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投稿时间：2024-01-12 网络发布日期：2024-10-20
投稿时间：2024-01-12 网络发布日期：2024-10-20
中文摘要: 目的 筛选宫颈癌细胞系（SiHa/HeLa）与正常宫颈上皮细胞系（H8）培养上清外泌体中miRNA表达差异，并通过生物信息分析确定靶基因。
方法 培养宫颈癌细胞系（SiHa/HeLa）与正常宫颈上皮细胞系（H8），收集细胞培养的上清液，超速离心法提取外泌体，透射电镜（TEM）观察外泌体的形态，蛋白质印迹实验鉴定外泌体标志蛋白。将提取的外泌体进行miRNA高通量测序，分别得出SiHa和H8外泌体中差异的miRNA，HeLa和H8外泌体中差异的miRNA，取二者交集。对于所得到的交集miRNA进行靶基因预测并进行基因本体（GO）、京都基因与基因组百科全书（KEGG）通路富集分析。
结果 TEM观察到外泌体呈典型的杯状结构，大小30～200 nm，蛋白质印迹实验显示所提取样本有外泌体标志蛋白的表达。测序结果显示HeLa与H8源外泌体表达差异的miRNA有235个，SiHa与H8源外泌体表达差异的miRNA有249个，两种细胞系源外泌体中表达差异的miRNA有166个。GO生物功能分析显示，宫颈癌细胞外泌体miRNA的差异主要分布于细胞核、胞质、囊泡和细胞骨架等，蛋白质结合主要在金属离子结合、转移酶活性、核苷酸结合等环节发生作用，主要参与RNA聚合酶Ⅱ对转录的调控、DNA模板、磷酸化作用、细胞周期等生物功能。KEGG通路富集分析显示，宫颈癌相关通路与代谢途径、肿瘤的发病途径、轴突导向、PI3K-Akt、钙离子、Rap1、Ras等信号通路的调控相关。
结论 miR-335-5p、miR-660、miR-155-5p、miR-339-5p、miR-345-3p等miRNA在宫颈癌源外泌体中差异表达，这些差异表达的miRNA通过代谢途径、肿瘤的发病途径、轴突导向、PI3K-Akt、钙离子、Rap1、Ras等信号通路在宫颈癌的进展中发挥关键作用。
Abstract:ObjectiveTo screen the expression differences of miRNAs in supernatant exosomes of cervical cancer cell line (SiHa/HeLa) and normal cervical epithelial cell line (H8) and to identify target genes by bioinformatics analysis.
Methods Cervical cancer cell line (SiHa/HeLa) and normal cervical epithelial cell line (H8) were cultured, the supernatant of cultured cells was collected, and exosomes were extracted by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscopy (TEM), and exosome marker proteins were identified by Western blot experiments. The extracted exosomes were subjected to high-throughput sequencing of miRNAs, and the differential miRNAs in SiHa and H8 exosomes, and the differential miRNAs in HeLa and H8 exosomes, respectively, were obtained. The target genes of the obtained intersecting miRNAs were predicted, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed.
Results TEM showed that the exosomes were typical cup-shaped structures with the size of 30~200 nm. Western blot showed that the extracted samples had the expression of exosomes marker proteins. Sequencing results revealed that there were 235 miRNAs with differential expression between HeLa and H8 exosomes, 249 miRNAs with differential expression between SiHa and H8 exosomes, and 166 miRNAs with differential expression in exosomes from two cell lines. GO biological function analysis revealed that the differences in cervical cancer exosomal miRNAs were mainly distributed in nucleus, cytoplasm, vesicles and cytoskeleton, and protein binding mainly played a role in metal ion binding, transferase activity, nucleotide binding and other links, and was mainly involved in RNA polymerase Ⅱs transcription regulation, DNA template, phosphorylation, cell cycle and other biological functions. KEGG pathway enrichment analysis displayed that cervical cancer-related pathways were related to the regulation of metabolic pathways, tumor pathogenesis pathways, axon guidance, PI3K-Akt, calcium ions, Rap1, Ras and other signaling pathways.
Conclusion miRNAs such as miR-335-5p, miR-660, miR-155-5p, miR-339-5p, and miR-345-3p are differentially expressed in exosomes derived from cervical cancer, and these differentially expressed miRNAs play a key role in the progression of cervical cancer through metabolic pathways, pathogenesis of cancer, axon guidance, PI3K-Akt, calcium ions, Rap1, Ras and other signaling pathways.
文章编号： 中图分类号：R735.7 文献标志码：A
基金项目:新疆维吾尔自治区重点实验室开放课题项目（2022D04023）
附件
| 作者 | 单位 |
| 娄邵升1 | 1. 新疆医科大学基础医学院，新疆 乌鲁木齐 830000； |
| 曹玲玲1 | 1. 新疆医科大学基础医学院，新疆 乌鲁木齐 830000； |
| 欧阳云珊1 | 1. 新疆医科大学基础医学院，新疆 乌鲁木齐 830000； |
| 吾米丹·阿不都热合曼1 | 1. 新疆医科大学基础医学院，新疆 乌鲁木齐 830000； |
| 林晨2 | 2. 新疆医科大学研究生学院，新疆 乌鲁木齐 830000 |
| Author Name | Affiliation |
| LOU Shaosheng*, CAO Lingling, OUYANG Yunshan, WUMIDAN Aburureheman, LIN Chen | *School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, Xinjiang 830000, China |
引用文本：
娄邵升,曹玲玲,欧阳云珊,等.宫颈癌源外泌体差异表达miRNA筛选及其关键靶基因的生物信息学分析[J].中国临床研究,2024,37(10):1567-1572.
娄邵升,曹玲玲,欧阳云珊,等.宫颈癌源外泌体差异表达miRNA筛选及其关键靶基因的生物信息学分析[J].中国临床研究,2024,37(10):1567-1572.