miR-128-3p 通过调节血管内皮生长因子 C 增加鼻咽癌的放射敏感性。

miR-128-3p increases the radiosensitivity in nasopharyngeal carcinoma via regulating vascular endothelial growth factor C.

机构信息

Department of Radiotherapy, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, China.

出版信息

Pathol Res Pract. 2023 Sep;249:154768. doi: 10.1016/j.prp.2023.154768. Epub 2023 Aug 16.

DOI:10.1016/j.prp.2023.154768

PMID:37639953

Abstract

PURPOSE

This study aims to investigate the role of miR-128-3p in the radiosensitivity of nasopharyngeal carcinoma (NPC) and its underlying mechanism.

METHODS

6-10B cells were transfected with miR-128-3p mimic, pcDNA-VEGFC, and the corresponding negative control. C666-1 cells were transfected with miR-128-3p inhibitor, sh-VEGFC, and the corresponding negative control. RT-qPCR was used to determine the miR-128-3p and VEGFC mRNA expression level. Dual-luciferase assay was used to investigate the relationship between miR-128-3p and VEGFC. The protein levels of VEGFC, H2AX, γ-H2AX, p-P50, p-P65, p-IκB, and the apoptosis markers Bcl-2, caspase3, caspase9, and Bax were detected by Western blot. The proliferation activity was detected by CCK-8, and cell DNA damage was assessed by comet assay. The apoptosis rate was detected by flow cytometry. The growth of NPC in vivo was observed in mice through xenotransplantation. TUNEL staining was used to detect cell apoptosis in tumor tissues.

RESULTS

miR-128-3p was targeted and was negatively regulated with VEGFC. Overexpression of miR-128-3p or knockdown VEGFC significantly inhibited the proliferation of 6-10B and C666-1 cells, induced DNA damage and apoptosis and promoted the radiosensitivity of cells. Knocking down miR-128-3p or up-regulated VEGFC promoted the proliferation of C666-1 and 6-10B cells, reduced cell DNA damage and apoptosis, and enhanced cell resistance to radiotherapy. Overexpression of miR-128-3p reversed the effect of VEGFC on 6-10B cells and inhibited P50/P65/IKB signal pathway. In vivo, experiments in mice confirmed that miR-128-3p significantly inhibited NPC proliferation and promoted DNA damage and apoptosis by targeting VEGFC.

CONCLUSION

The miR-128-3p pathway is a novel therapy target to overcome radiation resistance in NPC.

摘要

目的

本研究旨在探讨 miR-128-3p 在鼻咽癌（NPC）放射敏感性中的作用及其潜在机制。

方法

将 miR-128-3p 模拟物、pcDNA-VEGFC 及其相应的阴性对照转染至 6-10B 细胞中，将 miR-128-3p 抑制剂、sh-VEGFC 及其相应的阴性对照转染至 C666-1 细胞中。采用 RT-qPCR 检测 miR-128-3p 和 VEGFC mRNA 表达水平。采用双荧光素酶报告基因实验研究 miR-128-3p 与 VEGFC 的关系。采用 Western blot 检测 VEGFC、H2AX、γ-H2AX、p-P50、p-P65、p-IκB 以及凋亡标志物 Bcl-2、caspase3、caspase9 和 Bax 的蛋白水平。采用 CCK-8 检测细胞增殖活性，采用彗星实验评估细胞 DNA 损伤，采用流式细胞术检测细胞凋亡率。通过异种移植观察 NPC 在小鼠体内的生长情况，采用 TUNEL 染色检测肿瘤组织中的细胞凋亡。

结果

miR-128-3p 是 VEGFC 的靶向调节因子。过表达 miR-128-3p 或敲低 VEGFC 显著抑制 6-10B 和 C666-1 细胞的增殖，诱导 DNA 损伤和凋亡，并促进细胞的放射敏感性。敲低 miR-128-3p 或上调 VEGFC 促进 C666-1 和 6-10B 细胞的增殖，减少细胞 DNA 损伤和凋亡，增强细胞对放疗的抵抗能力。过表达 miR-128-3p 逆转了 VEGFC 对 6-10B 细胞的作用，并抑制了 P50/P65/IKB 信号通路。在体内实验中，小鼠实验证实 miR-128-3p 通过靶向 VEGFC 显著抑制 NPC 增殖，促进 DNA 损伤和凋亡。