Mechanisms of miR-448-Mediated Inhibition of Malignant Behaviors in Gastric Cancer Cells Through Downregulation of ADAM10 Expression
-
摘要:
目的 探究miR-448与ADAM10在MGC-803胃癌细胞恶性表型中的调控作用及机制。 方法 采用RT-qPCR比较miR-448与ADAM10在正常GES-1细胞与胃癌细胞系MGC-803、MKN-7、NCI-N87中的表达水平，经双荧光素酶报告基因实验验证二者靶向结合作用。干预MGC-803细胞miR-448表达后，通过克隆形成实验、划痕实验及Transwell实验评估细胞增殖、迁移和侵袭能力。共转染miR-448 mimics/inhibitor与ADAM10过表达/干扰质粒，通过流式细胞术、RT-qPCR和Western blot检测MGC-803细胞凋亡及EMT相关分子表达变化。 结果 与正常GES-1细胞相比，MGC-803细胞中miR-448显著下调，而ADAM10显著上调（P < 0.01）。双荧光素酶实验证实miR-448直接靶向ADAM10。此外，miR-448过表达可抑制MGC-803增殖、迁移与侵袭（P < 0.05），并通过下调ADAM10（P < 0.05）降低N-cadherin、Vimentin及MMP9水平（P < 0.05），进而抑制MGC-803迁移与侵袭；同时升高Bax、cleaved Caspase-3并降低Bcl2，促进MGC-803凋亡（P < 0.05）。 结论 miR-448可能通过靶向抑制ADAM10遏制MGC-803细胞相关恶性表型。 Abstract:Objective To investigate the regulatory role and mechanism of miR-448 and ADAM10 in the malignant phenotype of MGC-803 gastric cancer cells. Methods RT-qPCR was used to compare the expression levels of miR-448 and ADAM10 in normal GES-1 cells and the gastric cancer cell lines MGC-803, MKN-7, NCI-N87. The targeting binding interaction between the two was validated by dual-luciferase reporter assay. After intervening with miR-448 expression in MGC-803 cells, cell proliferation, migration, and invasion capabilities were assessed via colony formation assays, scratch assays and Transwell assays. MGC-803 cells were co-transfected with miR-448 mimics/inhibitors and ADAM10 overexpression/interference plasmids. Changes in apoptosis and EMT-related molecular expression in MGC-803 cells were detected by flow cytometry, RT-qPCR, and Western blot. Results Compared with normal GES-1 cells, miR-448 was significantly downregulated while ADAM10 was significantly upregulated in MGC-803 cells （P < 0.01）. Dual-luciferase assay confirmed that miR-448 directly targets ADAM10. Furthermore, miR-448 overexpression inhibited MGC-803 proliferation, migration and invasion （P < 0.05）, and downregulated ADAM10 （P < 0.05）, thereby reducing the levels of N-cadherin, vimentin and MMP9 （P < 0.05）, which in turn inhibited MGC-803 migration and invasion. Simultaneously, miR-448 overexpression increased Bax and cleaved Caspase-3 levels while decreasing Bcl-2 levels, promoting MGC-803 cell apoptosis （P < 0.05）. Conclusion miR-448 may suppress the malignant phenotype of MGC-803 cells by targeting and inhibiting ADAM10. -
Key words:
- Gastric cancer /
- miR-448 /
- ADAM10 /
- MGC-803 /
- Malignant phenotype
-
图 1 miR-448和ADAM10的表达水平以及靶向调控作用（$ \bar{x}\pm s $，n = 3）
A～B：miR-448、ADAM10 mRNA表达水平；C：双荧光素酶报告统计；与GES-1组比较，***P < 0.001；**P < 0.01；*P < 0.05。
Figure 1. Expression levels of miR-448 and ADAM10，and their targeted regulatory interactions（$ \bar{x}\pm s $，n = 3）
图 2 miR-448对MGC-803细胞增殖和侵袭的影响（$ \bar{x}\pm s $，n = 3）
A: MGC-803细胞增殖、侵袭变化；B～C：MGC-803细胞增殖、侵袭数量；与NC组比较，**P < 0.01；*P < 0.05。
Figure 2. Effects of miR-448 on the proliferation and invasion of MGC-803 cells（$ \bar{x}\pm s $，n = 3）
图 3 miR-448对MGC-803细胞迁移的影响（$ \bar{x}\pm s $，n = 3）
