Abstract
Long non-coding RNAs (lncRNAs) play an indispensable role in the occurrence and development of ovarian cancer (OC). However, the potential involvement of lncRNAs in the progression of OC is largely unknown. To investigate the detailed roles and mechanisms of RAD51 homolog B-antisense 1 (RAD51B-AS1), a novel lncRNA in OC, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to verify the expression of RAD51B-AS1. Cellular proliferation, metastasis, and apoptosis were detected using the cell counting kit-8 (CCK-8), colony-formation, transwell, and flow cytometry assays. Mouse xenograft models were established for the detection of tumorigenesis. The results revealed that RAD51B-AS1 was significantly upregulated in a highly metastatic human OC cell line and OC tissues. RAD51B-AS1 significantly increased the proliferation and metastasis of OC cells and enhanced their resistance to anoikis. Biogenetics prediction analysis revealed that the only target gene of RAD51B-AS1 was RAD51B. Subsequent gene function experiments revealed that RAD51B exerts the same biological effects as RAD51B-AS1. Rescue experiments demonstrated that the malignant biological behaviors promoted by RAD51B-AS1 overexpression were partially or completely reversed by RAD51B silencing in vitro and in vivo. Thus, RAD51B-AS1 promotes the malignant biological behaviors of OC and activates the protein kinase B (Akt)/B cell lymphoma protein-2 (Bcl-2) signaling pathway, and these effects may be associated with the positive regulation of RAD51B expression. RAD51B-AS1 is expected to serve as a novel molecular biomarker for the diagnosis and prediction of poor prognosis in OC, and as a potential therapeutic target for disease management.
摘要
长链非编码 RNA (lncRNA) 在卵巢癌的发生发展中起着不可或缺的作用, 但它们在卵巢癌进展中的潜在作用在很大程度上仍是未知. 为了研究新型 lncRNA RAD51B-AS1 在卵巢癌中的具体作用和机制, 我们通过逆转录-定量聚合酶链反应实验验证了 RAD51B-AS1 的表达; 使用 CCK-8 实验、 集落形成实验、 transwell 实验和流式细胞术检测细胞的增殖、 转移和凋亡水平; 建立小鼠异种移植瘤模型检测肿瘤发生情况. 结果显示: RAD51B-AS1 在人高转移卵巢癌细胞系和卵巢癌组织中显著上调; 同时, RAD51B-AS1 显著增强卵巢癌细胞的增殖、 转移和抵抗失巢凋亡的能力. 生物遗传学预测分析显示, RAD51B-AS1 的唯一靶基因为 RAD51B. 随后的基因功能实验表明, RAD51B 与 RAD51B-AS1 具有相同的生物学效应. 体外和体内实验均表明, 过表达 RAD51B-AS1 促进的恶性生物学行为可以通过沉默 RAD51B 的表达部分或完全逆转. 由此可见, RAD51B-AS1 可促进卵巢癌的恶性生物学行为, 并激活 Akt/Bcl-2 信号通路, 这些作用可能与其正向调节 RAD51B 的表达有关. RAD51B-AS1 有望作为一种新的分子生物标志物, 用于卵巢癌不良预后的诊断和预测, 并作为疾病管理的潜在治疗靶点.
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The data presented in this study are available from the corresponding author upon reasonable request.
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Acknowledgments
This research received no external funding. We thank all subjects who participated in this study and thank for the technical support by the core facilities, Zhejiang University, School of Medicine (Hangzhou, China).
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Weiguo LU, Junfen XU, Xiaodong CHENG, Xinyu WANG, and Xing XIE contributed to the study design. Xinyi WEI, Conghui WANG, Sangsang TANG, and Qian YANG performed the experimental research and data analysis. Xinyi WEI, Zhangjin SHEN, and Jiawei ZHU performed the establishment of animal models. Xinyi WEI, Weiguo LU, and Junfen XU wrote and edited the manuscript. Weiguo LU and Junfen XU revised the manuscript, and supervised and administrated the study. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Junfen XU and Weiguo LU serve as Young Scientist Committee Member and Editorial Board Member, respectively, for Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), and were not involved in the editorial review or the decision to publish this article. Xinyi WEI, Conghui WANG, Sangsang TANG, Qian YANG, Zhangjin SHEN, Jiawei ZHU, Xiaodong CHENG, Xinyu WANG, Xing XIE, Junfen XU, and Weiguo LU declare that they have no conflict of interest.
The study was approved by the Ethics Committee of Women’s Hospital, Zhejiang University School of Medicine, (approval number: IRB-20210147-R) for studies involving humans. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2013. Informed consent was obtained from all patients for being included in the study.
The animal study protocol was approved by Institutional Animal Care and Use Committee (approval number: IACUC-20200506-07) for studies involving animals. All institutional and national guidelines for the care and use of laboratory animals were followed.
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Xing XIE is deceased
Materials and methods
Detailed methods are provided in the supplemental materials of this paper. In addition, the patient information is provided in Table S5. Primers’ sequences are provided in Table S6. The sequences of probes for northern blotting are listed in Table S7. The sequences of siRNAs are listed in Table S8.
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Materials and methods; Tables S1–S8; Figs. S1–S3
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Wei, X., Wang, C., Tang, S. et al. RAD51B-AS1 promotes the malignant biological behavior of ovarian cancer through upregulation of RAD51B. J. Zhejiang Univ. Sci. B 25, 581–593 (2024). https://doi.org/10.1631/jzus.B2300154
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DOI: https://doi.org/10.1631/jzus.B2300154