Department of Neurology, Zhejiang Hospital, Hangzhou, Zhejiang 310013, P.R. China.
Int J Mol Med. 2018 Sep;42(3):1741-1755. doi: 10.3892/ijmm.2018.3736. Epub 2018 Jun 20.
The present study aimed to examine how the long non‑coding RNA (lncRNA) RP11‑543N12.1 interacted with microRNA (miR)‑324‑3p to modify microglials (MIs)‑induced neuroblastoma cell apoptosis, which may pose benefits to the treatment of Alzhemier's disease (AD). The cell model of AD was established by treating SH‑SY5Y cells with amyloid β (Aβ)25‑35, and MI were acquired using primary cell culture technology. The lncRNAs that were differentially expressed between SH‑SY5Y and control cells were screened through a microarray assay and confirmed via polymerase chain reaction. In addition, overexpression of RP11‑543N12.1 and miR‑324‑3p was established by transfection of SH‑SY5Y cells with pcDNA3.1(+)‑RP11‑543N12.1 and miR‑324‑3p mimics, respectively, while downregulation of RP11‑543N12.1 and miR‑324‑3p was achieved by transfection with RP11‑543N12.1‑small interfering RNA (siRNA) and miR‑324‑3p inhibitor, respectively. The interaction between RP11‑543N12.1 and miR‑324‑3p was confirmed with a dual‑luciferase reporter gene assay. The results revealed that the expression levels of total and phosphorylated tau in SH‑SY5Y cells were significantly elevated following Aβ25‑35 treatment (P<0.05), and RP11‑543N12.1 was found to be differentially expressed between the control and Aβ25‑35‑treated cells (P<0.05). Furthermore, the targeted association of RP11‑543N12.1 and miR‑324‑3p was predicted based on miRDB4.0 and PITA databases, and then validated via the dual‑luciferase reporter gene assay. SH‑SY5Y cells transfected with siRNA or inhibitor, and treated with Aβ25‑35 displayed cellular survival and apoptosis that were similar to the normal levels (P<0.05). Finally, co‑culture of MI and SH‑SY5Y cells transfected with RP11‑543N12.1‑siRNA/miR‑324‑3p inhibitor significantly enhanced cell apoptosis (P<0.05). In conclusion, RP11‑543N12.1 targeted miR‑324‑3p to suppress proliferation and promote apoptosis in the AD cell model, suggesting that RP11‑543N12.1 and miR‑324‑3p may be potential biomarkers and therapeutic targets for AD.
本研究旨在探讨长链非编码 RNA (lncRNA) RP11-543N12.1 如何与 microRNA (miR)-324-3p 相互作用，从而改变小胶质细胞 (MI)诱导的神经母细胞瘤细胞凋亡，这可能对阿尔茨海默病 (AD)的治疗有益。通过用淀粉样蛋白β (Aβ)25-35 处理 SH-SY5Y 细胞建立 AD 细胞模型，并通过原代细胞培养技术获得 MI。通过微阵列分析筛选出 SH-SY5Y 细胞和对照细胞之间差异表达的 lncRNA，并通过聚合酶链反应进行验证。此外，通过转染 pcDNA3.1(+) - RP11-543N12.1 和 miR-324-3p 模拟物分别建立 SH-SY5Y 细胞中 RP11-543N12.1 和 miR-324-3p 的过表达，通过转染 RP11-543N12.1-siRNA 和 miR-324-3p 抑制剂分别下调 RP11-543N12.1 和 miR-324-3p。通过双荧光素酶报告基因实验证实了 RP11-543N12.1 和 miR-324-3p 之间的相互作用。结果显示，SH-SY5Y 细胞中总tau 和磷酸化 tau 的表达水平在 Aβ25-35 处理后明显升高 (P<0.05)，并且在对照和 Aβ25-35 处理的细胞之间发现 RP11-543N12.1 差异表达 (P<0.05)。此外，基于 miRDB4.0 和 PITA 数据库预测了 RP11-543N12.1 与 miR-324-3p 的靶向关联，然后通过双荧光素酶报告基因实验进行验证。转染 siRNA 或抑制剂并用 Aβ25-35 处理的 SH-SY5Y 细胞的细胞存活和细胞凋亡与正常水平相似 (P<0.05)。最后，共培养转染 RP11-543N12.1-siRNA/miR-324-3p 抑制剂的 MI 和 SH-SY5Y 细胞显著增强了细胞凋亡 (P<0.05)。总之，RP11-543N12.1 通过靶向 miR-324-3p 抑制 AD 细胞模型中的增殖并促进凋亡，提示 RP11-543N12.1 和 miR-324-3p 可能是 AD 的潜在生物标志物和治疗靶点。
