Modulation of MicroRNA Expression During In Vitro Chondrogenesis.
Modulation of MicroRNA Expression During In Vitro Chondrogenesis.
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DOI：
10.1007/978-1-0716-2839-3_15
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发表时间：
2023
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自1993年发现以来，microRNAs（miRNAs）已被认为是哺乳动物细胞增殖、凋亡、代谢和分化等过程中重要的表观遗传调节因子。这些小的非编码RNA通过与mRNA的3 '-非翻译区中的特定区域相互作用而起作用，从而导致mRNA降解或翻译抑制。由于miRNA具有靶向给定细胞类型内的许多mRNA的能力，因此可以调节许多细胞途径和网络。为了研究miRNA的功能，可以使用多种方法来调节其在细胞中的活性，例如用于短期测定的合成模拟物或antagomirs，或用于长期实验（例如细胞分化测定）的基于病毒的方法。在本章中，我们提供了我们的方法，组成型过表达所需的miRNA在体外软骨形成的人软骨祖细胞（CPC）。具体来说，我们描述了我们如何从人关节软骨标本中获得CPC，我们如何产生和滴定慢病毒工程过表达前体miRNA，我们如何用慢病毒转染CPC并使其向软骨细胞谱系分化，以及我们如何提取RNA并测量体外软骨形成过程中感兴趣的miRNA的表达水平。我们还提供了一些来自我们实验室的数据，证明我们可以在软骨颗粒培养物中实现并维持miRNA过表达长达14天。我们预测，这些基于慢病毒的方法也将有助于研究祖细胞的miRNA调节如何影响三维生物材料支架内的细胞分化和细胞外基质产生。
Since their discovery in 1993, microRNAs (miRNAs) are now recognized as important epigenetic regulators of many mammalian cellular processes including proliferation, apoptosis, metabolism, and differentiation. These small non-coding RNAs function by interacting with specific regions in the 3’-untranslated region of mRNAs, thereby resulting in mRNA degradation or suppression of translation. Since miRNAs have the ability to target many mRNAs within a given cell type, a number of cellular pathways and networks may be regulated as a result. To study the function of miRNAs, a number of methods can be used to modulate their activity in cells such as synthetic mimics or antagomirs for short-term assays, or viral-based approaches for longer-term experiments such as cell differentiation assays. In this Chapter, we provide our methodology to constitutively over-express a desired miRNA during in vitro chondrogenesis of human cartilage progenitor cells (CPCs). Specifically, we describe how we obtain CPCs from human articular cartilage specimens, how we generate and titrate lentivirus engineered to over-express a precursor miRNA, how we transduce CPCs with lentivirus and differentiate them toward the chondrocyte lineage, and how we extract RNA and measure expression levels of the miRNA of interest during in vitro chondrogenesis. We also provide some data from our laboratory demonstrating that we can achieve and maintain miRNA over-expression for up to 14 days in cartilage pellet cultures. We predict that these lentiviral-based approaches will also be useful to study how miRNA modulation of progenitor cells affects cell differentiation and extracellular matrix production within three-dimensional biomaterial scaffolds.