骨髓间充质干细胞递送的 microRNA-30c 诱导 U-251 神经胶质瘤细胞系凋亡和减少细胞侵袭。

MicroRNA-30c delivered by bone marrow-mesenchymal stem cells induced apoptosis and diminished cell invasion in U-251 glioblastoma cell line.

机构信息

Department of Immunology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Department of Medical Microbiology, Faculty of Medicine, Shahed University of Medical Sciences, Tehran, Iran.

出版信息

Life Sci. 2021 Aug 15;279:119643. doi: 10.1016/j.lfs.2021.119643. Epub 2021 May 25.

DOI:10.1016/j.lfs.2021.119643

PMID:34048811

Abstract

BACKGROUND

Glioblastoma multiform (GBM) is the most belligerent and prevalent brain malignancy among adults. Due to the blood-brain barrier (BBB), drug administration is confronted by massive challenges, making resectional surgery the only treatment pipeline. MicroRNAs have recently absorbed the attention of studies for correlating with the progression of various malignancies. miR-30c has been reported to play a role in cell proliferation, metabolism, and apoptosis process. For instance, miR-30c has been reported to regulate apoptosis through the TNF-related apoptosis-inducing ligand (TRAIL). miR-30c also targets IL-6, which further induces apoptosis. Besides, miR-30c inhibits glioma proliferation and its migratory ability. Besides, the overexpression of miR-30c arrested cells at G0 as well as dampening their migration and invasion. However, it has been shown that the expression level of miR-30c was low in glioma. MSCs can migrate toward tumor cells which is called tumor-tropism, in which they are capable of delivering engineered miR-30c based on gap junction and non-intimacy mechanisms.

MATERIAL AND METHODS

MiR-30c was cloned into pCDH-CMV-MCS-EF1-copGFP vector utilizing XbaI and EcoRI in order to construct pCDH-miR-30c. Then psPAX2, pMD2.G, and pCDH-miR-30c were co-transfected into Hek-293T to yield lenti-miR-30c virus particles. Next, bone marrow-mesenchymal stem cells (BM-MSCs) were Transduced with lenti-miR-30c. Thereafter, we co-cultured U-251 cell line with BM-MCSs-miR-30c and evaluated the apoptosis rate and the relative expression level of IL-6, Klf4, Sox2, c-Myc, and Oct4 using Real-Time PCR and flow cytometry.

RESULTS

Wound healing assays represented low migratory ability in U-251 cells treated with BM-MSCs-miR-30c. Plus, apoptosis assay using Annexin V/7AAD showed an increased number of apoptotic U-251 cells following the treatment. miR-30 targeted IL-6 and induced apoptosis. It also impacted on the self-renewal and the anti-apoptotic cluster of genes, namely Klf4, Sox2, c-Myc, and Oct4, to induce apoptosis and dwindle the migration and invasion.

摘要

背景

多形性胶质母细胞瘤（GBM）是成年人中最具侵略性和最常见的脑恶性肿瘤。由于血脑屏障（BBB）的存在，药物给药面临着巨大的挑战，使得切除术成为唯一的治疗途径。MicroRNAs 最近引起了研究人员的关注，因为它们与各种恶性肿瘤的进展有关。miR-30c 已被报道在细胞增殖、代谢和细胞凋亡过程中发挥作用。例如，miR-30c 已被报道通过肿瘤坏死因子相关凋亡诱导配体（TRAIL）调节细胞凋亡。miR-30c 还靶向 IL-6，进一步诱导细胞凋亡。此外，miR-30c 抑制神经胶质瘤的增殖及其迁移能力。此外，miR-30c 的过表达使细胞停滞在 G0 期，并抑制其迁移和侵袭。然而，已经表明 miR-30c 在神经胶质瘤中的表达水平较低。间充质干细胞（MSCs）可以向肿瘤细胞迁移，称为肿瘤趋向性，它们能够通过缝隙连接和非亲密机制传递基于工程 miR-30c 的治疗方法。

材料和方法

利用 XbaI 和 EcoRI 将 miR-30c 克隆到 pCDH-CMV-MCS-EF1-copGFP 载体中，构建 pCDH-miR-30c。然后将 psPAX2、pMD2.G 和 pCDH-miR-30c 共转染到 Hek-293T 中，产生 lenti-miR-30c 病毒颗粒。接下来，用 lenti-miR-30c 转导骨髓间充质干细胞（BM-MSCs）。然后，我们将 U-251 细胞系与 BM-MCSs-miR-30c 共培养，并使用实时 PCR 和流式细胞术评估细胞凋亡率以及 IL-6、Klf4、Sox2、c-Myc 和 Oct4 的相对表达水平。

结果

U-251 细胞经 BM-MSCs-miR-30c 处理后，划痕愈合试验显示其迁移能力较低。此外，用 Annexin V/7AAD 进行的凋亡试验表明，经处理后 U-251 细胞的凋亡数量增加。miR-30c 靶向 IL-6 并诱导细胞凋亡。它还影响自我更新和抗凋亡基因簇，即 Klf4、Sox2、c-Myc 和 Oct4，以诱导细胞凋亡并减少迁移和侵袭。