南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (9): 1515-1524.doi: 10.12122/j.issn.1673-4254.2023.09.09

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Hsa-miR-148a-3p通过下调DUSP1基因促进乳腺癌细胞的恶性行为

许家铭，林 龙，陈琼慧，李 兰   

  1. 海南省中医院，海南 海口 570000；海口市人民医院 肿瘤化疗科，海南 海口 570208；海南医学院第一附属医院，海南 海口 570102
  • 出版日期:2023-09-20 发布日期:2023-09-28

Hsa-miR-148a-3p promotes malignant behavior of breast cancer cells by downregulating DUSP1

XU Jiaming, LIN Long, CHEN Qionghui, LI Lan   

  1. Hainan Provincial Hospital of Traditional Chinese Medicine, Haikou 570000, China; Department of Cancer Chemotherapy, Haikou People's Hospital, Haikou 570208, China; First Affiliated Hospital of Hainan Medical College, Haikou 570102, China
  • Online:2023-09-20 Published:2023-09-28

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摘要： 目的 通过生信分析结合体内外实验验证Hsa-miR-148a-3p调控双特异性磷酸酶1（DUSP1）对乳腺癌细胞生物学行为的影响。方法 TCGA数据库下载数据并鉴定差异微小RNA（miRNA)和信使RNA（mRNA）；数据库筛选差异miRNA靶基因；String和Cytoscape构建蛋白质互作网络及筛选TOP10 hub基因；TCGA数据库验证TOP10hub基因的表达；Cytoscape构建miRNA-TOP10hub基因网络。定量逆转录 PCR（RT-qPCR）实验检测Hsa-miR-148a-3p和DUSP1在人乳腺癌组织和细胞中的表达。采用Hsa-miR-148a-3p mimic、Hsa-miR-148a-3p inhibitor及对照NC转染MCF-7细胞。分为NC组，NC mimics组，Hsa-miR-148a-3p mimics组，NC inhibitor组，Hsa-miR-148a-3p inhibitor组；荧光素酶报告基因实验验证Hsa-miR-148a-3p和DUSP1的结合；CCK-8，划痕实验，Transwell和细胞凋亡实验验证Hsa-miR-148a-3p对乳腺癌细胞增殖，迁移，侵袭和凋亡的影响。建立裸鼠成瘤模型，测量小鼠肿瘤质量和体积；Kaplan-Meier生存曲线分析小鼠生存情况；免疫组化检测DUSP1的表达水平。结果 获得乳腺癌差异miRNA 54个（8个下调，46个上调），差异mRNA 799个（509个下调，290个上调）；54个差异miRNA预测靶基因3716个，与差异mRNA取交集，得到150个交集基因；蛋白-蛋白互作网络图中的TOP10hub基因为MET，LPAR1，LAMC1，FOS，EGFR，PIK3R1，DUSP1，GNAI1，LAMA4，NR3C1，这些基因在乳腺癌组织中均显著下调；双荧光素酶报告基因实验证实Hsa-miR-148a-3p能与DUSP1结合；RT-qPCR结果表明，Hsa-miR-148a-3p在乳腺癌组织和细胞中显著上调（P<0.01），而DUSP1则显著下调（P<0.01）。细胞实验结果表明，Hsa-miR-148a-3p促进乳腺癌细胞增殖，迁移和侵袭，抑制细胞的凋亡（P<0.01）。动物实验结果表明，Hsa-miR-148a-3p促进小鼠体内肿瘤质量和体积的增长（P<0.01），缩短小鼠的存活时间（P<0.01），免疫组化结果显示，Hsa-miR-148a-3p 抑制DUSP1的表达水平（P<0.01）。结论 Hsa-miR-148a-3p通过抑制DUSP1的表达而作为癌基因发挥作用，这有助于开发针对乳腺癌的新型治疗靶点。

关键词: Hsa-miR-148a-3p；DUSP1；乳腺癌；促癌

Abstract: Objective To investigate the role of Hsa-miR-148a-3p in regulating biological behaviors of breast cancer cells and explore the mechanism. Methods TCGA database was used to identify the differential miRNAs and mRNAs in breast cancer, and the protein-protein interaction (PPI) network was constructed using String and Cytoscape to screen the top 10 hub genes and construct the miRNA-TOP10hub network. RT-qPCR was used to detect the expressions of Hsa-miR-148a-3p and DUSP1 in breast cancer tissues and cell lines. The effects of Hsa-miR-148a-3p mimic and inhibitor on proliferation, migration, invasion and apoptosis of MCF-7 cells were analyzed, and luciferase reporter gene experiment was performed to verify the binding of Hsa-miR-148a-3p to DUSP1. The effect of Hsa-miR-148a-3p overexpression on breast cancer cell xenograft growth was evaluated in nude mice. Kaplan-Meier survival curve analysis was used to analyze the survival of the tumor-bearing mice, and the expression level of DUSP1 in the xenografts was detected using immunohistochemistry. Results A total of 54 differential miRNAs and 799 differential mRNAs were identified in breast cancer; 3716 target genes were intersected with the differential mRNA, resulting in 150 intersected genes. The top 10 hub genes were downregulated in breast cancer tissues in the PPI network. Double luciferase reporter gene experiment confirmed that Hsa-miR-148a-3p was capable of binding to DUSP1. Hsa-miR-148a-3p was up-regulated and DUSP1 was down-regulated significantly in breast cancer tissues and cells (P<0.01). In breast cancer cells, Hsa-miR-148a- 3p mimic strongly promoted cell proliferation, migration and invasion and inhibited cell apoptosis (P<0.01). Hsa-miR-148a-3p overexpression obviously promoted xenograft growth in nude mice (P<0.01), shortened survival time of the mice (P<0.01), and reduced the expression of DUSP1 in the xenografts (P<0.01). Conclusion Hsa-miR-148a-3p promotes malignant behavior of breast cancer cells by inhibiting the expression of DUSP1.

Key words: Hsa-miR-148a-3p; DUSP1; breast cancer; tumor promotion