Knockdown of TRIM47 Overcomes Paclitaxel Resistance in Ovarian Cancer by Suppressing the TGF-β Pathway via PPM1A

IF 2.5 3区医学 Q3 IMMUNOLOGY

American Journal of Reproductive Immunology Pub Date : 2025-05-28 DOI:10.1111/aji.70102

Hezhu Wang, Shengjun Chen, Feifei Xu, Xiaojing Chen

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引用次数: 0

Abstract

Objective

We investigated the role of tripartite motif 47 (TRIM47) in paclitaxel resistance in ovarian cancer, focusing on its regulation of protein phosphatase magnesium-dependent 1A (PPM1A), transforming growth factor-β (TGF-β) pathway activation, and methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification.

Methods

Bioinformatics analysis using Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan–Meier plotter (KM plot), Linkedomics, and sequence-based RNA adenosine methylation site predictor (SRAMP) databases identified TRIM47 and PPM1A expression patterns, prognostic significance, co-expression networks, and m6A modification sites. Paclitaxel-resistant ovarian cancer cell lines were generated. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot analyzed gene and protein expression. Co-immunoprecipitation (Co-IP) and GST pull-down assays assessed TRIM47-PPM1A interaction, while cycloheximide (CHX) chase, and IP assays examined PPM1A stability and ubiquitination. RNA immunoprecipitation (RIP) and dual-luciferase assays determined METTL3’s effect on TRIM47 m6A modification. Functional assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and flow cytometry) evaluated proliferation, apoptosis, and drug response. An in vivo xenograft model confirmed TRIM47’s role in chemoresistance.

Results

Bioinformatics analysis showed that TRIM47 was overexpressed in ovarian cancer and negatively correlated with the expression of PPM1A. Kaplan–Meier analysis showed that high TRIM47 and low PPM1A expression were correlated with poor prognosis. TRIM47 was upregulated in paclitaxel-resistant ovarian cancer cells. The knockdown of TRIM47 restored drug sensitivity, inhibited cell proliferation, and induced cell apoptosis. Mechanistically, TRIM47 functioned as an E3 ubiquitin ligase, targeting PPM1A for degradation, leading to sustained TGF-β signaling and enhanced chemoresistance. CHX chase assays demonstrated reduced PPM1A stability in the presence of TRIM47, while IP-WB confirmed increased PPM1A ubiquitination. METTL3-mediated m6A modification enhanced TRIM47 mRNA stability, further promoting its oncogenic role. In vivo, TRIM47 knockdown reduced tumor growth and improved paclitaxel efficacy, reinforcing its role in resistance.

Conclusion

TRIM47 promoted paclitaxel resistance in ovarian cancer by inducing PPM1A degradation and activating the TGF-β pathway.

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TRIM47敲低通过PPM1A抑制TGF-β通路克服卵巢癌紫杉醇耐药

目的探讨TRIM47在卵巢癌紫杉醇耐药中的作用，重点研究其对蛋白磷酸酶镁依赖性1A （PPM1A）、转化生长因子-β (TGF-β)通路激活、甲基转移酶样3 （METTL3）介导的n6 -甲基腺苷（m6A）修饰的调控作用。方法利用基因表达谱交互分析（GEPIA）、Kaplan-Meier绘图仪（KM plot）、Linkedomics和基于序列的RNA腺苷甲基化位点预测器（SRAMP）数据库进行生物信息学分析，确定TRIM47和PPM1A的表达模式、预后意义、共表达网络和m6A修饰位点。获得了紫杉醇耐药卵巢癌细胞系。定量逆转录聚合酶链反应（qRT-PCR）和western blot分析基因和蛋白的表达。共免疫沉淀（Co-IP）和GST下拉试验评估TRIM47-PPM1A相互作用，而环己亚胺（CHX）追踪和IP试验检测PPM1A的稳定性和泛素化。RNA免疫沉淀（RIP）和双荧光素酶测定METTL3对TRIM47 m6A修饰的影响。功能测定（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）、菌落形成和流式细胞术）评估增殖、凋亡和药物反应。体内异种移植模型证实了TRIM47在化疗耐药中的作用。结果生物信息学分析显示TRIM47在卵巢癌中过表达，与PPM1A的表达呈负相关。Kaplan-Meier分析显示TRIM47高表达和PPM1A低表达与预后不良相关。TRIM47在紫杉醇耐药卵巢癌细胞中上调。TRIM47基因敲低恢复药物敏感性，抑制细胞增殖，诱导细胞凋亡。机制上，TRIM47作为E3泛素连接酶，靶向PPM1A降解，导致持续的TGF-β信号传导和增强的化学耐药。CHX追踪实验显示TRIM47存在时PPM1A的稳定性降低，而IP-WB证实PPM1A泛素化增加。mettl3介导的m6A修饰增强了TRIM47 mRNA的稳定性，进一步促进了其致癌作用。在体内，TRIM47敲低可抑制肿瘤生长，提高紫杉醇疗效，增强其在耐药中的作用。结论TRIM47通过诱导PPM1A降解和激活TGF-β通路促进卵巢癌紫杉醇耐药。

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来源期刊

American Journal of Reproductive Immunology 医学-免疫学

CiteScore

6.20

自引率

5.60%

发文量

314

审稿时长

2 months

期刊介绍： The American Journal of Reproductive Immunology is an international journal devoted to the presentation of current information in all areas relating to Reproductive Immunology. The journal is directed toward both the basic scientist and the clinician, covering the whole process of reproduction as affected by immunological processes. The journal covers a variety of subspecialty topics, including fertility immunology, pregnancy immunology, immunogenetics, mucosal immunology, immunocontraception, endometriosis, abortion, tumor immunology of the reproductive tract, autoantibodies, infectious disease of the reproductive tract, and technical news.