| 徐月华,刘晓健,刘小江.下调miR-221-3p通过PTEN/PI3K/Akt增强胶质瘤U87-TMZ细胞对TMZ的敏感性.[J].中南医学科学杂志.,2025,(6):963-968. |
| 下调miR-221-3p通过PTEN/PI3K/Akt增强胶质瘤U87-TMZ细胞对TMZ的敏感性 |
| Downregulation of miR-221-3p enhances the sensitivity of glioma U87-TMZ cells to TMZ through PTEN/PI3K/Akt |
| 投稿时间：2025-07-18 修订日期：2025-10-11 |
| DOI：10.15972/j.cnki.43-1509/r.2025.06.005 |
| 中文关键词: miR-221-3p PTEN/PI3K/Akt信号通路 胶质瘤 替莫唑胺 化疗敏感性 U87-TMZ细胞 |
| 英文关键词:miR-221-3p PTEN/PI3K/Akt signaling pathway glioma temozolomide chemotherapy sensitivity U87-TMZ cells |
| 基金项目:南通市药学会-常州四药医院药学科研基金项目(ntyx2105) |
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| 中文摘要: |
| 目的探讨下调微小RNA(miR)-221-3p增强胶质瘤U87-替莫唑胺(TMZ)细胞对TMZ敏感性的作用机制。 方法将培养的人胶质瘤细胞TMZ耐药株U87-TMZ根据不同载体转染分为阴性对照(NC)组(常规培养)、miR-221-3p抑制剂(i-miR-221-3p)组、i-miR-221-3p阴性对照(i-NC)组、i-miR-221-3p+磷酸酶与张力蛋白同源物(PTEN)小干扰RNA(si-PTEN)组、i-miR-221-3p+si-PTEN阴性对照(si-PTEN-NC)组。采用qRT-PCR检测各组细胞miR-221-3p、PTEN mRNA表达水平。双荧光素酶报告实验验证miR-221-3p和PTEN之间的靶向关系。WST-1和集落形成实验、流式细胞术分别检测各组细胞增殖、细胞凋亡情况；Western blotting实验测定各组细胞增殖细胞核抗原(PCNA)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)及PTEN/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)通路相关蛋白的表达水平；WST-1实验评估各组细胞对TMZ的化疗敏感性。 结果与NC组或i-NC组比较,i-miR-221-3p组miR-221-3p、PCNA、Bcl-2、磷酸化(p)-PI3K/PI3K、p-Akt/Akt蛋白表达下调(P＜0.05),PTEN mRNA及其蛋白、Bax蛋白表达上调(P＜0.05),细胞存活率、集落数及半抑制浓度(IC50)值降低(P＜0.05),细胞凋亡率升高(P＜0.05)。与i-miR-221-3p组或i-miR-221-3p+si-PTEN-NC组比较,i-miR-221-3p+si-PTEN组PTEN mRNA及其蛋白、Bax蛋白表达下调(P＜0.05),PCNA、Bcl-2、p-PI3K/PI3K、p-Akt/Akt表达上调(P＜0.05),细胞存活率、集落数及IC50值升高(P＜0.05),细胞凋亡率降低(P＜0.05)。双荧光素酶报告实验验证miR-221-3p和PTEN存在互补的结合位点。过表达miR-221-3p后,转染野生型PTEN报告基因质粒的细胞荧光素酶活性下降(P＜0.05)。 结论下调miR-221-3p可增强胶质瘤U87-TMZ细胞对TMZ的敏感性,其机制可能是通过上调PTEN表达,抑制PI3K/Akt通路,进而抑制细胞增殖并促进细胞凋亡。 |
| 英文摘要: |
| AimTo explore the mechanism by which downregulation of microRNA (miR) -221-3p enhances the sensitivity of glioma U87 temozolomide (TMZ) cells to TMZ. MethodsThe cultured human glioma cell line U87-TMZ resistant to TMZ was transfected with different vectors and divided into the negative control (NC) group (conventional culture), the miR-221-3p inhibitor (i-miR-221-3p) group, the i-miR-221-3p negative control (i-NC) group, the i-miR-221-3p+phosphatase and tensin homolog (PTEN) small interfering RNA (si-PTEN) group, and the i-miR-221-3p+si-PTEN negative control (si-PTEN-NC) group. qRT-PCR was used to detect the expression levels of miR-221-3p and PTEN mRNA in each group of cells; The dual luciferase reporter assay was used to verify the targeting relationship between miR-221-3p and PTEN; WST-1 and colony formation experiments were conducted to determine the proliferation of cells in each group; Flow cytometry was used to determine the apoptosis status of cells in each group; Western blotting was used to measure the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and PTEN/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway related proteins in each group of cells; The WST-1 experiment was used to evaluate the chemotherapy sensitivity of each group of cells to TMZ. ResultsCompared with the NC group or i-NC group, the i-miR-221-3p group showed downregulation of miR-221-3p, PCNA, Bcl-2, phosphorylated (p)-PI3K/PI3K, and p-Akt/Akt protein expression (P＜0.05), upregulation of PTEN mRNA and its protein, Bax protein expression (P＜0.05), decreased cell survival rate, colony number, and half maximal inhibitory concentration (IC50) values (P＜0.05), and increased cell apoptosis rate (P＜0.05). Compared with the i-miR-221-3p group or the i-miR-221-3p+si-PTEN-NC group, the i-miR-221-3p+si-PTEN group showed downregulation of PTEN mRNA and protein, as well as Bax protein expression (P＜0.05), upregulation of PCNA, Bcl-2, p-PI3K/PI3K, and p-Akt/Akt expression (P＜0.05), increased cell survival rate, colony number, and IC50 value (P＜0.05), and decreased cell apoptosis rate (P＜0.05). The dual luciferase reporter assay confirmed that miR-221-3p and PTEN have complementary binding sites. After overexpression of miR-221-3p, the luciferase activity of cells transfected with wild-type PTEN reporter gene plasmid was decreased (P＜0.05). ConclusionDownregulation of miR-221-3p can enhance the sensitivity of glioma U87-TMZ cells to TMZ. The mechanism may be through the downregulation of PTEN expression and inhibition of the PI3K/Akt pathway, thereby inhibiting cell proliferation and promoting cell apoptosis. |
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