PO.MCB10.01 · 分子与细胞生物学
Auro-Liposome miR-195递送系统阻碍卵巢癌进展并增强顺铂敏感性
Auro-Liposome miR-195 delivery system impedes ovarian cancer progression and enhances cisplatin-sensitivity
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：卵巢癌（OvCa）在所有妇科恶性肿瘤中死亡率最高。这主要归因于晚期检出、早期转移和耐药性。虽然大多数患者最初对化疗有反应，但癌症干样细胞（CSC）促进化疗耐药和肿瘤复发。WNT7A作为β-catenin信号的关键激活因子，在OvCa中过表达并促进CSC特性、上皮-间质转化（EMT）和化疗耐药，而抑癌性的miR-195则下调。在本研究中，我们探讨了恢复miR-195表达是否能通过靶向WNT7A介导的β-catenin信号来抑制癌症进展并改善药物敏感性。
方法：使用非贴壁球体培养条件富集CSC。通过qRT-PCR评估miR-195表达水平。开展功能实验以确定miR-195对CSC标志物、EMT和药物敏感性的影响。利用荧光素酶报告基因实验验证miR-195在WNT7A 3'UTR内的直接结合。通过免疫印迹、免疫荧光分析β-catenin的激活和核定位。为模拟肿瘤微环境，使用OvCa细胞系（A2780-CP20）、癌症相关成纤维细胞（CAF19）和人微血管内皮细胞（HMEC1）建立多细胞聚集体模型，并用于评估AuLP作为miR-195递送系统的潜力。
结果：球体来源的CSC显示干细胞标志物NANOG、OCT4、cMYC、KLF4表达增强，同时miR-195水平显著降低。miR-195表达的恢复抑制了球体生长，导致干性标志物下调并增强了顺铂敏感性。miR-195直接靶向WNT7A，导致活性β-catenin的核定位减少以及WNT/β-catenin信号和EMT通路的抑制。此外，体内归巢实验显示，稳定的miR-195重新表达显著减少了A2780-CP20对小鼠网膜的黏附，凸显其抗转移作用。值得注意的是，与商业试剂相比，我们新型的AuLP系统赋予了更优的miR-195细胞内递送，对致癌WNT7A/β-catenin通路的抑制更为显著，并对多细胞聚集体产生了深远的抗肿瘤效应。
结论：miR-195通过直接调控WNT7A/β-catenin通路，靶向癌症干性、EMT并增强顺铂敏感性来抑制OvCa进展，而新型AuLP递送系统的配制提供了一种有前景的基于miRNA的治疗策略。
致谢：授予SKD的PHF和DOD卵巢癌学院基金（HT94252310772）。
查看英文原文 English abstract
Background: Ovarian cancer (OvCa) accounts for the highest mortality rate among all gynecological malignancy. This is mainly due to late-stage detection, early metastasis and drug-resistance. While most patients initially respond to chemotherapy, Cancer Stem-like Cells (CSCs) facilitate chemoresistance and tumor relapse. WNT7A, a key activator of beta-catenin signaling, is overexpressed in OvCa and promotes CSC characteristics, epithelial-mesenchymal transition (EMT), chemoresistance, whereas the tumor-suppressive miR-195 is downregulated. In this study, we investigated whether restoring miR-195 expression could inhibit cancer progression and improve drug-sensitivity by targeting WNT7A mediated beta-catenin signaling.
Methods: CSCs were enriched using anchorage-independent spheroid culture conditions. miR-195 expression levels were assessed by qRT-PCR. Functional assays were performed to determine the effect of miR-195 on CSC markers, EMT, drug-sensitivity. A luciferase reporter assay was utilized to validate the direct miR-195 binding within the WNT7A 3'UTR. beta-catenin activation and nuclear localization were analyzed by immunoblot, immunofluorescence. To mimic tumor microenvironment, multicellular aggregate model was generated using OvCa cell line (A2780-CP20), cancer-associated fibroblast (CAF19) and human microvascular endothelial cells (HMEC1) and for assessing AuLPs's potential as a miR-195 delivery system.
Results: Spheroid-derived CSCs displayed enhanced expression of stem cell markers NANOG, OCT4, cMYC, KLF4, accompanied by a significant reduction in miR-195 levels. Restoration of miR-195 expression inhibited spheroid growth, caused downregulation of stemness markers and enhanced cisplatin-sensitivity. miR-195 directly targeted WNT7A, leading to decreased nuclear localization of active beta-catenin and inhibition of WNT/beta-catenin signaling, EMT pathway. Furthermore, in vivo homing assay revealed that stable miR-195 re-expression significantly reduced A2780-CP20 adhesion to the mice omentum, highlighting its anti-metastatic role. Noticeably, our novel AuLPs system conferred superior intracellular delivery of miR-195 compared to commercial agents and more striking inhibition of oncogenic WNT7A/beta-catenin pathway and profound anti-tumor effect against multicellular aggregate.
Conclusions: miR-195 suppress OvCa progression by targeting cancer stemness, EMT and enhance cisplatin-sensitivity via directly modulating WNT7A/beta-catenin pathway and formulations of novel AuLPs delivery system offers a promising miRNA-based therapeutic strategy.
Acknowledgments: PHF and DOD'S Ovarian Cancer Academy grant (HT94252310772) to SKD.
利益披露 Disclosure
A. Dey Bhowmik, None..
P. Shaw, None..
P. Panja, None..
G. Rao, None..
R. Bhattacharya, None..
P. Mukherjee, None..
S. K. Dwivedi, None.