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文章题目：RNA Sequencing Identifies Upregulated KyphoscoliosisPeptidase and Phosphatidic Acid Signaling Pathways in Muscle HypertrophyGenerated by Transgenic Expression of Myostatin Propeptide

杂志：International journal of molecular sciences

发表时间：2015年                  IF：2.862

研究方法：RNA测序-solexa sequencing

摘要：

本研究通过MSTN N末端肽酶cDNA转入老鼠体内，获得肌肉肥大的老鼠模型。同时对转基因鼠和其同时出生的正常对照组进行RNA测序，比较表达谱，结果发现有132个基因在两组间骨骼肌中的表达量差异显著，97个基因上调，35个下调。其中上调的肌浆球蛋白结合蛋白H(mybph)和锌金属肽酶STE24 (Zmpste24)第一次被报道与肌肉肥大有关。另外脊柱后侧凸肽酶（Ky）在转基因鼠中也是上调的。心肌症有关的信号通路和磷脂酸信号通路（PA）也是上调的，增强的PA信号可能会增加mTOR信号，导致骨骼肌生长。

研究背景：

肌肉生长抑制素(MSTN)是转化生长因子β超家族中的一员，主要在骨骼肌组织中表达，之前有研究证实MSTN的突变或抑制可促进哺乳动物肌肉生长。为了进一步阐明被MSTN抑制导致的肌肉肥大机制，比较转基因鼠和对照组的表达谱。

实验流程：

实验结果：

通过转基因组和对照组基因表达谱比较，有132个基因在两组间骨骼肌中的表达量差异显著，其中97个基因上调，35个下调。117个基因能在数据库中被注释。其中上调的肌浆球蛋白结合蛋白H(mybph)，beta，gamma，alpha actin (Actb, actc1, actg1)、蛋白酪氨酸磷酸酶受体C (Ptprc)、锌金属肽酶STE24 (Zmpste24)以及与磷脂酸通路有关的基因（Dgki, Dgkz ,Plcd4）第一次被报道与骨骼肌肥大有关（图2）。

Figure 2. Differentially expressed genes between MSTN propeptide transgenic mice and their littermate control mice. (A) A heatmap of FPKM expression values in the five samples. Each row represents a gene ID and the column represents a sample; (B) The volcano plots reveal genes that differ significantly between two groups. The black dots represent the expression levels of genes that do not differ and the red dots represent the expression level differences.   

GO注释结果显示多部分基因富集在两种类型的细胞组成中：胞外区和胞外间隙；在生物过程中富集基因最多的是生物粘附和细胞粘附；在分子功能中显著富集的是钙离子结合通路（图3）。

Figure 3. Overview of enriched gene ontology functional classifications of muscle differential expression genes between MSTN propeptide transgenic and wild-type mice.  

通过信号通路分析鉴定出肥厚型心肌（HCM）和扩张型心肌的信号通路，均可调节肌肉生长和发育。另外，维持肌肉结构与肌细胞的信号通路也被鉴定出，包括ECM受体相互作用和焦点粘连通路。尤其是磷脂酸信号系统中编码3种关键酶（Dgki, Dgkz,Plcd4）的基因在两组中的表达量显著差异（图6、表4）。

Figure 6. A theoretical model of the negative regulation of the PA signal pathway by myostatin in skeletal muscle. Plcd4 can catalyze hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and DAG [32–34]. Diacylglycerol (DAG) can then be phosphorylated by Dgkz to produce phosphatidic acid (PA). Subsequently, PA binds to the FKBP12-rapamycine binding (FRB) domain of mTOR to induce muscle fiber hypertrophy [35,36].

作者随机选择5个差异基因进行RT-qPCR验证，基因表达模式与测序结果一致（图4/图5）。

Figure 4. Q-PCR validation of genes from RNA-seq results between MSTN propeptide transgenic and control mice. All samples were normalized to 18S RNA. (A) Tnfaip2, Mybph, Cbr2 and MSTN propeptide were highly expressed in transgenic mice (TN) compared with control mice (CN). Mss51 had lower expression in transgenic mice (TN) compared with control mice (CN); (B) Endogenous myostatin (C-terminal mature peptide) mRNA levels in transgenic mice was slightly higher than the controls, but not significantly different from the control (p > 0.05). The error bars show the SD. Two-tailed t-test was used to calculate the significance of differentially expressed genes. Mark * in Figure 4A represent the expression level significantly different between two group (p < 0.05).

Figure 5. Line fit plot of Q-PCR results and RNA-Seq data for selected genes difference expression between MSTN propeptide transgenic and control mice. Linear regression model and R-Squ ared shown in the figure.     

参考文献：Miao Y, Yang J, Xu Z, et al. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide[J]. International journal of molecular sciences, 2015, 16(4): 7976-7994.