[1]任瑾卓,张 华,吴海永.肿瘤源外泌体miR-3173-5p靶向抑制FBXW11促进CAFs活化调控NSCLC进展的机制研究[J].现代检验医学杂志,2025,40(04):67-72,78.[doi:10.3969/j.issn.1671-7414.2025.04.012]
　REN Jinzhuo,ZHANG Hua,WU Haiyong.Study on the Mechanism of Targeted Inhibition of FBXW11 by Tumor- Derived Exosome miR-3173-5p to Promote CAFs Activation and Regulate NSCLC Progression[J].Journal of Modern Laboratory Medicine,2025,40(04):67-72,78.[doi:10.3969/j.issn.1671-7414.2025.04.012]
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肿瘤源外泌体miR-3173-5p靶向抑制FBXW11促进CAFs活化调控NSCLC进展的机制研究()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第40卷
期数:
2025年04期
页码:
67-72,78
栏目:
论著
出版日期:
2025-07-15

文章信息/Info

Title:
Study on the Mechanism of Targeted Inhibition of FBXW11 by Tumor- Derived Exosome miR-3173-5p to Promote CAFs Activation and Regulate NSCLC Progression
文章编号:
1671-7414（2025）04-067-07
作者:
任瑾卓a，张 华a，吴海永b
（张家口市第一医院a. 呼吸与危重症医学三科；b. 胸外科，河北张家口 075000）
Author(s):
REN Jinzhuoa, ZHANG Huaa,WU Haiyongb
(a. Department of Respiratory and Critical Care Medicine; b. Thoracic Surgery，Zhangjiakou First Hospital , Hebei Zhangjiakou 075000, China)
关键词:
非小细胞肺癌; 外泌体; miR-3173-5p; F-box/WD-40 结构域蛋白11; 癌相关成纤维细胞
分类号:
R730.43
DOI:
10.3969/j.issn.1671-7414.2025.04.012
文献标志码:
A
摘要:
目的 研究肿瘤源外泌体miR-3173-5p 促进肿瘤相关成纤维细胞（CAFs）活化调控非小细胞肺癌（NSCLC）细胞增殖、侵袭、凋亡的潜在作用机制。方法 提取NSCLC 细胞A549 的外泌体（Exo），透射电镜观察Exo 形态，Western blot 法检测Exo 标志蛋白表达。实时荧光定量PCR（qRT-PCR）检测miR-3173-5p 在A549 细胞和Exo 中的表达。利用Starbase 数据库预测miR-3173-5p 与F-box/WD-40 结构域蛋白11（FBXW11）的结合，通过荧光素酶报告基因分析进行验证。采用Exo 与Exo 抑制剂GW4869 或NC inhibitor/mimic 或miR-3173-5p inhibitor/mimic 和（或）FBXW11 过表达/ 空载体共处理人肺成纤维细胞系（MRC-5），分别设置NC 组、Exo 组、Exo+GW4869 组、Exo-NC inhibitor 组、Exo-miR-3173-5p inhibitor 组、Exo-NC mimic 组、Exo-miR-3173-5p mimic 组、Exo-miR-3173-5p mimic+Vector 组及ExomiR-3173-5p mimic+FBXW11 组。通过Western blot 检测各组细胞中CAFs 标志蛋白[ 基质金属蛋白酶9（MMP-9）、α-平滑肌肌动蛋白（α-SMA）、趋化因子12（CXCL12）、纤维连接蛋白（Fibronectin）、波形蛋白（Vimentin）、白细胞介素1β（IL-1β）、白细胞介素6（IL-6）和白细胞介素8（IL-8）] 水平，分析外泌体miR-3173-5p 及FBXW11 表达对CAFs 活化的影响。分离Exo 及Exo-miR-3173-5p mimic，Exo-FBXW11 孵育MRC-5 细胞的培养上清液作为条件培养液（CM），培养A549 细胞，分别设置NC-CM 组、Exo-CM 组、Exo-miR-3173-5p mimic-CM 组及Exo-FBXW11-CM 组。采用MTT 法、Transwell 实验及流式细胞术分别检测CAFs 活化对NSCLC 细胞增殖、侵袭和凋亡的影响。结果 透射电镜显示，NSCLC 细胞来源外泌体囊泡直径30~100 nm；Western blot 检测外泌体标记蛋白均呈阳性。NSCLC来源外泌体中miR-3173-5p（33.45 ± 3.16）表达显著高于肿瘤细胞（1.01±0.07），差异具有统计学意义（t=1.263，P<0.001）。miR-3173-5p 靶向结合FBXW11 并抑制其表达。与NC 组相比，Exo 组细胞中CAFs 标志蛋白水平均显著升高（t=12.214~24.908），Exo+GW4869 组CAFs 标志蛋白表达均较Exo 组显著抑制（t=13.160~25.143），差异具有统计学意义（均P<0.01）。与Exo 组相比，miR-3173-5p inhibitor 可抑制Exo 诱导的CAFs 标志蛋白表达（t=11.059~21.094），miR-3173-5p mimic 则促进CAFs 标志蛋白表达（t=12.943~18.671），差异具有统计学意义（均P<0.01）。过表达FBXW11 能够逆转miR-3173-5p mimic 对CAFs 活化的诱导作用。与NC-CM 组相比，Exo-CM 组A549 细胞增殖活力（168.57%±8.14% vs 100.18%±7.26%）、侵袭率（49.69%±7.17% vs 38.52%±3.18%）能力明显增强，细胞凋亡率（3.15%±0.43% vs 6.03%±0.61%） 明显抑制（t=10.207，2.359，3.001），miR-3173-5p mimic 可增强Exo-CM 对A549 细胞增殖、侵袭的促进作用和对细胞凋亡的抑制作用（t=9.399，3.438，3.208），过表达FBXW11 可拮抗Exo-CM 对A549 细胞增殖、侵袭的促进作用和对细胞凋亡的抑制作用（t=18.868，7.070，9.813)，差异具有统计学意义（均P<0.05）。结论 肿瘤源外泌体miR-3173-5p 通过靶向抑制FBXW11 表达促进CAFs 活化，进一步促进NSCLC 细胞增殖和侵袭，抑制细胞凋亡，调控NSCLC 的发生发展。
Abstract:
Objective To investigate the potential mechanism of tumor-derived exosome miR-3173-5p promoting the activation of cancer-associated fibroblasts (CAFs) and regulating the proliferation, invasion and apoptosis of non-small cell lung cancer (NSCLC) cells. Methods Exosome (Exo) of NSCLC cell A549 was extracted, the morphology of Exo was observed by transmission electron microscopy, and the expression of Exo marker protein was detected by Western blot. Real-time quantitative fluorescent PCR (qRT-PCR) was used to detect the expression of miR-3173-5p in A549 cells and Exo. The binding