沉默乙酰肝素酶联合那屈肝素对肺癌细胞的抑制作用及其机制

doi:10.3969/j.issn.1004-616x.2022.06.002

摘要/Abstract

摘要： 目的：研究慢病毒转染沉默乙酰肝素酶(HPA)联合那屈肝素(nadroparin)的协同抗肿瘤作用，并探讨其潜在的分子机制。方法：以肺癌A549细胞系为研究对象，探讨利用慢病毒介导沉默HPA和/或联合那屈肝素处理后对A549细胞的影响。抑制试验共设6组，分别为空白对照组（A549细胞）、阴性对照组（转染阴性对照shRNA序列）、沉默HPA组（转染HPA shRNA）、空白对照+那屈肝素组、阴性对照+那屈肝素组和shRNA+那屈肝素联合组。分别采用CCK-8检测各组细胞活力，酶联免疫吸附法(ELISA)测定细胞培养液中转化生长因子(TGF-β1)含量，体外侵袭实验(transwell)检测各组细胞迁移和侵袭能力的差异，Western blot检测各组细胞TGF-β信号通路中主要蛋白TGF-β1、磷酸化Smad2/3、锌指E盒结合同源框1(ZEB-1)、锌指转录因子SNAIL、TWIST相关蛋白、E钙黏蛋白(E-cadherin)、N钙黏蛋白(N-cadherin)和基质金属蛋白酶-2(MMP-2)的表达情况。结果：通过慢病毒转染shRNA，成功建立了HPA沉默的A549细胞株。与2个对照组相比，各组细胞经沉默HPA和/或联合那屈肝素处理后，CCK-8检测结果显示，单独抑制HPA或那屈肝素处理可轻微抑制细胞活力，而联合处理会显著抑制细胞活力(P<0.05)；单独处理后TGF-β1的表达减少(P均<0.05)，联合组TGF-β1表达减少更显著(P<0.05)；单独HPA沉默或那屈肝素均能抑制细胞迁移和侵袭能力，两者联合时效果更显著(P<0.05)。Western blot同样证实了HPA沉默或那屈肝素均可不同程度诱导TGF-β1、p-Smad2/3、ZEB-1、TWIST、SNAIL、N-cadherin和MMP-2表达下调(P均<0.05)，E-cadherin表达上调(P<0.05)，且联合处理组效果更显著(P<0.05)。结论：沉默HPA联合那屈肝素可显著抑制肺癌细胞侵袭和迁移，并上调E-cadherin表达。此过程可能与TGF-β1介导上皮细胞转化过程中关键转录因子ZEB-1、TWIST和SNAIL的抑制有关。

关键词: 乙酰肝素酶, 那屈肝素, 转化生长因子-β1, 上皮-间质转化, 肺癌

Abstract: OBJECTIVE:To investigate synergistic antitumor effects between lentivirus-mediated short hairpin RNAs (shRNA) targeting HPA and nadroparin in A549 cells,and to determine their underlying molecular mechanisms. METHODS:The lung cancer A549 cell line with down-regulated HPA expression by shRNA was used. These cells were also treated with six different conditions and then evaluated. CCK-8 assay was used to detect cell viability,enzyme-linked immunosorbent assay (ELISA) to measure concentrations of transforming growth factor (TGF-β1) in the culture medium,and the transwell assay to determine cell migration and invasion ability. Western Blot was used to detect expression of TGF-β1,phosphorylated- Smad2/3,ZEB-1,SNAIL,TWIST,E-cadherin,N-cadherin and MMP-2 in the TGF-β signaling pathway. RESULTS:HPA-silenced A549 cell line was successfully established by transfection of shRNA with lentivirus. Results from the CCK-8 assay demonstrated that shRNA inhibiting HPA or nadroparin alone slightly inhibited cell viability(all P<0.05),while the combined treatments significantly inhibited cell viability (P<0.05). ELISA results demonstrated that secretion of transforming growth factor (TGF)-β1 was decreased with inhibition of HPA and/or treatment with nadroparin (all P<0.05),particularly in the combined treatment group (P<0.05). Results from the transwell assay demonstrated that both HPA-silencing and nadroparin suppressed cell migration and invasive abilities (all P<0.05),these effects from the combined treatments were significant (all P<0.05). Western blot analyses demonstrated the similar results. Down-regulation of TGF-β1,phosphorylated-Smad2/3,Zinc-finger E-box-binding 1 (ZEB-1),twist-related protein (TWIST),snail family transcriptional repressor (SNAIL),N-cadherin and MMP-2,and upregulation of E-cadherin were induced to varying degrees by HPA-silencing or nadroparin alone or in combinations (all P<0.05). CONCLUSION:HPA silencing,especially when combined with nadroparin significantly inhibited cell invasion and migration,and up-regulated E-cadherin expression which was associated with inhibition of ZEB-1,TWIST and SNAIL,and the TGF-β1-mediated EMT process.

Key words: heparanase, nadroparin, transforming growth factor-β1, epithelial-to-mesenchymal transition, lung cancer

中图分类号:

R730.2

庄喜兵, 袁素娟, 张琪, 吕名鹤, 程韵枫, 乔田奎. 沉默乙酰肝素酶联合那屈肝素对肺癌细胞的抑制作用及其机制[J]. 癌变·畸变·突变, 2022, 34(6): 413-420.

ZHUANG Xibing, YUAN Sujuan, ZHANG Qi, LU Minghe, CHENG Yunfeng, QIAO Tiankui. Antitumor effects of heparanase silencing plus nadroparin and the mechanisms in lung cancer cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2022, 34(6): 413-420.

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