1.山东第一医科大学附属人民医院病理科,济南 271199;2.济南市第二妇幼保健院妇产科,济南 271100
1. Department of Pathology, People’s Hospital Affiliated to Shandong First Medical University, Jinan 271199, China. 2. Jinan Second Maternal and Child Health Hospital, Obstetrics and Gynecology, Jinan 271100
目的 探究miR-486-5p 对子宫内膜癌(EC)细胞恶性生物学行为的影响及对PTEN 的靶向调节作用。 方法 qRT-PCR 检测EC 组织和细胞中miR-486-5p、PTEN mRNA 表达水平,分析EC 组织中miR-486-5p、PTEN mRNA 表达与患者临床特征的相关性。双荧光素酶报告基因实验验证miR-486-5p 与PTEN 的靶向关系。Ishikawa细胞分为NC 组、NC inhibitor 组、miR-486-5p inhibitor 组、miR-486-5p inhibitor+si-NC 组、miR-486-5p inhibitor+si-PTEN 组,Western blot 检测细胞中PTEN 蛋白及PI3K/ AKT 通路相关蛋白表达,CCK-8 检测细胞活力,集落形成实验检测细胞集落形成能力,流式细胞术检测细胞凋亡情况,划痕愈合实验、Transwell 实验检测细胞迁移、侵袭能力。 结果 EC 组织和细胞中miR-486-5p 高表达,而PTEN mRNA 低表达(P<0. 05)。EC 组织中miR-486-5p、PTEN mRNA 表达均与患者肿瘤大小、分化程度、国际妇产科联盟(FIGO)分期、淋巴结转移有关(P<0. 05)。经验证,Ishikawa 细胞中miR-486-5p 可能负靶向调节PTEN 表达。与NC 组、NC inhibitor 组比较,miR-486-5p inhibitor 组Ishikawa 细胞的48 h、72 h 细胞活力、划痕愈合率及p-PI3K/ PI3K、p-AKT/ AKT 比值降低(P<0. 05),集落形成数、迁移细胞数和侵袭细胞数减少(P<0. 05),凋亡率升高(P<0. 05);与miR-486-5p inhibitor 组、miR-486-5p inhibitor+si-NC 组比较,miR-486-5p inhibitor+si-PTEN 组Ishikawa 细胞的48 h、72 h 细胞活力、划痕愈合率及p-PI3K/ PI3K、p-AKT/ AKT 比值升高(P<0. 05),集落形成数、迁移细胞数和侵袭细胞数增加(P<0. 05),凋亡率降低(P<0. 05)。 结论 抑制miR-486-5p 可能通过负靶向调节PTEN 抑制PI3K/ AKT 通路活化,进而抑制EC 细胞增殖、迁移和侵袭,并诱导其凋亡。
Objective To explore the influence of miR-486-5p on the malignant behavior of endometrial cancer(EC) cells and its targeted regulation of PTEN. Methods The expression miR-486-5p and PTEN mRNA in EC tissues and cells was detected by qRT-PCR. The correlation between miR-486-5p and PTEN mRNA expression in EC tissues and the clinical characteristics of patients were analyzed. A dual luciferase reporter assay was performed to verify the relationship between miR-486-5p and PTEN. Ishikawa cells were separated into NC, NC inhibitor, miR-486-5p inhibitor, miR-486-5p inhibitor+si-NC, and miR-486-5p inhibitor+si-PTEN groups. Western blot was performed to detect expression of PTEN and PI3K/ AKT pathway-related proteins in cells. CCK-8 assays were performed to assess cell viability. Colony formation assays were used to examine the ability of cells to form colonies. Flow cytometry was performed to assess apoptosis, Scratch healing and transwell assays were applied to assess cell migration and invasion. Results The miR-486-5p was highly expressed in EC tissues and cells, while PTEN mRNA was expressed at a low level (P<0. 05). miR-486-5p and PTEN mRNA expression in EC tissues was related to the tumor size, differentiation degree, International Federation of Gynecology and Obstetrics stage, and lymph node metastasis (P<0. 05). miR-486-5p in Ishikawa cells might negatively target and regulate PTEN expression. Compared with NC and NC inhibitor groups, cell viability at 48 and 72 h, scratch healing rate, and ratios of p-PI3K/ PI3K and p-AKT/ AKT of Ishikawa cells in the miR-486-5p inhibitor group were decreased (P<0. 05), the number of colonies formed and the numbers of migrating and invasive cells were decreased (P<0. 05), and the apoptosis rate was increased (P<0. 05). Compared with miR-486-5p inhibitor and miR-486-5p inhibitor+si-NC groups, cell viability at 48 and 72 h, scratch healing rate, and ratios of p-PI3K/ PI3K and p-AKT/ AKT of Ishikawa cells in the miR-486-5p inhibitor+si-PTEN group were increased (P< 0. 05), the number of colonies formed and the numbers of migrating and invasive cells were increased (P<0. 05), and the apoptosis rate was decreased (P<0. 05). Conclusions Inhibition of miR-486-5p may suppress activation of the PI3K/ AKT pathway by negatively targeting PTEN, thereby inhibiting the proliferation, migration, and invasion of EC cells and inducing their apoptosis.
DENG Yu, WEI Pixi, REN Shuangshuang, LYU Guixue, WEI Wenwen. miR-486-5p 靶向调节PTEN 对子宫内膜癌细胞恶性生物学行为的影响[J].中国比较医学杂志,2023,33(7):55~66.复制