Zhu Changyu, Jiang Xiaolei, Xiao Hua, Guan Jianmei
Personalized Drug Therapy Key Laboratory of Sichuan Province, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, P. R. China.
Department of Pharmacy, Sichuan Academy of Medical Science & Sichuan Provincial People's Hospital, Chengdu, 610072, P. R. China.
Cell Death Discov. 2022 Jan 10;8(1):17. doi: 10.1038/s41420-021-00794-5.
Radioresistance prevails as one of the largest obstacles in the clinical treatment of nasopharyngeal carcinoma (NPC). Meanwhile, tumor-derived extracellular vesicles (TEVs) possess the ability to manipulate radioresistance in NPC. However, its mechanism remains to be further explored. Therefore, the current study set out to explore the mechanism of microRNA (miR)-142-5p delivered by TEVs in regard to the radiosensitivity of NPC. Firstly, peripheral blood samples were collected from patients with radioresistance and radiosensitivity, followed by RT-qPCR detection of miR-142-5p expression. A dual-luciferase reporter assay was carried out to elucidate the targeting relationship of miR-142-5p with HGF and EGF. In addition, radiotherapy-resistant NPC cell models were established by screening NPC cells with gradient increasing radiation exposure, and co-incubated with EVs isolated from miR-142-5p mimic-transfected NPC cells, followed by overexpression of HGF and EGF. Moreover, cell viability was detected by means of MTS, cell proliferation with a colony formation assay, cell apoptosis with flow cytometry, and expression patterns of related genes with the help of Western blot analysis. NPC xenotransplantation models in nude mice were also established by subcutaneous injection of 5-8FR cells to determine apoptosis, tumorigenicity, and radiosensitivity in nude mice. It was found that miR-142-5p was poorly expressed in peripheral blood from NPC patients with radioresistance. Mechanistic experimentation illustrated that miR-142-5p inversely targeted HGF and EGF to inactivate the HGF/c-Met and EGF/EGFR pathways, respectively. NPC cell apoptosis was observed to be augmented, while their radioresistance and proliferation were restricted by EVs-miR-142-5p or HGF silencing, or EGF silencing. Furthermore, EVs-miR-142-5p inhibited growth and radioresistance and accelerated the apoptosis of radiotherapy-resistant NPC cells in nude mice by inhibiting the HGF/c-Met and EGF/EGFR pathways. Collectively, our findings indicated that TEVs might inhibit the HGF/c-Met and EGF/EGFR pathways by delivering miR-142-5p into radiotherapy-resistant NPC cells to enhance radiosensitivity in NPC.
放射抗性是鼻咽癌(NPC)临床治疗中最大的障碍之一。同时,肿瘤来源的细胞外囊泡(TEV)具有调控NPC放射抗性的能力。然而,其机制仍有待进一步探索。因此,本研究旨在探讨TEV传递的微小RNA(miR)-142-5p对NPC放射敏感性的影响机制。首先,收集放射抗性和放射敏感性患者的外周血样本,随后通过RT-qPCR检测miR-142-5p的表达。进行双荧光素酶报告基因检测以阐明miR-142-5p与HGF和EGF的靶向关系。此外,通过对NPC细胞进行梯度增加辐射暴露筛选建立放射抗性NPC细胞模型,并与从miR-142-5p模拟物转染的NPC细胞中分离的细胞外囊泡(EV)共同孵育,随后过表达HGF和EGF。此外,通过MTS检测细胞活力,采用集落形成试验检测细胞增殖,利用流式细胞术检测细胞凋亡,并借助蛋白质印迹分析检测相关基因的表达模式。还通过皮下注射5-8FR细胞建立裸鼠NPC异种移植模型,以确定裸鼠的细胞凋亡、致瘤性和放射敏感性。研究发现,miR-142-5p在放射抗性NPC患者的外周血中表达较低。机制实验表明,miR-142-5p分别通过反向靶向HGF和EGF使HGF/c-Met和EGF/EGFR通路失活。观察到EV-miR-142-5p或HGF沉默、EGF沉默可增强NPC细胞凋亡,同时限制其放射抗性和增殖。此外,EV-miR-142-5p通过抑制HGF/c-Met和EGF/EGFR通路,抑制裸鼠中放射抗性NPC细胞的生长和放射抗性并加速其凋亡。总体而言,我们的研究结果表明,TEV可能通过将miR-142-5p传递到放射抗性NPC细胞中抑制HGF/c-Met和EGF/EGFR通路,从而增强NPC的放射敏感性。