Department of Oncology, The First Affiliated Hospital of Nanchang University, China.
Department of Oncology, Jiangxi Lushan People's Hospital, Jiujiang, China.
Adv Clin Exp Med. 2022 Jun;31(6):671-687. doi: 10.17219/acem/146580.
Radiotherapy is the main treatment for nasopharyngeal carcinoma. The radioresistance mechanism of cells is related to miRNAs.
To investigate the miRNA profiling of HONE1 and CNE2 after X-ray therapy.
The HONE1 and CNE2 cells were treated with X-ray at 4 Gy, 8 Gy, 16 Gy, and 20 Gy doses. The cell lines CNE2 with the best therapy effects and HONE1 with the worst therapy effects were screened out. Apoptosis and cell viability were detected with flow cytometry and Cell Counting Kit-8 (CCK-8). High-throughput sequencing was performed. A miRNA library was constructed. The miRNA annotation expression distribution, family prediction and target gene interaction, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted.
The 24-hour 20 Gy dose X-rays were selected as the optimal therapy conditions. The CNE2_C, CNE2_M, HONE1_C and HONE1_M miRNAs accounted for 26.5%, 31.7%, 21.3%, and 22.9% of the Cleandata reads count, respectively, and the contents of rRNAs accounted for 24.9%, 14.7%, 25.1%, and 25.1% of the Cleandata reads count, respectively. The miRNAs with differential expression between the HONE1 and CNE2 cell lines including hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-125a-5p, hsa-miR-26a-5p, hsa-let-7f-5p, hsa-miR-20a-5p, and hsa-miR-24a-3p. There were also differentially expressed miRNAs in HONE1_C vs. HONE1_M, such as hsa-miR-21-5p and hsa-let-7i-5p. The differentially expressed miRNA in CNE2_C vs. CNE2_M was hsa-miR-148b-3p. The Gene Ontology analysis showed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were mainly enriched in biological process such as negative and positive regulation of transcription from RNA polymerase II promoter, cellular component such as cytosol and molecular function such as protein binding factor. The KEGG pathway analysis revealed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were enriched in the cancer-related pathways, such as pathways in cancer, MAPK signaling pathway and Wnt signaling pathway.
Twelve miRNAs and 9 genes which contribute to X-ray radiation resistance were identified. Among those with differential expression between the HONE1 and CNE2 cell lines, which played a regulatory role in multiple pathways, were hsa-miR-20a-5p, hsa-let-7a-5p, hsa-let-7f5p, hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-148b-3p, and hsa-miR-200c-3p. The corresponding genes were MAPK1, SOS1, TGFBR1, TGFBR2, TP53, CASP3, CCNE2, PTEN, and CDK2.
放射治疗是鼻咽癌的主要治疗方法。细胞的放射抵抗机制与 miRNAs 有关。
研究 X 射线治疗后 HONE1 和 CNE2 细胞的 miRNA 谱。
用 4 Gy、8 Gy、16 Gy 和 20 Gy 剂量的 X 射线照射 HONE1 和 CNE2 细胞。筛选出治疗效果最好的 CNE2 细胞系和治疗效果最差的 HONE1 细胞系。用流式细胞术和细胞计数试剂盒-8（CCK-8）检测细胞凋亡和细胞活力。进行高通量测序。构建 miRNA 文库。进行 miRNA 注释表达分布、家族预测和靶基因相互作用、基因本体论和京都基因与基因组百科全书（KEGG）通路分析。
选择 24 小时 20 Gy 剂量 X 射线作为最佳治疗条件。CNE2_C、CNE2_M、HONE1_C 和 HONE1_M 的 miRNAs 分别占 CleanData 读数的 26.5%、31.7%、21.3%和 22.9%，rRNAs 的含量分别占 CleanData 读数的 24.9%、14.7%、25.1%和 25.1%。在 HONE1 和 CNE2 细胞系之间差异表达的 miRNAs 包括 hsa-miR-21-5p、hsa-let-7a-5p、hsa-miR-125a-5p、hsa-miR-26a-5p、hsa-let-7f-5p、hsa-miR-20a-5p 和 hsa-miR-24a-3p。在 HONE1_C 与 HONE1_M 之间也存在差异表达的 miRNAs，如 hsa-miR-21-5p 和 hsa-let-7i-5p。在 CNE2_C 与 CNE2_M 之间差异表达的 miRNA 是 hsa-miR-148b-3p。基因本体论分析显示，HONE1_M 与 CNE2_M 之间差异表达的 miRNA 相互作用基因主要富集在 RNA 聚合酶 II 启动子转录的负调控和正调控等生物学过程、细胞溶质等细胞成分以及蛋白结合因子等分子功能。KEGG 通路分析表明，HONE1_M 与 CNE2_M 之间差异表达的 miRNA 相互作用基因富集在癌症相关通路，如癌症通路、MAPK 信号通路和 Wnt 信号通路。
鉴定出 12 个与 X 射线辐射抵抗相关的 miRNAs 和 9 个基因。在 HONE1 和 CNE2 细胞系之间差异表达的 miRNA 中，hsa-miR-20a-5p、hsa-let-7a-5p、hsa-let-7f5p、hsa-let-7i-5p、hsa-miR-30e-5p、hsa-miR-148b-3p 和 hsa-miR-200c-3p 等 miRNA 在多个通路中发挥调节作用。相应的基因是 MAPK1、SOS1、TGFBR1、TGFBR2、TP53、CASP3、CCNE2、PTEN 和 CDK2。