A：MGC-803细胞迁移能力变化；B～C：MGC-803细胞12 h、24 h迁移率；与NC组比较，*P < 0.05。
Figure 3. Effects of miR-448 on MGC-803 cell migration（$ \bar{x}\pm s $，n = 3）
图 4 miR-448对EMT相关分子的蛋白表达水平的影响（$ \bar{x}\pm s $，n = 3）
A：Western blot代表性条带；B～E：ADAM10、N-cadherin、Vimentin和MMP9蛋白表达水平；与NC/miR-448 mimics/miR-448 inhibitor组比较，*P < 0.05；**P < 0.01。
Figure 4. Effects of miR-448 on the protein expression levels of EMT-related molecules（$ \bar{x}\pm s $，n = 3）
图 5 miR-448对MGC-803细胞凋亡的影响（$ \bar{x}\pm s $，n = 3）
A～E：流式细胞术代表图；F：MGC-803细胞凋亡率；与NC/miR-448 mimics/miR-448 inhibitor组比较，**P < 0.01；*P < 0.05。
Figure 5. Effects of miR-448 on apoptosis in MGC-803 cells$ \bar{x}\pm s $，n = 3）
图 6 miR-448对MGC-803细胞凋亡分子水平的影响（$ \bar{x}\pm s $，n = 3）
A：Western blot代表性条带；B～D：Bax、Bcl2和cleaved caspase3蛋白表达水平；与NC/miR-448 mimics/miR-448 inhibitor组比较，***P < 0.001;**P < 0.01;*P < 0.05。
Figure 6. Effects of miR-448 on apoptosis in MGC-803 cells at the molecular level（$ \bar{x}\pm s $，n = 3）
表 1 引物序列信息
Table 1. Primer sequences
基因 序列（5′→3′） ADAM10 Forward Primer CGGAACACGAGAAGCTGTGA Reverse Primer TCCGGAGAAGTCTGTGGTCT miR-448 Forward Primer GTGATCATGTGCACCGCACACGTG Reverse Primer CAGACGTTGCACGTGCGTCGTGTC GAPDH Forward Primer GTCAAGGCTGAGAACGGGAA Reverse Primer AAATGAGCCCCAGCCTTCTC 
下载: 导出CSV
表 2 质粒转染效率验证（$ \bar{x}\pm s $，n = 3）
Table 2. Validation of plasmid transfection efficiency（$ \bar{x}\pm s $，n = 3）
分组 miR-448/ ADAM10 mRNA mimics NC 0.98 ± 0.12 miR-448 mimics 21.16 ± 4.13 anti-NC 1.02 ± 0.15 miR-448 inhibitor 0.21 ± 0.02 vector 1.10 ± 0.11 OE-ADAM10 6.54 ± 0.82 Sh-NC 0.97 ± 0.13 Sh-ADAM10 0.23 ± 0.05
下载: 导出CSV
-
[1] Joshi S S, Badgwell B D. Current treatment and recent progress in gastric cancer[J]. CA A Cancer J Clin, 2021, 71(3): 264-279. doi: 10.3322/caac.21657 [2] 王海伟, 张小辉, 田云霄, 等. TPI-1通过糖酵解重编程促进胃癌细胞增殖和侵袭的机制[J]. 昆明医科大学学报, 2026, 47(2): 35-42. doi: 10.12259/j.issn.2095-610X.S20260204 [3] Ho P T B, Clark I M, Le L T T. microRNA-based diagnosis and therapy[J]. Int J Mol Sci, 2022, 23(13): 7167. doi: 10.3390/ijms23137167 [4] 薛富森, 陈大为, 孙江华. miR-145-5p、FSCN1在胃癌组织中的表达及生存预测价值[J]. 昆明医科大学学报, 2025, 46(9): 114-120. doi: 10.12259/j.issn.2095-610X.S20250913 [5] Gou R, Chen S, Lei Y, et al. Hypoxia inhibitor improves iodine uptake disorder in thyroid cancer through the hsa_circ_0023990/miR-448/DNMT1/NIS axis[J]. Cancer Sci, 2025, 116(8): 2113-2124. [6] Wu H, Dong D, Wang J, et al. LncRNA NEAT1 promotes the malignant progression of colorectal cancer by targeting ZEB1 via miR-448[J]. Technol Cancer Res Treat, 2022, 21: 15330338221085348. [7] Yan Z, Li J, Guo J, et al. LncRNA XIST sponges microRNA-448 to promote malignant behaviors of colorectal cancer cells via regulating GRHL2[J]. Funct Integr Genom, 2022, 22(5): 977-988. doi: 10.1007/s10142-022-00873-5 [8] Liu D C, Song L L, Li X Z, et al. Circular RNA circHIPK3 modulates prostate cancer progression via targeting miR-448/MTDH signaling[J]. Clin Transl Oncol, 2021, 23(12): 2497-2506. doi: 10.1007/s12094-021-02650-5 [9] Li