ABSTRACT

This study aimed to investigate the influence of miR-221-3p and O⁶-methylguanine-DNA methyltransferase (MGMT) interaction in human hepatocellular carcinoma (HCC), thereby revealing a novel molecular mechanism of hepatic carcinogenesis involving miR-221-3p and MGMT.

Fluorescence qPCR and immunoblot assays were performed to determine the expression of RNA and protein in HCC tissues and cell lines. We also employed the firefly and Renilla luciferase assay to verify the target relationship between miR-221-3p and MGMT mRNA. Assessments including the MTT assay, wound-healing assay, transwell assay, colony foci formation experiment, and flow cytometric experiment were carried out to determine the viability, migration, invasion, proliferation, cell cycle progression, and apoptosis of SMMC-7721 and BEL-7404 cell lines with the modulated expression of miR-221-3p and MGMT. Compared to healthy tissues and cell line HL7702, miR-221-3p was significantly upregulated but MGMT was significantly downregulated in carcinomas and cancerous cell lines. Forced miR-221-3p overexpression was found to enhance the proliferation, migration, invasion, and clonogenicity of cell lines, but it suppressed cell apoptosis. Findings also revealed that forced miR-221-3p overexpression had little effect on cell cycle progression. After MGMT was confirmed to be atarget gene of miR-221-3p, it was found that the forced upregulation of miR-221-3p downregulated MGMT mRNA and protein levels significantly. MiR-221-3p was identified as an HCC promoting factor, and it specifically inhibited the expression of the MGMT. Besides, the upregulation of miR-221-3p had apositive influence on HCC pathogenesis by inhibiting MGMT expression.

摘要

本研究旨在探讨miR-221-3p和O ^{6 -}甲基鸟嘌呤-DNA甲基转移酶（MGMT）相互作用对人肝细胞癌（HCC）的影响，从而揭示miR-221-3p和MGMT参与肝癌发生的新分子机制。 .

进行荧光 qPCR 和免疫印迹分析以确定 HCC 组织和细胞系中 RNA 和蛋白质的表达。我们还采用萤火虫和海肾荧光素酶测定来验证 miR-221-3p 和 MGMT mRNA 之间的靶标关系。进行了包括 MTT 测定、伤口愈合测定、transwell 测定、集落灶形成实验和流式细胞术实验在内的评估，以确定 SMMC-7721 和 BEL-的活力、迁移、侵袭、增殖、细胞周期进程和凋亡。 miR-221-3p 和 MGMT 表达调节的 7404 细胞系。与健康组织和细胞系 HL7702 相比，miR-221-3p 在癌和癌细胞系中显着上调，但 MGMT 显着下调。发现强制 miR-221-3p 过表达可增强增殖，细胞系的迁移、侵袭和克隆形成，但它抑制细胞凋亡。研究结果还表明，强制 miR-221-3p 过表达对细胞周期进程几乎没有影响。在证实MGMT是miR-221-3p的靶基因后，发现miR-221-3p的强制上调显着下调MGMT mRNA和蛋白质水平。MiR-221-3p 被鉴定为 HCC 促进因子，它特异性抑制 MGMT 的表达。此外，miR-221-3p的上调通过抑制MGMT表达对HCC发病机制产生积极影响。发现 miR-221-3p 的强制上调显着下调了 MGMT mRNA 和蛋白质水平。MiR-221-3p 被鉴定为 HCC 促进因子，它特异性抑制 MGMT 的表达。此外，miR-221-3p的上调通过抑制MGMT表达对HCC发病机制产生积极影响。发现 miR-221-3p 的强制上调显着下调了 MGMT mRNA 和蛋白质水平。MiR-221-3p 被鉴定为 HCC 促进因子，它特异性抑制 MGMT 的表达。此外，miR-221-3p的上调通过抑制MGMT表达对HCC发病机制产生积极影响。