Department of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
Bone. 2011 Mar 1;48(3):543-51. doi: 10.1016/j.bone.2010.11.006. Epub 2010 Nov 21.
It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to evaluate the extent of alternative splicing in a bone subjected to mechanical loading and subsequent bone formation. We used the rat forelimb loading model, in which the right forelimb was loaded axially for 3 min, while the left forearm served as a non-loaded control. Animals were subjected to loading sessions every day, with 24 h between sessions. Ulnae were sampled at 11 time points, from 4 h to 32days after beginning loading. RNA was isolated and mRNA abundance was measured at each time point using Affymetrix exon arrays (GeneChip® Rat Exon 1.0 ST Arrays). An ANOVA model was used to identify potential alternatively spliced genes across the time course, and five alternatively spliced genes were validated with qPCR: Akap12, Fn1, Pcolce, Sfrp4, and Tpm1. The number of alternatively spliced genes varied with time, ranging from a low of 68 at 12h to a high of 992 at 16d. We identified genes across the time course that encoded proteins with known functions in bone formation, including collagens, matrix proteins, and components of the Wnt/β-catenin and TGF-β signaling pathways. We also identified alternatively spliced genes encoding cytokines, ion channels, muscle-related genes, and solute carriers that do not have a known function in bone formation and represent potentially novel findings. In addition, a functional characterization was performed to categorize the global functions of the alternatively spliced genes in our data set. In conclusion, mechanical loading induces alternative splicing in bone, which may play an important role in the response of bone to mechanical loading.
据估计，超过 90%的人类基因由于选择性剪接而表达多个 mRNA 转录本。因此，不同剪接变体产生的蛋白质可能具有不同的功能和表达水平。骨骼中有几个具有剪接变体的基因，其功能影响成骨细胞功能和骨形成。本研究的主要目的是评估在经受机械加载和随后骨形成的骨骼中选择性剪接的程度。我们使用大鼠前肢加载模型，其中右侧前肢轴向加载 3 分钟，而左侧前肢作为未加载对照。动物每天接受加载，两次加载之间间隔 24 小时。在开始加载后 4 小时至 32 天，从尺骨中采集 RNA，并在每个时间点使用 Affymetrix 外显子阵列（GeneChip® Rat Exon 1.0 ST Arrays）测量 mRNA 丰度。使用方差分析模型来识别整个时间过程中的潜在选择性剪接基因，并用 qPCR 验证了五个选择性剪接基因：Akap12、Fn1、Pcolce、Sfrp4 和 Tpm1。选择性剪接基因的数量随时间而变化，从 12 小时的 68 个到 16 天的 992 个不等。我们在整个时间过程中鉴定了编码已知在骨形成中具有功能的蛋白质的基因，包括胶原蛋白、基质蛋白以及 Wnt/β-catenin 和 TGF-β 信号通路的成分。我们还鉴定了编码细胞因子、离子通道、肌肉相关基因和溶质载体的选择性剪接基因，这些基因在骨形成中没有已知功能，代表潜在的新发现。此外，还进行了功能特征分析，以分类我们数据集中文本的选择性剪接基因的全局功能。总之，机械加载会诱导骨骼中的选择性剪接，这可能在骨骼对机械加载的反应中发挥重要作用。
