与GRP78相关的miR-15a-5p在鼻咽癌放疗抵抗中的作用

The role of miR-15a-5p associated with GRP78 in radiotherapy resistance of nasopharyngeal carcinoma

摘要:

目的 探究与葡萄糖调节蛋白78(GRP78)相关的非编码微小RNA-15a-5p(miR-15a-5p)在鼻咽癌放疗抵抗中的作用并初步探索二者之间的调控机制。方法 应用shRNA技术干扰鼻咽癌细胞GRP78表达，通过miRNA芯片筛选与GRP78相关miRNA，并结合RT-PCR实验验证敲低后miR-15a-5p表达水平；在鼻咽癌细胞系中瞬转miR-15a-5p相应的模拟物和抑制剂后验证miR-15a-5p在鼻咽癌放疗抵抗中的作用；通过生物信息学初步分析GRP78调控miR-15a-5p的可能分子机制。结果 建立的鼻咽癌放射抵抗细胞系C666-IR对放射的抵抗性强于母本细胞C666-1，且GRP78蛋白的表达更高，分布更广; C666-IR细胞中miR-15a-5p的表达高于C666-1细胞，且GRP78高表达的鼻咽癌细胞经射线照射后miR-15a-5p仍维持较高水平，而敲低GRP78可以降低鼻咽癌细胞的miR-15a-5p水平；瞬转miR-15a-5p模拟物增强C666-1细胞的迁移能力和放射抵抗性，瞬转miR-15a-5p抑制剂降低C666-IR细胞的迁移能力和放射抵抗性;生物信息学分析预测miR-15a-5p基因启动子460位点左右区域与转录因子结合可能性最大，且预测到13个可能的转录因子，其中STAT4和FOXP3可能性最大。结论 GRP78介导miR-15a-5p表达上调促进鼻咽癌放疗抵抗，其调控机制可能为GRP78通过驱动转录因子STAT4/FOXP3提高miR-15a-5p表达，靶向miR-15a-5p可能为放疗抵抗的鼻咽癌提供新的治疗策略。

Abstract:

Objective To investigate the role of non-coding microRNA-15a-5p (miR-15a-5p) associated with glucose-regulated protein 78 (GRP78) in radioresistance of nasopharyngeal carcinoma and to preliminarily explore the regulatory mechanism between them. Methods The expression of GRP78 in nasopharyngeal carcinoma cells was interfered by shRNA technology. The miRNA related to GRP78 was screened by miRNA chip, and the expression level of miR-15a-5p after knockdown was verified by reverse transcription-polymerase chain reaction. The role of miR-15a-5p in nasopharyngeal carcinoma radiotherapy resistance was verified by transient transfection of miR-15a-5p corresponding mimics and inhibitors in nasopharyngeal carcinoma cell lines. The possible molecular mechanism of GRP78 regulating miR-15a-5p was preliminarily analyzed by bioinformatics. Results The established radioresistant nasopharyngeal carcinoma cell line C666-IR showed stronger resistance to radiation than its parental cell C666-1, and had higher expression and wider distribution of GRP78 protein. The expression of miR-15a-5p in C666-IR cells was higher than that in C666-1 cells. After irradiation, the level of miR-15a-5p remained high in nasopharyngeal carcinoma cells with high expression of GRP78, while knockdown of GRP78 could reduce the level of miR-15a-5p in nasopharyngeal carcinoma cells. Transient transfection of miR-15a-5p mimics enhanced the migration ability and radioresistance of C666-1 cells, while transient transfection of miR-15a-5p inhibitors reduced the migration ability and radioresistance of C666-IR cells. Bioinformatics analysis predicted that the region around the 460 site of the miR-15a-5p gene promoter was most likely to bind to transcription factors, and 13 possible transcription factors were predicted, of which STAT4 and FOXP3 had the greatest possibility. Conclusions GRP78 mediates the up-regulation of miR-15a-5p expression to promote radiotherapy resistance of nasopharyngeal carcinoma. The regulatory mechanism may be that GRP78 increases the expression of miR-15a-5p by driving the transcription factor STAT4/FOXP3. Targeting miR-15a-5p may provide a new therapeutic strategy for radiotherapy-resistant nasopharyngeal carcinoma.