Department of Orthopedic, Nantong First People's Hospital & The Second Affiliated Hospital of Nantong University, No. 6 Haierxiangbei Road, Nantong, 226001, Jiangsu Province, China.
J Orthop Surg Res. 2021 Jan 6;16(1):6. doi: 10.1186/s13018-020-02041-6.
Spinal cord injury (SCI) is associated with health burden both at personal and societal levels. Recent assessments on the role of lncRNAs in SCI regulation have matured. Therefore, to comprehensively explore the function of lncRNA LEF1-AS1 in SCI, there is an urgent need to understand its occurrence and development.
Using in vitro experiments, we used lipopolysaccharide (LPS) to treat and establish the SCI model primarily on microglial cells. Gain- and loss of function assays of LEF1-AS1 and miR-222-5p were conducted. Cell viability and apoptosis of microglial cells were assessed via CCK8 assay and flow cytometry, respectively. Adult Sprague-Dawley (SD) rats were randomly divided into four groups: Control, SCI, sh-NC, and sh-LEF-AS1 groups. ELISA test was used to determine the expression of TNF-α and IL-6, whereas the protein level of apoptotic-related markers (Bcl-2, Bax, and cleaved caspase-3) was assessed using Western blot technique.
We revealed that LncRNA LEF1-AS1 was distinctly upregulated, whereas miR-222-5p was significantly downregulated in LPS-treated SCI and microglial cells. However, LEF1-AS1 knockdown enhanced cell viability, inhibited apoptosis, as well as inflammation of LPS-mediated microglial cells. On the contrary, miR-222-5p upregulation decreased cell viability, promoted apoptosis, and inflammation of microglial cells. Mechanistically, LEF1-AS1 served as a competitive endogenous RNA (ceRNA) by sponging miR-222-5p, targeting RAMP3. RAMP3 overexpression attenuated LEF1-AS1-mediated protective effects on LPS-mediated microglial cells from apoptosis and inflammation.
In summary, these findings ascertain that knockdown of LEF1-AS1 impedes SCI progression via the miR-222-5p/RAMP3 axis.
脊髓损伤（SCI）给个人和社会都带来了沉重的健康负担。最近对长链非编码 RNA（lncRNA）在 SCI 调控中的作用的评估已经成熟。因此，要全面探索 lncRNA LEF1-AS1 在 SCI 中的功能，就迫切需要了解其发生和发展。
我们使用脂多糖（LPS）处理微胶质细胞，建立 SCI 模型，进行 LEF1-AS1 和 miR-222-5p 的功能增益和功能丧失实验。CCK8 法和流式细胞术分别评估微胶质细胞的活力和凋亡。将成年 Sprague-Dawley（SD）大鼠随机分为四组：对照组、SCI 组、sh-NC 组和 sh-LEF1-AS1 组。酶联免疫吸附试验（ELISA）用于检测 TNF-α 和 IL-6 的表达，Western blot 技术用于评估凋亡相关标志物（Bcl-2、Bax 和 cleaved caspase-3）的蛋白水平。
我们发现，LPS 处理的 SCI 和微胶质细胞中，LncRNA LEF1-AS1 明显上调，而 miR-222-5p 明显下调。然而，LEF1-AS1 敲低增强了 LPS 介导的微胶质细胞的活力，抑制了细胞凋亡和炎症。相反，miR-222-5p 的上调降低了细胞活力，促进了微胶质细胞的凋亡和炎症。机制上，LEF1-AS1 通过海绵 miR-222-5p 作为竞争性内源 RNA（ceRNA），靶向 RAMP3。RAMP3 的过表达减弱了 LEF1-AS1 对 LPS 介导的微胶质细胞凋亡和炎症的保护作用。
总之，这些发现证实，通过 miR-222-5p/RAMP3 轴，敲低 LEF1-AS1 可抑制 SCI 的进展。
