Li Feifei, Mi Rui, Fan Chuling, Zhang Ping, Zhu Ting, Wang Qian, Lu Yaojuan, Gu Junxia, Zheng Qiping
Department of Pathophysiology, Anhui Medical University Hefei 230032, China.
Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University Zhenjiang 212013, China.
Am J Transl Res. 2016 Dec 15;8(12):5465-5474. eCollection 2016.
Runx2, a member of the Runt domain family, is a well-known master transcription factor for osteoblast differentiation. Runx2 has also been shown to play essential roles during chondrocyte hypertrophy, an important late stage of endochondral ossification linking both bone and cartilage development. To identify the co-factors that may interact with Runx2 together to regulate this critical process, we have performed yeast two-hybrid (Y2H) screening using Runx2 as a bait to screen a cDNA library of hypertrophic chondrocytes. The bait expressing cassette was constructed by fusing Runx2 with the pGBKT7 vector containing the Gal4 DNA binding domain (BD). The Mate & Plate libraries were constructed using pGADT7-Rec and cDNAs derived from hypertrophic chondrocytes enriched limb tissues or hypertrophic MCT cells. After co-transformation of pGBKT7-Runx2 and the cDNA libraries, colonies that grew in nutrition deficient medium were selected and subjected to PCR and sequencing analysis. We successfully identified more than 30 candidate genes, including Lectin-1 (Lgals1), Col1a2, Edf1 and Timp-2. We have performed literature review and bioinformatics analysis of these genes using GenePaint. Most of them show ubiquitous expression with Lgals1 show enhanced expression in hypertrophic chondrocytes. We further performed preliminary expression analysis by quantitative PCR and detected differential expression of these candidate genes in proliferative and hypertrophic MCT cells, with Timp-2 significantly (around 3-fold) and Lgals1 moderately (around 1.5 fold) upregulated in hypertrophic MCT cells. Our results suggest that, candidate gene Timp-2 is very likely to interact with Runx2 and together to play essential function during cartilage development, and possibly its homeostasis.
Runx2是Runt结构域家族的成员之一，是一种众所周知的成骨细胞分化主转录因子。Runx2还被证明在软骨细胞肥大过程中发挥重要作用，软骨细胞肥大是软骨内骨化的一个重要晚期阶段，连接着骨骼和软骨的发育。为了鉴定可能与Runx2相互作用以调节这一关键过程的辅助因子，我们以Runx2为诱饵进行了酵母双杂交（Y2H）筛选，以筛选肥大软骨细胞的cDNA文库。诱饵表达盒是通过将Runx2与含有Gal4 DNA结合结构域（BD）的pGBKT7载体融合构建而成。Mate & Plate文库是使用pGADT7-Rec和来自富含肥大软骨细胞的肢体组织或肥大MCT细胞的cDNA构建的。在将pGBKT7-Runx2和cDNA文库共转化后，选择在营养缺陷培养基中生长的菌落，并进行PCR和测序分析。我们成功鉴定了30多个候选基因，包括凝集素-1（Lgals1）、Col1a2、Edf1和Timp-2。我们使用GenePaint对这些基因进行了文献综述和生物信息学分析。它们中的大多数显示出普遍表达，其中Lgals1在肥大软骨细胞中表达增强。我们进一步通过定量PCR进行了初步表达分析，并检测了这些候选基因在增殖性和肥大性MCT细胞中的差异表达，其中Timp-2在肥大性MCT细胞中显著上调（约3倍），Lgals1中度上调（约1.5倍）。我们的结果表明，候选基因Timp-2很可能与Runx2相互作用，并在软骨发育过程中共同发挥重要作用，可能还参与其稳态维持。
