Lian Xin, Zhou Honglan, Liu Si
Department of Urology, The First Hospital of Jilin University, Changchun, China.
Biofactors. 2024 Nov-Dec;50(6):1251-1267. doi: 10.1002/biof.2083. Epub 2024 May 31.
Despite advancements in cancer research, the prognostic implications of competing endogenous RNA (ceRNA) networks in prostate cancer (PCa) remain incompletely understood. This study aimed to elucidate the prognostic relevance of ceRNA networks in PCa, utilizing a comprehensive bioinformatics approach alongside experimental validation. After searching The Cancer Genome Atlas (TCGA) database, RNA sequencing (RNA-Seq) data were extracted to identify differentially expressed RNAs (DERs) between 491 PCa samples and 51 normal prostate tissues, following which a comprehensive bioinformatics strategy was implemented to construct a ceRNA network. An optimal prognostic signature comprising these DERs was then established and validated using TCGA data. In addition, functional validation was performed through RNA pull-down, dual-luciferase reporter assays, quantitative real-time PCR, and western blot analysis conducted in PC-3 and DU145 cell lines, thereby complementing the bioinformatics analysis. A total of 613 DERs, comprising 103 long noncoding RNAs (lncRNAs), 60 microRNAs (miRNAs), and 450 messenger RNAs (mRNAs), were identified and utilized in constructing a ceRNA network, which encompassed 23 lncRNAs, 9 miRNAs, and 52 mRNAs. An optimal prognostic signature was established, including VPS9D1 antisense RNA 1 (VPS9D1-AS1), miR-449a, cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1), targeting protein for Xklp2 (TPX2), solute carrier family 7 member 11 (SLC7A11), copine7 (CPNE7), and maternal embryonic leucine zipper kinase (MELK), yielding area under the curve (AUC) values exceeding 0.8 across training, validation, and entire datasets. Our experiments results revealed an interaction between lncRNA TRHDE antisense RNA 1 (TRHDE-AS1) and miR-449a and that miR-449a could target the ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) mRNA. Knockdown of miR-449a significantly impeded cell proliferation, G1/S transition, migration and invasion, and promoted apoptosis in PC-3 and DU145 cells. Furthermore, knockdown of miR-449a notably downregulated protein expression of CDK4, cyclin D1, N-cadherin and vimentin, while upregulating protein expression of cleaved caspase-3 and E-cadherin. This study contributes to a deeper understanding of the prognostic-linked ceRNA network in PCa, providing fundamental insights that could improve diagnostic and therapeutic approaches for PCa management.
尽管癌症研究取得了进展，但竞争内源性RNA（ceRNA）网络在前列腺癌（PCa）中的预后意义仍未完全明确。本研究旨在利用综合生物信息学方法并结合实验验证，阐明ceRNA网络在PCa中的预后相关性。在搜索癌症基因组图谱（TCGA）数据库后，提取RNA测序（RNA-Seq）数据，以鉴定491例PCa样本与51例正常前列腺组织之间的差异表达RNA（DER），随后实施综合生物信息学策略构建ceRNA网络。然后使用TCGA数据建立并验证了包含这些DER的最佳预后特征。此外，通过在PC-3和DU145细胞系中进行RNA下拉、双荧光素酶报告基因检测、定量实时PCR和蛋白质印迹分析进行功能验证，从而补充生物信息学分析。共鉴定出613个DER，包括103个长链非编码RNA（lncRNA）、60个微小RNA（miRNA）和450个信使RNA（mRNA），并用于构建ceRNA网络，该网络包含23个lncRNA、9个miRNA和52个mRNA。建立了一个最佳预后特征，包括VPS9D1反义RNA 1（VPS9D1-AS1）、miR-449a、细胞周期蛋白依赖性激酶5调节亚基1（CDK5R1）、Xklp2靶向蛋白（TPX2）、溶质载体家族7成员11（SLC7A11）、copine7（CPNE7）和母体胚胎亮氨酸拉链激酶（MELK），在训练、验证和整个数据集中的曲线下面积（AUC）值均超过0.8。我们的实验结果揭示了lncRNA促甲状腺素释放激素脱碘酶反义RNA 1（TRHDE-AS1）与miR-449a之间的相互作用，并且miR-449a可以靶向含血小板反应蛋白基序的金属蛋白酶5（ADAMTS5）mRNA。敲低miR-449a显著抑制PC-3和DU145细胞的增殖、G1/S期转换、迁移和侵袭，并促进细胞凋亡。此外，敲低miR-449a显著下调CDK4、细胞周期蛋白D1、N-钙黏蛋白和波形蛋白的蛋白表达，同时上调裂解的半胱天冬酶-3和E-钙黏蛋白的蛋白表达。本研究有助于更深入地了解PCa中与预后相关的ceRNA网络，为改善PCa管理的诊断和治疗方法提供了重要见解。
