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目的 探讨缺氧条件下外泌体微小RNA-148-3p(microRNA-148-3p, miR-148-3p)对SW480细胞增殖、侵袭等生物学行为的影响及其对DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)与细胞因子信号转导抑制因子3(suppressor of cytokine signaling 3,SOCS3)表达的调控作用机制。方法 CoCl2处理SW480细胞构建缺氧模型，细胞计数试剂盒(cell counting kit-8,CCK-8)法检测细胞活力；采用差异超速离心法提取细胞外泌体，通过透射电镜(transmission electron microscopy, TEM)与纳米流式分析其形态、粒径、浓度及标志蛋白CD9(cluster of differentiation 9)、CD63的表达；收集常氧与缺氧SW480细胞的外泌体，分别与常氧SW480细胞共培养24 h;采用Transwell和划痕实验评估细胞侵袭与迁移，流式细胞术检测细胞周期与凋亡；实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)检测miR-148-3p、DNMT1及SOCS3信使RNA(messenger RNA,mRNA)表达，蛋白质印迹法检测DNMT1和SOCS3蛋白表达。结果 与对照组相比，缺氧来源外泌体促进SW480细胞的增殖、迁移和侵袭(P<0.05),下调miR-148-3p与SOCS3 mRNA表达(P<0.05),同时降低DNMT1和SOCS3蛋白水平(P<0.05)。结论 缺氧状态下SW480细胞外泌体促进SW480细胞的增殖、迁移、侵袭及凋亡，可能与抑制miR-148-3p表达来调控DNMT1/SOCS3通路有关。
Abstract：Objective To investigate the regulatory effect of exosomal microRNA-148-3p(miR-148-3p) on the expression of DNA methyltransferase 1(DNMT1) and suppressor of cytokine signaling 3(SOCS3) under hypoxic conditions, and its impact on the proliferation, invasion, and other biological behaviors of SW480 cells. Methods A hypoxic model was established by treating SW480 cells with CoCl2. Cell viability was detected by the CCK-8 assay. Exosomes were isolated from cell culture supernatants via differential ultracentrifugation and characterized by using transmission electron microscopy(TEM) and nano-flow cytometry for morphology, particle size, concentration, and expression of the marker proteins CD9 and CD63. Exosomes derived from normoxic and hypoxic SW480 cells were collected and co-cultured with normoxic SW480 cells for 24 hours. Cell invasion and migration were assessed using Transwell and wound healing assays, respectively. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The mRNA expression levels of miR-148-3p, DNMT1, and SOCS3 were measured by quantitative real-time PCR(qPCR), and the protein expression levels of DNMT1 and SOCS3 were determined by Western blot. Results Compared with the control group, exosomes derived from hypoxic SW480 cells significantly promoted the proliferation, migration, and invasion of recipient SW480 cells. Furthermore, these hypoxic exosomes downregulated the mRNA expression of miR-148-3p and SOCS3, and concurrently reduced the protein levels of both DNMT1 and SOCS3. Conclusion Under hypoxic conditions, exosomes secreted by SW480 cells may promote the migration, invasion, and apoptosis of recipient SW480 cells by inhibiting miR-148-3p expression and regulating the DNMT1/SOCS3 pathway.
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基本信息:
DOI：10.19367/j.cnki.2096-8388.2025.12.001
中图分类号:R735.35
引用信息:
[1]黄玉荣,王迪迪,陈冬梅,等.缺氧诱导的外泌体miR-148a-3p对结肠癌SW480细胞增殖、迁移、侵袭及凋亡的影响及机制[J].贵州医科大学学报,2025,50(12):1717-1725+1739.DOI:10.19367/j.cnki.2096-8388.2025.12.001.
基金信息:
国家自然科学基金项目(82060541); 贵州省卫生健康委员会科学技术基金项目(gzwjkj2020-1-074); 六盘水市人民医院院士工作站培育项目(52020-2020-0911)
2025-12-18
2025-12-18