Department of Traumatology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People's Republic of China.
Department of Neurology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People's Republic of China.
Comb Chem High Throughput Screen. 2023;26(14):2476-2486. doi: 10.2174/1386207326666230417103137.
Long intergenic non-coding RNA-p21 (lincRNA-p21) plays a critical role in various senescence-associated physiological and pathological conditions. We aimed to explore the senescence-associated effects of lincRNA-p21 in 1-methyl-4-phenylpyridinium (MPP) treated neuroblastoma SH-SY5Y cell line as a therapeutic target.
The RNA expression levels of lincRNA-p21, p53, p16, and telomere length were examined with reverse transcription-quantitative polymerase chain reaction (RTqPCR). The Telo TAGGG™ Telomerase PCR ELISA PLUS Kit was used to determine telomerase activity. Cellular viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay. Western blot was performed to analyze β-catenin protein expression. Besides, oxidative stress was evaluated by Jaggregate- forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolocarbocyanine++ + iodide (JC‑1) stain, fluorescence spectrophotometry, colorimetric assay, and malondialdehyde (MDA) formation.
This research demonstrated that MPP caused a distinct increase in the expression of LincRNA- p21 in SH-SY5Y cells. MPP induced cellular senescence with decreasing cellular proliferation and viability, increasing expression levels of senescence-associated makers such as genes p53 and p16, accompanied by significantly decreasing telomere length and telomerase activity. At the same time, these effects were abolished by silencing lincRNA-p21 with small interfering RNA (siRNA). On the contrary, β-catenin silencing contributes to reversing anti-senescent effects caused by lincRNA-p21 silencing. Moreover, modifying lincRNA-p21 exerted an anti-senescent influence depending on decreasing oxidant stress.
Our study showed that in the treatment of MPP, lincRNA-p21 might serve a role in the SH-SY5Y cell senescence by modulating the Wnt/β-catenin pathway, as well as increasing oxidant stress. Thus, trying to target lincRNA-p21 may have important therapeutic and practical implications for PD.
长链非编码 RNA-p21(lincRNA-p21) 在各种与衰老相关的生理和病理条件中发挥关键作用。我们旨在探讨 lincRNA-p21 在 1-甲基-4-苯基吡啶(MPP) 处理的神经母细胞瘤 SH-SY5Y 细胞系中的衰老相关作用，作为一种治疗靶点。
采用逆转录定量聚合酶链反应(RTqPCR)检测 lincRNA-p21、p53、p16 和端粒长度的 RNA 表达水平。使用 TeloTAGGG ™ 端粒酶 PCR ELISA PLUS 试剂盒测定端粒酶活性。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐(MTT)测定和乳酸脱氢酶(LDH)测定评估细胞活力。通过 Western blot 分析 β-连环蛋白蛋白表达。此外，通过 Jaggregate 形成定位的亲脂阳离子，5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑碳菁碘化物( JC-1)染色、荧光分光光度法、比色法和丙二醛(MDA)形成评估氧化应激。
本研究表明，MPP 导致 SH-SY5Y 细胞中 lincRNA-p21 的表达明显增加。MPP 诱导细胞衰老，导致细胞增殖和活力下降，衰老相关标志物如基因 p53 和 p16 的表达水平增加，同时端粒长度和端粒酶活性显著降低。与此同时，这些效应被用小干扰 RNA(siRNA)沉默 lincRNA-p21 所消除。相反，β-连环蛋白沉默有助于逆转由 lincRNA-p21 沉默引起的抗衰老效应。此外，修饰 lincRNA-p21 可以通过降低氧化应激来发挥抗衰老作用。
我们的研究表明，在 MPP 治疗中，lincRNA-p21 可能通过调节 Wnt/β-连环蛋白通路以及增加氧化应激在 SH-SY5Y 细胞衰老中发挥作用。因此，靶向 lincRNA-p21 可能对 PD 具有重要的治疗和实际意义。
