Department of Gastroenterology, Shanxi Provincial People's Hospital, Shanxi, China.
Department of Lymphoma, Shanxi Bethune Hospital, Shanxi, China.
Balkan Med J. 2022 Jan 25;39(1):55-65. doi: 10.4274/balkanmedj.galenos.2021.2021-8-62.
BACKGROUND: MiR-196a is particularly noticeable in the development of liver cancer. However, the rapid degradation by ribonuclease (RNase) imposes a limit on the miRNA gene therapy applications. AIMS: To design a novel gene-targeting nano system for liver cancer treatment. STUDY DESIGN: Cell culture study and animal experimentation. METHODS: Deacetylated (DEAC)-poly-N-acetylglucosamine (PNAG)-folic acid (FA) was prepared via ethyl (dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide reaction, and miR-196a inhibitor (miR-196a I)/DEAC-PNAG-FA was prepared through self-assembly. The characterization and nucleic acid protection of the self-assembly system were also determined. The biological function and related mechanism of the prepared system were studied at cellular and molecular levels. Mice were established as a xenotransplantation model to evaluate the anticancer capacity of miR-196a I/DEAC-PNAG-FA in vivo. RESULTS: The morphology of miR-196a I/DEAC-PNAG-FA was uniform, and its particle size was approximately 70-100 nm. A nanocarrier with an N/P ratio of 200:1 can maximize the nucleic acid carrying capacity of the self-assembly system. The nanosystem can protect miRNA from RNase degradation and could be internalized rapidly within 4 h. The self-assembly system significantly enhanced the apoptosis-inducing effect of miR-196a I on HepG2 cells ( = 0.003). Molecular biological analyses confirmed that the apoptosis-inducing effect of the nanosystem was due to the inhibition of miR-196a gene expression in HepG2 cells, which upregulate the expression of pro-apoptotic proteins FOXO1 ( < 0.001), Bax ( < 0.001), Ki67 ( < 0.001), and proliferating cell nuclear antigen ( < 0.001), and inhibit the expression of apoptosis inhibitory protein Bcl-2 ( < 0.001). Moreover, compared with free miR-196a inhibitor or miR-196a I/DEAC-PNAG, miR-196a I/DEAC-PNAG-FA can more effectively inhibit tumor growth in vivo ( = 0.026). CONCLUSION: The newly prepared self-assembly targeting system can effectively induce apoptosis and abrogate tumor growth, which may open a new approach for liver cancer treatment.
背景：miR-196a 在肝癌的发展中尤为明显。然而，核糖核酸酶（RNase）的快速降解对 miRNA 基因治疗的应用构成了限制。
目的：设计一种用于肝癌治疗的新型基因靶向纳米系统。
研究设计：细胞培养研究和动物实验。
方法：通过乙基（二甲基氨基丙基）碳二亚胺/N-羟基琥珀酰亚胺反应制备去乙酰化（DEAC）-聚-N-乙酰葡萄糖胺（PNAG）-叶酸（FA），并通过自组装制备 miR-196a 抑制剂（miR-196a I）/DEAC-PNAG-FA。还确定了自组装系统的特征和核酸保护。在细胞和分子水平上研究了制备系统的生物学功能和相关机制。建立了小鼠异种移植模型，以评估 miR-196a I/DEAC-PNAG-FA 在体内的抗癌能力。
结果：miR-196a I/DEAC-PNAG-FA 的形态均匀，粒径约为 70-100nm。N/P 比为 200:1 的纳米载体可以使自组装系统的核酸携带能力最大化。该纳米系统可以保护 miRNA 免受 RNase 降解，并能在 4 小时内迅速内化。自组装系统可显著增强 miR-196a I 对 HepG2 细胞的诱导凋亡作用（=0.003）。分子生物学分析证实，纳米系统的诱导凋亡作用是由于 miR-196a 基因在 HepG2 细胞中的表达受到抑制，上调了促凋亡蛋白 FOXO1（<0.001）、Bax（<0.001）、Ki67（<0.001）和增殖细胞核抗原（<0.001）的表达，并抑制了凋亡抑制蛋白 Bcl-2 的表达（<0.001）。此外，与游离 miR-196a 抑制剂或 miR-196a I/DEAC-PNAG 相比，miR-196a I/DEAC-PNAG-FA 能更有效地抑制体内肿瘤生长（=0.026）。
结论：新制备的靶向自组装系统能有效诱导细胞凋亡，抑制肿瘤生长，为肝癌治疗开辟新途径。
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