W, Ma X, Wang F, et al. SNHG3 affects gastric cancer development by regulating SEPT9 methylation[J]. J Oncol, 2022, 2022(1): 3433406. [10] Xu X, Li Y, Zhang R, et al. Jianpi Yangzheng decoction suppresses gastric cancer progression via modulating the miR-448/CLDN18.2 mediated YAP/TAZ signaling[J]. J Ethnopharmacol, 2023, 311: 116450. doi: 10.1016/j.jep.2023.116450 [11] Zhu H, Wang J, Nie W, et al. ADAMs family in kidney physiology and pathology[J]. eBioMedicine, 2021, 72: 103628. doi: 10.1016/j.ebiom.2021.103628 [12] Cai C, Zhang M, Liu L, et al. ADAM10-cleaved ephrin-A5 contributes to prostate cancer metastasis[J]. Cell Death Dis, 2022, 13: 453. doi: 10.1038/s41419-022-04893-8 [13] Oh S, Lee S S, Jin H, et al. A disintegrin and metallopeptidase domain (ADAM) 12, ADAM 17 mRNA and ADAM10 protein hold potential as biomarkers for detection of early gastric cancer[J]. Sci Rep, 2025, 15: 763. doi: 10.1038/s41598-024-84237-y [14] Wu X, Tang H, Liu G, et al. miR-448 suppressed gastric cancer proliferation and invasion by regulating ADAM10[J]. Tumor Biol, 2016, 37(8): 10545-10551. doi: 10.1007/s13277-016-4942-0 [15] Shui B, Beyett T S, Chen Z, et al. Oncogenic K-Ras suppresses global miRNA function[J]. Mol Cell, 2023, 83(14): 2509-2523. e13. [16] 何蕴灏, 李青科, 张云丽, 等. 环状RNA sirt1对胃癌细胞焦亡和肿瘤血管生成的影响研究[J]. 河北医科大学学报, 2025, 46(6): 642-648. [17] Hill M, Tran N. miRNA interplay: Mechanisms and consequences in cancer[J]. Dis Models Mech, 2021, 14(4): dmm047662. doi: 10.1242/dmm.047662 [18] 项婷婷, 孙铭, 潘莉, 等. 辛伐他汀对胃癌细胞增殖、侵袭、迁移及凋亡的影响及机制[J]. 贵州医科大学学报, 2025, 50(10): 1405-1414. [19] Chen Y, Zhu T, Chen F, et al. Exosomal microRNA-448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis[J]. Oncol Lett, 2026, 31(3): 1-12. doi: 10.3892/ol.2026.15450 [20] Saha N, Baek D S, Mendoza R P, et al. Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells[J]. Biomed Pharmacother, 2023, 161: 114494. doi: 10.1016/j.biopha.2023.114494 [21] Wang K, Liao J, Yuan Y, et al. Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling[J]. Signal Transduct Target Ther, 2025, 10: 334. doi: 10.1038/s41392-025-02432-3 [22] Badhai C, Singh M R, Dwivedi S D, et al. ADAM and ADAMTS proteases in breast cancer: Molecular mechanisms and therapeutic implications[J]. Clin Breast Cancer, 2026, 26(2): 17-35. doi: 10.1016/j.clbc.2025.12.001 [23] Yang X, Han F, Guo Y, et al. ADAM10 promotes uveal melanoma development by regulating the Wnt/β-catenin pathway[J]. Exp Cell Res, 2026, 454(1): 114522. doi: 10.1016/j.yexcr.2025.114522 [24] 樊华, 邵淑丽, 何孟奇, 等. 不同浓度桦木酸对人胃癌MGC-803细胞凋亡的影响[J]. 中国应用生理学杂志, 2021, 37(4): 419-422+448. doi: 10.12047/j.cjap.6078.2021.039 [25] Wang H, Zhang X, Liu Y, et al. Celastrus orbiculatus extract inhibits the epithelial-mesenchymal transition processby transforming growth factor-β signaling pathway in gastric cancer[J]. Anti Cancer Agents Med Chem, 2022, 22(12): 2282-2291. doi: 10.2174/1871520621666211210145011 -
点击查看大图
计量
- 文章访问数: 2
- HTML全文浏览量: 3
- PDF下载量: 1
- 被引次数: 0