of miR-3173- 5p to F-box/WD-40 domain protein11 (FBXW11) was predicted by Starbase database, and verified by luciferase reporter gene analysis. Human lung fibroblast cell line (MRC-5) was treated with Exo and Exo inhibitor GW4869 or NC inhibitor/mimic or miR-3173-5p inhibitor/mimic and/or FBXW11 overexpression/empty vector, NC group, Exo group, Exo+GW4869 group, Exo- NC inhibitor group, Exo-miR-3173-5p inhibitor group, Exo-NC mimic group, Exo-miR-3173-5p mimic group,Exo-miR-3173-5p mimic+Vector group and Exo-miR-3173-5p mimic+FBXW11 group were set up respectively. The level of CAFs marker protein [Matrix metalloproteinase-9(MMP-9), α-Smooth muscle aorta(α-SMA), Chemokine (C-X-C motif) ligand 12(CXCL12), Fibronectin, Vimentin, Interleukin-1β(IL-1β), Interleukin-6(IL-6), Interleukin-8(IL-8)] in cells of each group was detected by Western blot analysis, and the influence of exosome miR-3173-5p and FBXW11 expression on CAFs activation was analyzed. Exo and Exo-miR-3173-5P mimic were separated and the supernatant of MRC-5 cells incubated with Exo-FBXW11 was used as conditioned medium (CM) to culture A549 cells. The NC-CM group, Exo-CM group, Exo-miR-3173-5p mimic-CM group and Exo-FBXW11-CM group were set up respectively. MTT assay, Transwell assay and flow cytometry were used to detect the effects of CAFs activation on proliferation, invasion and apoptosis of NSCLC cells. Results Transmission electron microscopy showed that the diameter of exosomal vesicles derived from NSCLC cells was about 30~100 nm. Western blot analysis showed that all exosome labeled proteins were positive. The expression of miR-3173-5p (33.45 ± 3.16) in NSCLC-derived exosomes was significantly higher than that in tumor cells (1.01±0.07), and the difference was statistically significant(t=1.263，P<0.001). MiR-3173-5p targets FBXW11 and inhibits its expression. Compared with NC group, CAFs marker protein levels in Exo group were significantly increased (t=12.214~24.908), the expression of CAFs marker protein in Exo+GW4869 group was significantly inhibited compared with Exo group (t=13.160~25.143)，the differences were statistically significant (all P<0.01), respetively. Compared with Exo group, miR-3173-5p inhibitor inhibited EXO-induced CAFs marker protein expression(t=11.059~21.094), and miR-3173-5p mimic promoted the expression of CAFs marker protein (t=12.943~18.671),the differences were statistically significant (all P<0.01), respectively. Overexpression of FBXW11 reversed the induction of CAFs activation by miR-3173-5p mimic. Compared with the NC-CM group, the proliferation activity and invasion rate of A549 cells in the Exo-CM group were significantly enhanced, and the apoptosis rate was significantly inhibited (t=10.207, 2.359, 3.001), miR-3173-5p mimic enhanced the promoting effect of Exo-CM on proliferation and invasion of A549 cells and the inhibitory effect on apoptosis (t=9.399， 3.438，3.208), while overexpression of FBXW11 can antagonize the promotion effect of Exo-CM on proliferation and invasion of A549 cells and the inhibition effect on apoptosis(t=18.868，7.070，9.813)，the differences were statistically significant (all P<0.05), respectively. Conclusion Tumor-derived exosome miR-3173-5p promotes CAFs activation through targeted inhibition of FBXW11 expression, further promotes proliferation and invasion of NSCLC cells, inhibits cell apoptosis, and regulates the occurrence and development of NSCLC.

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备注/Memo

备注/Memo:
基金项目：张家口市重点研发项目（2322126D）。
作者简介：任瑾卓（1986-），男，本科，研究方向：肺癌，E-mail：QiZH10328@163.com。
通讯作者：吴海永（1987-），男，本科，研究方向：肺癌，E-mail：LLF200411@163.com。
更新日期/Last Update: 2025-07-15