School of Stomatology, Stomatological Hospital, Southern Medical University, Guangzhou, China.
Guangdong Academy of Stomatology, Guangzhou, Guangdong, China.
J Cell Mol Med. 2024 Sep;28(17):e70051. doi: 10.1111/jcmm.70051.
Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.
开发策略以增强间充质干细胞中的软骨分化并保留细胞外基质对于成功的软骨组织重建至关重要。缺氧诱导因子-1α（HIF-1α）在维持细胞外基质和软骨细胞表型方面发挥着关键作用，因此是软骨组织工程策略中的关键调节剂。最近的研究表明，泛素 C 端水解酶 L1（UCHL1）参与 HIF-1α的去泛素化。然而，UCHL1 在软骨分化中的调节作用尚未得到研究。在本研究中，我们首先验证了 UCHL1 表达对脂肪来源干细胞（ADSCs）软骨形成的促进作用。随后，设计了一种杂交杆状病毒系统，并利用三种 CRISPR 激活（CRISPRa）系统，使用来自三种不同细菌来源的死 Cas9（dCas9）靶向 UCHL1。然后，使用 UCHL1 和 HIF-1α抑制剂以及靶向 SRY 盒转录因子 9（SOX9）的 siRNA 分别阻断 UCHL1、HIF-1α 和 SOX9。通过 qRT-PCR、免疫荧光和组织学染色来测量软骨分化和软骨形成。我们观察到，来自金黄色葡萄球菌的 CRISPRa 系统在激活 UCHL1 方面比常用的来自化脓性链球菌的 CRISPRa 系统具有更高的效率。此外，通过利用 Cre/loxP 基于的杂交杆状病毒延长了激活时间。此外，我们的研究结果表明，UCHL1 通过调节 HIF-1α的稳定性和定位来增强 SOX9 的表达，从而促进 ADSC 中的软骨生成。这些发现表明，使用 CRISPRa 系统激活 UCHL1 在软骨再生应用中具有重要潜力。
