MiRNA-218 对PAG1 的调控及其对卵巢癌干细胞的影响
| 作者: |
1贺敏,
2陈勇伟,
1曾康康,
1郑蓉
1 十堰市太和医院(湖北医药学院附属医院)妇产科，湖北 十堰 442700 2 十堰市太和医院(湖北医药学院附属医院)泌尿外科，湖北 十堰 442700 |
| 通讯: |
贺敏
Email: 3036420578@qq.com |
| DOI: | 10.3978/j.issn.2095-6959.2019.04.003 |
摘要
目的：研究miR-218在卵巢癌干细胞(ovarian cancer stem cells，OCSCs)中的表达，并探讨其对PAG1的调控作用和对OCSCs的影响及作用机制。方法：收集卵巢癌患者新鲜肿瘤组织，分离培养原代卵巢癌细胞，用流式细胞仪筛选出CD133+表型的OCSCs。采用Western印迹法检测干性特异相关基因Nanog和Sox2蛋白的表达。qRT-PCR检测miR-218在OCSCs中的表达。miR-218 mimic转染OCSCs，分为miR-218 mimic组和miR-218 NC组，MTS检测各组OCSCs增殖；软琼脂克隆实验检测各组OCSCs肿瘤球形成能力的影响；miR-218过表达慢病毒质粒及对照质粒vector感染OCSCs，经1.0 μg/mL嘌呤霉素筛选稳定过表达miR-218或vector的OCSCs细胞，注射到BALB/c裸鼠中，观察miR-218对OCSCs体内成瘤能力的影响。Targetscan 7.2预测软件及双荧光素酶报道基因试验，分析miR-218对PAG1基因的靶向作用，qRT-PCR检测miR-218 mimic组和miR-218 NC组OCSCs中PAG1 mRNA的表达。Western印迹法检测miR-218 mimic组和miR-218 NC组OCSCs中pSrc和pAKT蛋白的表达。结果：Nanog和Sox2蛋白在CD133+表型OCSCs中的表达显著高于CD133−表型OCSCs中的表达；miR-218在CD133+表型OCSCs中的表达显著低于CD133−表型OCSCs中的表达(P<0.05)。miR-218 mimic组OCSCs增殖减慢，OCSCs肿瘤球形成能力下降。OCSCsmiR-218裸鼠成瘤体积显著低于OCSCsvector组(P<0.05)。TargetScan7.2软件预测和双荧光素酶报道基因试验证实PAG1为miR-218的靶基因，miR-218 mimic组PAG1 mRNA的表达减少。Western印迹法检测miR-218 mimic组OCSCs中pSrc和pAKT蛋白的表达显著减少。结论：MiR-218在OCSCs中低表达，可能通过靶向下调PAG1，抑制pSrc和pAKT蛋白的表达影响OCSCs的干性。
关键词：
卵巢癌；miRNA-218；PAG1；干性
Regulation of miRNA-218 on PAG1 and its effect on ovarian cancer stem cells
CorrespondingAuthor: HE Min Email: 3036420578@qq.com
DOI: 10.3978/j.issn.2095-6959.2019.04.003
Abstract
Objective: To investigate the expression of miR-218 in ovarian cancer stem cells (OCSCs), and to explore its regulation on PAG1 and its effect on OCSCs. Methods: Fresh tumor tissues of ovarian cancer patients were collected, and primary ovarian cancer cells were isolated and cultured. The OCSCs of CD133+ phenotype were selected by flow cytometry. Western blot was used to detect the expression of stem specific genes Nanog and Sox2. qRT-PCR was used to detect the expression of miR-218 in OCSCs. miR-218 mimic was transfected into OCSCs and divided into miR-218 mimic group and miR-218 NC group. MTS was used to detect the proliferation of OCSCs in each group. Soft agar clone assay was used to detect the effect of tumor formation ability of each group; OCSCs were infected with viral plasmid and control plasmid vector, and OCSCs cells stably overexpressing miR-218 or vector were screened by 1.0 μg/mL puromycin and injected into BALB/c nude mice to observe the tumorigenic ability of miR-218 in OCSCs. Targetscan 7.2 predictive software and dual luciferase reporter gene assay were used to analyze the targeting effect of miR-218 on PAG1 gene. qRT-PCR was used to detect the expression of PAG1 mRNA in miR-218 mimic group and miR-218 NC group. Western blot was used to detect the expression of pSrc and pAKT proteins in miR-218 mimic group and miR-218 NC group. Results: The expression of Nanog and Sox2 protein in CD133+ phenotype OCSCs was significantly higher than that in CD133− phenotype OCSCs; the expression of miR-218 mRNA in CD133+ phenotype OCSCs was significantly lower than that in CD133− phenotype OCSCs (P<0.05); the proliferation of OCSCs in the miR-218 mimic group was slowed down, and the tumor formation ability of OCSCs was decreased. The tumor formation volume of OCSCsmiR-218 nude mice was significantly lower than that of OCSCsvector group (P<0.05). TargetScan 7.2 software prediction and dual luciferase reporter gene assay confirmed that PAG1 is a target gene of miR-218, and the expression of PAG1 mRNA is decreased in miR-218 mimic group. The expression of pSrc and pAKT protein in OCSCs of miR-218 mimic group was significantly reduced by Western blot. Conclusion: MiR-218 is down-regulated in OCSCs, which may affect the dryness of OCSCs by down-regulating PAG1 and inhibiting the expression of pSrc and pAKT proteins.
Keywords：
ovarian cancer; miR-218; PAG1; stem