GAS5作为miR-223-3p的ceRNA介导U87细胞的焦亡抑制脑胶质细胞的增殖, 槲皮素可以增强GAS5的作用

doi:10.5246/jcps.2024.03.018

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (3): 230-240.DOI: 10.5246/jcps.2024.03.018

GAS5作为miR-223-3p的ceRNA介导U87细胞的焦亡抑制脑胶质细胞的增殖, 槲皮素可以增强GAS5的作用

孙艳强¹, 李虹良², 陈康雪³, 李杰¹, 孙中磊¹^,³^,^*()

^1. 中国人民武装警察部队特色医学中心急诊科, 天津 300162
^2. 成都医学院, 四川成都 611137
^3. 四川泰康医院, 四川成都 610213

收稿日期:2023-10-11 修回日期:2023-11-16 接受日期:2023-12-09 出版日期:2024-03-31 发布日期:2024-03-31
通讯作者: 孙中磊

GAS5, as the ceRNA of miR-223-3p, mediates pyroptosis and inhibits the proliferation of brain glial cells U87, and quercetin can enhance its effect

Yanqiang Sun¹, Hongliang Li², Kangxue Chen³, Jie Li¹, Zhonglei Sun¹^,³^,^*()

¹ Department of Special Medical Center of Chinese People’s Armed Police Force, Emergency Department, Tianjin 300162, China
² Chengdu Medical University, Chengdu 611137, Sichuan, China
³ Sichuan Taikang Hospital, Chengdu 610213, Sichuan, China

Received:2023-10-11 Revised:2023-11-16 Accepted:2023-12-09 Online:2024-03-31 Published:2024-03-31
Contact: Zhonglei Sun
Supported by:
National Key Research and Development Project (Grant No. 2016YFC1101503).

摘要/Abstract

摘要：

本研究旨在探讨生长抑制特异性基因5(GAS5)作为miR-223-3p对脑胶质瘤生长的抑制作用及其可能机制。通过starbase工具预测GAS5与miR-223-3p 结合情况, 双荧光素酶报告实验和RNA免疫沉淀测定GAS5和miR-223-3p之间的结合关系。体外培养脑胶质细胞U87, 分为对照组(control)、槲皮素组(quercetin)、GAS5抑制组(si-GAS5)和槲皮素+si-GAS5组(quercetin + si-GAS5)组, 对比分析槲皮素对GAS5/miR-223-3p及焦亡蛋白(NLRP3、ASC、caspase-1、GSDMD和GSDME)的影响, 以及U87细胞增殖能力的影响。结果显示, Starbase工具预测GAS5与miR-223-3p 结合, 双荧光素酶报告实验和RNA免疫沉淀测定揭示了GAS5和miR-223-3p之间的直接结合。槲皮素促进GAS5的表达, 抑制miR-223-3p和焦亡相关蛋白(NLRP3、ASC、caspase-1、GSDMD和GSDME)的表达, 抑制U87细胞的增殖和侵袭(P < 0.05); 下调GAS5, miR-223-3p和焦亡相关蛋白(NLRP3、ASC、caspase-1、GSDMD和GSDME)的表达增多, 促进RC-4B/C细胞的增殖和侵袭(P < 0.05); 槲皮素可以逆转GAS5下调引起的miR-223-3p和焦亡相关蛋白(NLRP3、ASC、caspase-1、GSDMD和GSDME)的表达增加, 抑制U87细胞的增殖和侵袭(P < 0.05)。槲皮素抑制脑胶质瘤细胞U87的增殖,可能是通过促进GAS5表达竞争性抑制miR-223-3p, 抑制焦亡信号途径实现的。

关键词: 脑胶质瘤, U87, GAS5, miR-223-3p, 焦亡

Abstract:

