微小RNA-155-5p通过雷帕霉素靶蛋白相互作用蛋白调控胶质瘤细胞的自噬和凋亡。

MiR-155-5p regulates autophagy and apoptosis of glioma cells through RICTOR.

作者信息

Guo Zhao, Tian Jing-Jie, Wang Yao, Jiang Lei, Chen Yang, Dai He-Jun, Wang Lei, Zhang Yi

机构信息

Department of Neurosurgery, The Second Affiliated Hospital of Nantong University, Nantong, China.

Department of Neurosurgery, Danyang Hospital Affiliated to Nantong University, Zhenjiang, China.

出版信息

Transl Cancer Res. 2024 Oct 31;13(10):5509-5521. doi: 10.21037/tcr-24-543. Epub 2024 Oct 18.

DOI:10.21037/tcr-24-543

PMID:39525027

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11543026/

Abstract

BACKGROUND

Glioma characterized by the high degree of drug resistance and the poor prognosis is the most common primary malignant tumors of the brain. And miRNA is involved in a variety of biological behaviors of tumors, enhancing or inhibiting the occurrence and development of tumors. Therefore, the present study aims to explore whether miR-155-5p can regulate autophagy and apoptosis of glioma through RICTOR.

METHODS

The significantly differential gene miR-155-5p was identified from the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) databases GSE165937 and GSE138764 using bioinformatics analysis, and its expression was validated by quantitative real-time polymerase chain reaction (qRT-PCR). The putative target genes of miR-155-5p were predicted through interrogation of relevant databases, followed by identification of key target genes. Subsequently, core target genes were selected for functional enrichment analysis. The U87MG cell line was utilized as the experimental model and divided into Negative Control1 (NC1) group, Mimic group, Negative Control2 (NC2) group, Inhibitor group, and NC + 3-methyladenine (3-MA) group. The expression levels of miR-155-5p, RICTOR, P62, LC-3, Bax, Bcl-2, and Caspase-3 were assessed using qRT-PCR, cellular fluorescence imaging, and Western blotting; while apoptosis in the U87MG cell line was evaluated via flow cytometry.

RESULTS

The results showed that miR-155-5P was highly expressed in glioma cells, which could inhibit the expression of Bax, Caspase-3, LCII/LCI and Beclin-1, and increase the expression of Bcl2 and P62. Flow cytometry and cell fluorescence were used to verify the above results. Moreover, when U87MG cells treated with miR-155-5p inhibitor were inhibited by 3-MA, the results showed that miR-155-5p enhanced the anti-apoptotic ability of U87MG cells by regulating autophagy. In addition, the bioinformatics results show that miR-155-5p survival prognosis in glioma into a strong negative correlation, while the survival prognosis of RICTOR in glioma showed a strong positive correlation. The core target genes Kyoto Encyclopedia of Genes and Genomes (KEGG) mainly occurred in PI3K-AKT signaling pathway; in addition, qRT-PCR and Western blot confirmed the regulatory effect of miR-155-5P on RICTOR.

CONCLUSIONS

MiR-155-5p regulates autophagy and apoptosis-related proteins in glioma cells through RICTOR, affecting the occurrence and development of glioma.

摘要

背景

胶质瘤是最常见的原发性脑恶性肿瘤，具有高度耐药性和不良预后。微小RNA（miRNA）参与肿瘤的多种生物学行为，促进或抑制肿瘤的发生发展。因此，本研究旨在探讨miR-155-5p是否能通过雷帕霉素靶蛋白相互作用蛋白（RICTOR）调节胶质瘤的自噬和凋亡。

方法

利用生物信息学分析从基因表达综合数据库（GEO；http://www.ncbi.nlm.nih.gov/geo）GSE165937和GSE138764中鉴定出差异显著的基因miR-155-5p，并通过定量实时聚合酶链反应（qRT-PCR）验证其表达。通过查询相关数据库预测miR-155-5p的潜在靶基因，随后鉴定关键靶基因。随后，选择核心靶基因进行功能富集分析。将U87MG细胞系作为实验模型，分为阴性对照1（NC1）组、模拟物组、阴性对照2（NC2）组、抑制剂组和NC + 3-甲基腺嘌呤（3-MA）组。采用qRT-PCR、细胞荧光成像和蛋白质印迹法检测miR-155-5p、RICTOR、P62、微管相关蛋白轻链3（LC-3）、凋亡相关蛋白Bax、Bcl-2和半胱天冬酶-3（Caspase-3）的表达水平；同时通过流式细胞术评估U87MG细胞系中的凋亡情况。

结果

结果显示，miR-155-5P在胶质瘤细胞中高表达，可抑制Bax、Caspase-3、LCII/LCI和Beclin-1的表达，并增加Bcl2和P62的表达。采用流式细胞术和细胞荧光法验证上述结果。此外，当用3-MA抑制经miR-155-5p抑制剂处理的U87MG细胞时，结果表明miR-155-5p通过调节自噬增强了U87MG细胞的抗凋亡能力。此外，生物信息学结果显示，miR-155-5p在胶质瘤中的生存预后呈强负相关，而RICTOR在胶质瘤中的生存预后呈强正相关。京都基因与基因组百科全书（KEGG）核心靶基因主要发生在磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-AKT）信号通路；此外，qRT-PCR和蛋白质印迹法证实了miR-155-5P对RICTOR的调节作用。