[摘 要] 目的：探讨lncRNA核内富集丰富转录本1（NEAT1）调控miR-1287-5p/DNA损伤诱导转录本4（DDIT4）轴对卵巢癌SKOV3细胞增殖、凋亡和侵袭的影响及其机制。方法：收集2023年6月至2024年6月期间湖北医药学院附属随州医院手术切除的27例卵巢癌患者的癌及癌旁组织标本，以及正常人卵巢上皮细胞IOSE80和卵巢癌细胞系SKOV3、CAOV3和A2780。RT-qPCR检测卵巢癌组织与细胞中lncRNA NEAT1、miR-1287-5p及DDIT4 mRNA表达。将SKOV3细胞分为Ctrl组、si-NC组、si-NEAT1组、si-NEAT1+anti-miR-NC组、si-NEAT1+anti-miR-1287-5p组、si-NEAT1+vector组、si-NEAT1+OE-DDIT4组。CCK-8法、克隆形成实验、流式细胞术、Transwell实验分别检测各组细胞增殖、凋亡及侵袭能力，WB法检测细胞中DDIT4、细胞周期蛋白D1（cyclin D1）、p53、迁移侵袭增强子1（MIEN1）蛋白水平。双荧光素酶报告基因验证lncRNA NEAT1与miR-1287-5p/DDIT4靶向结合关系，RNA pull-down实验、RNA免疫沉淀实验分别验证lncRNA NEAT1与miR-1287-5p、miR-1287-5p与DDIT4的靶向结合关系。另设pcDNA组（转染空载体pcDNA3.1）和pc-NEAT1组（转染pcDNA-NEAT1）以验证NEAT1过表达效应。结果：卵巢癌组织中lncRNA NEAT1、DDIT4 mRNA表达水平显著高于癌旁组织（P < 0.05），而miR-1287-5p表达显著低于癌旁组织（P < 0.05）。敲低lncRNA NEAT1后，与Ctrl组和si-NC组相比，si-NEAT1组lncRNA NEAT1表达、DDIT4 mRNA表达均显著降低（P < 0.05），miR-1287-5p表达显著升高（P < 0.05）；而过表达lncRNA NEAT1则下调miR-1287-5p表达并上调DDIT4表达，呈现与敲低实验相反的调控效应；敲低lncRNA NEAT1后，克隆形成数、细胞增殖活性、细胞侵袭数均显著降低（P < 0.05），细胞凋亡率显著升高（P < 0.05）；DDIT4、cyclin D1、MIEN1蛋白表达均显著降低（P < 0.05），p53蛋白表达显著升高（P < 0.05）。进一步实验证实，anti-miR-1287-5p或OE-DDIT4均可减弱si-NEAT1对SKOV3细胞增殖和侵袭的抑制作用，同时减弱其对细胞凋亡的促进作用。lncRNA NEAT1靶向调控miR-1287-5p/DDIT4。结论：lncRNA NEAT1通过靶向调控miR-1287-5p/DDIT4轴促进SKOV3细胞增殖和侵袭，抑制细胞凋亡。

[Abstract] Objective: To explore the effects and mechanisms by which lncRNA nuclear enriched transcript 1 (NEAT1) regulating the miR-1287-5p/DNA damage inducible transcript 4 (DDIT4) axis on the proliferation, apoptosis and invasion of ovarian cancer SKOV3 cells. Methods: Tissue samples of cancer and adjacent tissues from 27 patients with ovarian cancer who underwent surgery at Suizhou Hospital Affiliated to Hubei University of Medicine between June 2023 and June 2024 were collected. Normal human ovarian epithelial cells IOSE80 and ovarian cancer cells SKOV3, CAOV3 and A2780 were also collected. RT-qPCR was used to detect the expressions of lncRNA NEAT1, miR-1287-5p and DDIT4 mRNA in ovarian cancer tissues and cells. SKOV3 cells were divided into the Ctrl group, the si-NC group, the si-NEAT1 group, the si-NEAT1 + anti-miR-NC group, the si-NEAT1 + anti-miR-1287-5p group, the si-NEAT1 + vector group, and the si-NEAT1 + OE-DDIT4 group. The CCK-8 assay, colony formation assay, flow cytometry, and Transwell assay were used to detect the proliferation, apoptosis, and invasion abilities of cells in each group. The WB method was used to detect the protein levels of DDIT4, cyclin D1, p53, and migration-invasion enhancer 1 (MIEN1) in the cells. The dual-luciferase reporter gene was used to verify the targeted binding relationship between lncRNA NEAT1 and miR-1287-5p/DDIT4. The RNA pull-down experiment and RNA immunoprecipitation experiment were used respectively to verify the target binding relationships between lncRNA NEAT1 and miR-1287-5p, and between miR-1287-5p and DDIT4. Additionally, the pcDNA group (transfected with empty vector pcDNA3.1) and the pc-NEAT1 group (transfected with pcDNA-NEAT1) were established to validate the overexpression effects of NEAT1. Results: The expression levels of lncRNA NEAT1 and DDIT4 mRNA in ovarian cancer tissues were significantly higher than those in adjacent tissues (P < 0.05), while the expression level of miR-1287-5p was significantly lower than that in adjacent tissues (P < 0.05). After knockdown of lncRNA NEAT1, compared with those in the Ctrl group and the si-NC group, the expressions of lncRNA NEAT1 and DDIT4 mRNA, the number of colony formation, the cell proliferation activity, the number of cell invasion, and the expressions of DDIT4, cyclin D1, and MIEN1 proteins in the si-NEAT1 group all decreased significantly (all P < 0.05), while the expression of miR-1287-5p, the apoptosis rate, and the expression of p53 protein increased significantly (all P < 0.05). Conversely, overexpression of lncRNA NEAT1 decreased miR-1287-5p expression and increased DDIT4 expression, showing opposite regulatory effects to the knockdown experiment. Further experiments proved that anti-miR-1287-5p or OE-DDIT4 might weaken the inhibitory effect of si-NEAT1 on the proliferation and invasion of SKOV3 cells, as well as its promoting effect on cell apoptosis. lncRNA NEAT1 targetedly regulates miR-1287-5p/DDIT4. Conclusion: lncRNA NEAT1 enhances SKOV3 cell proliferation and invasion, and inhibits cell apoptosis by targeting regulating the miR-1287-5p/DDIT4 axis.

随州市卫生健康委员会科技项目（2018SZ32008）