This study explored the inhibitory effect of miR-223-3p on the proliferation of brain glioma cells and its potential mechanism in relation to growth arrest-specific transcripts (GAS5). We utilized the StarBase tool to predict the binding of GAS5 to miR-223-3p, further confirming the binding relationship between GAS5 and miR-223-3p through a dual luciferase reporter assay and RNA immunoprecipitation. In vitro cultured U87 brain glial cells were categorized into four groups: the control group, quercetin group, GAS5 inhibition group, and quercetin + si-GAS5 group. We analyzed and compared the impacts of quercetin on GAS5/miR-223-3p and pyroptotic proteins (NLRP3, ASC, caspase-1, GSDMD, and GSDME), as well as the proliferative capacity of U87 cells. StarBase’s predictions indicated that GAS5 bound to miR-223-3p. This was corroborated by the dual luciferase reporter assay and RNA immunoprecipitation assay, which demonstrated a direct binding between GAS5 and miR-223-3p. Quercetin was observed to enhance GAS5 expression, inhibit the expression of miR-223-3p and pyroptotic proteins (NLRP3, ASC, caspase-1, GSDMD, and GSDME), and suppress the proliferation and invasion of U87 cells (P < 0.05). Downregulation of GAS5 expression correlated with an increase in the expression of miR-223-3p and pyroptotic proteins (NLRP3, ASC, caspase-1, GSDMD, and GSDME), promoting the proliferation and invasion of RC-4B/C cells (P < 0.05). Quercetin was able to reverse the increase in miR-223-3p expression and pyroptotic proteins (NLRP3, ASC, caspase-1, GSDMD, and GSDME) induced by the downregulation of GAS5, thereby inhibiting the proliferation and invasion of U87 cells (P < 0.05). In conclusion, quercetin might inhibit the proliferation of U87 brain glioma cells by promoting the expression of GAS5, which in turn competitively inhibited miR-223-3p and suppressed the pyroptotic signaling pathway.

Key words: Glimoa, U87, GAS5, miR-223-3p, Pyroptosis

Supporting:

孙艳强, 李虹良, 陈康雪, 李杰, 孙中磊. GAS5作为miR-223-3p的ceRNA介导U87细胞的焦亡抑制脑胶质细胞的增殖, 槲皮素可以增强GAS5的作用[J]. 中国药学(英文版), 2024, 33(3): 230-240.

Yanqiang Sun, Hongliang Li, Kangxue Chen, Jie Li, Zhonglei Sun. GAS5, as the ceRNA of miR-223-3p, mediates pyroptosis and inhibits the proliferation of brain glial cells U87, and quercetin can enhance its effect[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(3): 230-240.

图/表 5

Table 1. Primers used in real-time PCR.

Figure 1. GAS5 expression levels. Compared with the control group, *P＜0.05, **P＜0.01; Compared with the si-GAS5 group, #P＜0.05.

Figure 2. GAS5 directly binds to miR‑223‑3p lncRNA GAS5 as the ceRNA of miR-223-3p. (A) RegRNA website was used to analyze the interaction sites between GAS5 and miR-223-3p; (B) The interaction activity between lncRNA GAS5 and miR-223-3p by luciferase reporter assay; (C) RIP assay confirmed the interaction between lncRNA GAS5 and miR-223-3p in U87 cells. Compared with miR-223-3p mimics, *P＜0.05.

Figure 3. Quercetin reverses the increased proliferation and invasion of U87 cells caused by lncRNA GAS5 silencing. (A) MTT assay of U87 cells; (B) U87 cell transwell assay. Compared with the control group, *P＜0.05, compared with the si-GAS5 group, #P＜0.05.

Figure 4. Quercetin reverses the increase of miR-223-3p and pyroptosis-related proteins caused by lncRNA GAS5 silencing. (A) Expression of miR-223-3p; (B) Pyroptotic protein immunoblotting bands; (C) Expression of NLRP3 compared with GAPDH; (D) Expression of ACS compared with GAPDH; (E) Expression of caspase-1 compared with GAPDH; (F) Compared with the control group, the expression of GSDMD and GSDME, *P＜0.05, **P＜0.01, compared with the si-GAS5 group, #P＜0.05.

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GAS5作为miR-223-3p的ceRNA介导U87细胞的焦亡抑制脑胶质细胞的增殖, 槲皮素可以增强GAS5的作用

GAS5, as the ceRNA of miR-223-3p, mediates pyroptosis and inhibits the proliferation of brain glial cells U87, and quercetin can enhance its effect

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