Du Shanmei, Liu Jiaqi, Ning Yanfeng, Yin Mengmei, Xu Miao, Liu Zhong, Liu Kui
College of Medical Technology, Zibo Vocational Institute, Zibo, Shandong Province 255300, China.
Department of Breast and Thyroid Surgery, Zibo Central Hospital, Zibo, Shandong Province 255036, China.
Gene. 2025 Mar 20;942:149255. doi: 10.1016/j.gene.2025.149255. Epub 2025 Jan 20.
P-element-induced wimpy testis (PIWI) proteins bind to PIWI-interactingRNAs (piRNAs) to form the piRNA/PIWI complex, which affects protein regulation. PIWIL4, a member of the PIWI family, has been demonstrated in recent studies to promote the migration of triple-negative breast cancer (TNBC) cell line MDA-MB-231. However, the molecular mechanisms underlying cell migration remain obscure.
RNA immunoprecipitation and real-time PCR assays were conducted to detect piRNAs binding to PIWIL4. piRNA mimics and inhibitors were employed to modify piRNA expression in MDA-MB-231 cells. Cell migration assays were carried out using transwell inserts. Co-immunoprecipitation (co-IP) combined with mass spectrometry (MS) was performed to identify the proteins that interacted with PIWIL4 under the regulation of piRNA. Western blotting (WB) was utilised to detect the regulatory relationship between the piRNA/PIWIL4 complexes and the mutually-binding proteins.
RNA Immunoprecipitation (RIP) results revealed that PIWIL4 bound to piR-31115 in the MDA-MB-231 cells. Transwell assays demonstrated that piR-31115 promoted the migration of MDA-MB-231 cells via PIWIL4. Co-IP coupled with MS results showed that piR-31115 promoted the binding of PIWIL4 to HSP90AA1 in MDA-MB-231 cells, and this interaction protected HSP90AA1 from degradation. Knockdown of HSP90AA1 in MDA-MB-231 cells attenuated the promoting effects of piR-31115/PIWIL4 on cell migration.
Our findings cast light on a novel molecular pathway through which piR-31115 promotes the migration of MDA-MB-231 TNBC cells by regulating the interaction between PIWIL4 and HSP90AA1.
P 元件诱导的弱精睾丸（PIWI）蛋白与 PIWI 相互作用 RNA（piRNA）结合形成 piRNA/PIWI 复合物，影响蛋白质调控。PIWIL4 是 PIWI 家族成员，近期研究表明其可促进三阴性乳腺癌（TNBC）细胞系 MDA-MB-231 的迁移。然而，细胞迁移背后的分子机制仍不清楚。
进行 RNA 免疫沉淀和实时 PCR 检测与 PIWIL4 结合的 piRNA。使用 piRNA 模拟物和抑制剂改变 MDA-MB-231 细胞中的 piRNA 表达。使用 Transwell 小室进行细胞迁移检测。进行免疫共沉淀（co-IP）结合质谱（MS）以鉴定在 piRNA 调控下与 PIWIL4 相互作用的蛋白质。利用蛋白质免疫印迹（WB）检测 piRNA/PIWIL4 复合物与相互结合蛋白之间的调控关系。
RNA 免疫沉淀（RIP）结果显示，PIWIL4 在 MDA-MB-231 细胞中与 piR-31115 结合。Transwell 检测表明，piR-31115 通过 PIWIL4 促进 MDA-MB-231 细胞的迁移。免疫共沉淀结合质谱结果表明，piR-31115 促进 MDA-MB-231 细胞中 PIWIL4 与 HSP90AA1 的结合，且这种相互作用保护 HSP90AA1 不被降解。在 MDA-MB-231 细胞中敲低 HSP90AA1 减弱了 piR-31115/PIWIL4 对细胞迁移的促进作用。
我们的研究结果揭示了一种新的分子途径，即 piR-31115 通过调节 PIWIL4 与 HSP90AA1 之间的相互作用促进 MDA-MB-231 TNBC 细胞的迁移。
