Department of Obstetrics and Gynecology, Affiliated Hospital of Nantong University, 19 Xisi Road, Nantong, Jiangsu, 226000, People's Republic of China.
Department of Pediatrics, Affiliated Hospital of Nantong University, Nantong, People's Republic of China.
Stem Cell Res Ther. 2020 Dec 1;11(1):520. doi: 10.1186/s13287-020-02027-5.
Accumulating evidence indicates that mesenchymal stem cells (MSCs) exert tissue repair effects and therapeutic angiogenesis through their noncoding RNAs (ncRNAs). Our previous studies showed that MSCs derived from Wharton's jelly (WJ-MSCs) can ameliorate damaged human endometrium by promoting angiogenesis. There is limited information on the functions and mechanism of ncRNAs in MSC-induced endometrial repair, and additional studies are needed for more insights.
Here, WJ-MSCs were cocultured with or without endometrial stromal cells (ESCs) damaged by mifepristone (cocultured group versus non-cocultured group). TUNEL staining assays, EdU proliferation assays, flow cytometry apoptosis assays, and western blot assays were performed to observe the reparative effect of WJ-MSCs on damaged ESCs. Subsequently, circular RNA (circRNA) and microRNA microarrays were performed between the two groups. A subset of top upregulated circRNAs was validated by qRT-PCR. The functions of circ6401 (hsa_circ_0006401) in WJ-MSCs were investigated using lentivirus-mediated circRNA overexpression assays. The subcellular localization of circ6401 and miR-29b-1-5p in WJ-MSCs was identified by double RNA fluorescence in situ hybridization. Dual-luciferase reporter assays and western blot assays were performed to elucidate the regulatory mechanisms among circ6401, miR-29b-1-5p, and RAP1B.
WJ-MSCs significantly improved ESC proliferation and upregulated the expression of vascular angiogenesis markers. Circ6401 was upregulated in WJ-MSCs cocultured with damaged ESCs, while miR-29b-1-5p was significantly downregulated. Furthermore, circ6401 was found to bind to miR-29b-1-5p and prevent it from decreasing the level of RAP1B, a crucial protein involved in the VEGF signaling pathway, which promoted angiogenesis and stimulated the proliferation of ESCs.
Our results showed the abundance and regulation profiles of ncRNAs of WJ-MSCs during repair of damaged ESCs and, for the first time, clarified the underlying mechanism by which circ6401 promotes endometrial repair by WJ-MSCs; thus, demonstrating that circ6401 may serve as a potential therapeutic target.
越来越多的证据表明，间充质干细胞（MSCs）通过其非编码 RNA（ncRNA）发挥组织修复作用和治疗性血管生成作用。我们之前的研究表明，源自华通氏胶（WJ-MSCs）的 MSCs 通过促进血管生成来改善受损的人子宫内膜。关于 ncRNA 在 MSC 诱导的子宫内膜修复中的功能和机制的信息有限，需要更多的研究来深入了解。
在这里，WJ-MSCs 与米非司酮损伤的子宫内膜基质细胞（ESCs）共培养（共培养组与非共培养组）或不共培养。通过 TUNEL 染色、EdU 增殖、流式细胞术凋亡检测和 Western blot 检测观察 WJ-MSCs 对受损 ESCs 的修复作用。随后，对两组之间的环状 RNA（circRNA）和 microRNA 微阵列进行分析。通过 qRT-PCR 验证一组 top 上调的 circRNA。使用慢病毒介导的 circRNA 过表达实验研究 circ6401（hsa_circ_0006401）在 WJ-MSCs 中的功能。通过双 RNA 荧光原位杂交鉴定 circ6401 和 miR-29b-1-5p 在 WJ-MSCs 中的亚细胞定位。通过双荧光素酶报告基因检测和 Western blot 检测阐明 circ6401、miR-29b-1-5p 和 RAP1B 之间的调控机制。
WJ-MSCs 显著改善了 ESC 的增殖并上调了血管生成标志物的表达。在与受损 ESCs 共培养的 WJ-MSCs 中，circ6401 上调，而 miR-29b-1-5p 显著下调。此外，circ6401 被发现与 miR-29b-1-5p 结合，防止其降低参与 VEGF 信号通路的关键蛋白 RAP1B 的水平，从而促进血管生成并刺激 ESCs 的增殖。
我们的研究结果显示了 WJ-MSCs 在修复受损 ESCs 过程中 ncRNA 的丰度和调控谱，并且首次阐明了 circ6401 通过促进 WJ-MSCs 子宫内膜修复的潜在机制；因此，证明 circ6401 可能成为一种潜在的治疗靶点。
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