果显示tandem 3’UTR在脂肪细胞分化过程中有变长的趋势。将Fold Change≥3且FDR<0.01作为筛选标准，在牛肌内和皮下前脂肪细胞分化过程中分别发现611和825个显着的差异表达基因。结合上述结果，重点选择BBOX1、AAMDC、MSMO1和NSDHL等基因进行了研究；（2）获得了BBOX1基因的3'UTR的3个不同APA变异体。在研究群体中检测到BBOX1基因3’UTR中存在10个多态性座位。关联分析显示c.512_513insTGC 和 c.650T>C两个多态性座位与肌纤维直径、剪切力、净肉重和胴体重显著相关；（3）研究了AAMDC基因3’UTR的不同APA变异体对表达的影响，与长3’UTR相比，AAMDC基因的短3’UTR的APA变异体明显增加其蛋白表达量并提高mRNA的稳定性和翻译效率。更重要的发现是miR-2428/664a能与长3’UTR的APA变异体独有区序列结合抑制其表达量，进而揭示短3’UTR的APA变异体表达上调的机制是逃避了miR-2428/664a的调控。研究显示AAMDC 短的APA变异体促进前脂肪细胞分化；（4）研究发现MSMO1在3T3-L1脂肪细胞分化过程中是显著下调的。推测MSMO1可能对脂肪细胞分化具有调控作用，研究证实MSMO1基因是脂肪细胞分化的重要调控者，并发现NSDHL是MSMO1的下游基因，是调控脂肪生成的重要通路。通过该项目的资助，获得了15项成果，包括发表论文11篇，其中SCI论文5篇；获得山东省高等学校科学技术奖三等奖1项，培养毕业研究生3名。圆满完成了基金任务书规定的目标任务。