载 miR-29a 的细胞外囊泡通过阻断自噬有效地诱导 HCC 细胞凋亡。

MiR-29a-laden extracellular vesicles efficiently induced apoptosis through autophagy blockage in HCC cells.

机构信息

Department of Developmental Biology, University of Science and Culture, ACECR, Tehran 14155-4364, Iran; Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 14155-4364, Iran; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 14155-4364, Iran.

Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 14155-4364, Iran; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 14155-4364, Iran; Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

出版信息

Eur J Pharm Biopharm. 2024 Oct;203:114470. doi: 10.1016/j.ejpb.2024.114470. Epub 2024 Aug 26.

DOI:10.1016/j.ejpb.2024.114470

PMID:39197541

Abstract

BACKGROUND

In spite of significant advancements in theraputic modalities for hepatocellular carcinoma (HCC), there is still a high annual mortality rate with a rising incidence. Major challenges in the HCC clinical managment are related to the development of therapy resistance, and evasion of tumor cells apoptosis which leading unsatisfactory outcomes in HCC patients. Previous investigations have shown that autophagy plays crucial role in contributing to drug resistance development in HCC. Although, miR-29a is known to counteract authophagy, increasing evidence revealed a down-regulation of miR-29a in HCC patients which correlates with poor prognosis. Beside, evidences showed that miR-29a serves as a negative regulator of autophagy in other cancers. In the current study, we aim to investigate the impact of miR-29a on the autophagy and apoptosis in HCC cells using extracellular vesicles (EVs) as a natural delivery system given their potential in the miRNA delivery both in vitro and in vivo.

METHOD

Human Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles were lately isolated through 20,000 or 110,000 × g centrifugation (EV20K or EV110K, respectively), characterized by western blot (WB), scanning electron microscopy (SEM), and dynamic light scattering (DLS). miR-29a was subsequently loaded into these EVs and its loading efficiency was evaluated via RT-qPCR. Comprehensive in vitro and in vivo assessments were then performed on Huh-7 and HepG2 cell lines.

RESULTS

EV20K-miR-29a treatment significantly induces cell apoptosis and reduces both cell proliferation and colony formation in Huh-7 and HepG2 cell lines. In addition, LC3-II/LC3-I ratio was increased while the expression of key autophagy regulators TFEB and ATG9A were downregulated by this treatment. These findings suggest an effective blockade of autophagy by EV20K-miR-29a leading to apoptosis in the HCC cell lines through concomitant targeting of critical mediators within each pathway.

摘要

背景

尽管肝细胞癌（HCC）的治疗方法有了显著进展，但发病率仍在上升，年死亡率仍然很高。HCC 临床管理中的主要挑战与治疗耐药性的发展以及肿瘤细胞凋亡的逃避有关，这导致 HCC 患者的治疗效果不理想。先前的研究表明，自噬在促进 HCC 耐药性发展中起关键作用。尽管 miR-29a 已知可以拮抗自噬，但越来越多的证据表明 HCC 患者中 miR-29a 的下调与预后不良相关。此外，有证据表明，miR-29a 在其他癌症中作为自噬的负调节剂发挥作用。在本研究中，我们旨在通过细胞外囊泡（EVs）作为一种天然的递送系统，研究 miR-29a 对 HCC 细胞自噬和凋亡的影响，因为它们在 miRNA 递送方面具有在体外和体内的潜力。

方法

通过 20,000 或 110,000×g 离心（分别为 EV20K 或 EV110K）最近分离出人脐带来源间充质基质细胞衍生的细胞外囊泡，通过 Western blot（WB）、扫描电子显微镜（SEM）和动态光散射（DLS）进行表征。随后将 miR-29a 加载到这些 EV 中，并通过 RT-qPCR 评估其加载效率。然后在 Huh-7 和 HepG2 细胞系上进行全面的体外和体内评估。

结果

EV20K-miR-29a 处理显著诱导细胞凋亡，并降低 Huh-7 和 HepG2 细胞系的细胞增殖和集落形成。此外，LC3-II/LC3-I 比值增加，同时该处理下调关键自噬调节剂 TFEB 和 ATG9A 的表达。这些发现表明，EV20K-miR-29a 通过同时靶向每个途径中的关键介质，有效阻断自噬，导致 HCC 细胞系凋亡。