南方医科大学学报2024，Vol.44Issue(5)：818-826,9.DOI:10.12122/j.issn.1673-4254.2024.05.03
表达TGF-βⅡ受体的腺相关病毒载体抑制小鼠三阴性乳腺癌4T1细胞的增殖和肺转移
A recombinant adeno-associated virus expressing secretory TGF-β type Ⅱ receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice
摘要
Abstract
Objective To investigate the effects of an adeno-associated virus(AAV2)vector expressing secretory transforming growth factor-β(TGF-β)type Ⅱ receptor(sTβRⅡ)extracellular domain-IgG2a Fc fusion protein(sTβRⅡ-Fc)on proliferation and migration of triple-negative murine breast cancer 4T1 cells in mice.Methods The pAAV-sTβRⅡ-Fc vector expressing sTβRⅡ-Fc fusion protein constructed by molecular cloning,the capsid protein-expressing vector pAAV2 and the helper vector were co-transfected into HEK 293T cells to prepare the recombinant AAV2-sTβRⅡ virus,which was purified by density gradient centrifugation with iodixanol.Western blotting was used to examine the effects of AAV-sTβRⅡ virus on Smad2/3 phosphorylation in 4T1 cells and on expression levels of E-cadherin,vimentin and p-Smad2/3 in 4T1 cell xenografts in mice.BALB/c mice bearing subcutaneous xenografts of luciferase-expressing 4T1 cells received intravenous injections of AAV-sTβRⅡ virus,AAV-GFP virus or PBS(n=6)through the tail vein,and the proliferation and migration of 4T1 cells were analyzed with in vivo imaging.Ki67 expression in the tumor tissues and sTβRⅡ protein expressions in mouse livers were detected with immunohistochemistry and immunofluorescence staining,and tumor metastases in the vital organs were examined with HE staining.Results The recombinant pAAV-sTβRⅡ-Fc vector successfully expressed sTβRⅡ in HEK 293T cells.Infection with AAV2-sTβRⅡ virus significantly reduced TGF-β1-induced Smad2/3 phosphorylation in 4T1 cells and effectively inhibited proliferation and lung metastasis of 4T1 xenografts in mice(P<0.05).In the tumor-bearing mice,intravenous injection of AAV-sTβRⅡ virus significantly increased E-cadherin expression,reduced vimentin and Ki67 protein expressions and Smad2/3 phosphorylation level in the tumor tissues(P<0.05 or 0.01),and induced liver-specific sTβRⅡ expression without causing body weight loss or heart,liver,spleen or kidney pathologies.Conclusion The recombinant AVV2 vector encoding sTβRⅡ extracellular domain is capable of blocking the TGF-β signaling pathway to inhibit the proliferation and lung metastasis of 4T1 cells in mice.关键词
三阴性乳腺癌/TGF-βⅡ/迁移/TGF-β信号通路/腺相关病毒Key words
triple negative breast cancer/TGF-βⅡ/migration/transforming growth factor-β signaling pathway/adeno-associated virus引用本文复制引用
崔芝,马萃娇,王倩茹,陈金豪,严子阳,杨建林,吕亚丰,曹春雨..表达TGF-βⅡ受体的腺相关病毒载体抑制小鼠三阴性乳腺癌4T1细胞的增殖和肺转移[J].南方医科大学学报,2024,44(5):818-826,9.基金项目
国家自然科学基金(81772833) （81772833）
湖北省自然科学基金(2022CFB320) Supported by National Natural Science Foundation of China(81772833). （2022CFB320）
