长链非编码RNA STMN1P2在乳腺癌中的作用及机制研究

背景与目的：长链非编码RNA（long non-coding RNA，lncRNA）在肿瘤进展中发挥重要调控作用，我们的前期研究通过lncRNA表达谱芯片筛选发现，微管不稳定蛋白1假基因2（stathmin 1 pseudogene 2，STMN1P2）在乳腺癌中高表达，但其生物学功能以及在乳腺癌中的作用尚未见报道。探讨lncRNA STMN1P2在乳腺癌中的作用及其机制。方法：采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）在复旦大学附属肿瘤医院收治的27例乳腺癌患者的乳腺癌组织、癌旁组织以及3种乳腺癌细胞系MDA-MB-231、BT-549、MCF-7和人乳腺上皮细胞系MCF-10A中验证STMN1P2的表达水平。下调或上调STMN1P2的表达后，进行transwell、细胞凋亡和细胞周期检测，研究STMN1P2的生物学功能。通过RNA pull-down实验结合蛋白质印迹法（Western blot）挖掘STMN1P2的结合蛋白，探究潜在的分子机制。结果：STMN1P2在乳腺癌组织和细胞系中的表达显著上调（P<0.05）。干扰STMN1P2后，BT-549细胞的迁移能力显著减弱（P<0.05）；过表达STMN1P2后，MDA-MB-231细胞的迁移能力显著增强（P<0.05）。而STMN1P2对细胞凋亡和细胞周期影响不明显。RNA pull-down实验结果显示，核内不均一核糖核蛋白U（heterogeneous nuclear ribonucleoprotein U，hnRNPU）可能为STMN1P2结合蛋白，且hnRNPU的mRNA和蛋白水平均受STMN1P2调控（P<0.05）。挽救实验结果显示，抑制hnRNPU表达能够逆转STMN1P2的促细胞迁移作用（P<0.05）。结论：STMN1P2可能通过结合hnRNPU发挥促进乳腺癌细胞迁移的作用。

A study on the role of long non-coding RNA STMN1P2 in breast cancer and its mechanism

Background and purpose:?Long non-coding RNA (LncRNA) plays a critical role in the malignant progression of tumors. Former study identified lncRNA stathmin 1 pseudogene 2 (STMN1P2) was aberrantly expressed in breast cancer tissues with microarray. However, the role it executes in breast cancer is still unclear. This study aimed to investigate the expression and function of STMN1P2 in breast cancer cells and its underlying mechanism. Methods: Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to verify the expression of STMN1P2 in breast cancer tissues from 27 breast cancer patients who were treated in Fudan University Shanghai Cancer Center and paracancerous tissues, 3 breast cancer cell lines of MDA-MB-231, BT-549, MCF-7 and human mammary epithelial cell line MCF-10A. STMN1P2 levels were downregulated or upregulated, and transwell, cell apoptosis and cell cycle detection assays were performed to study the biological functions of STMN1P2. The binding protein was figured out through RNA pull-down experiment combined with Western blot to discover the underlying mechanism. Results: The expression of STMN1P2 was significantly upregulated in breast cancer tissues and cells (P<0.05). Interference with STMN1P2 decreased the migration of BT-549 cells (P<0.05), and overexpression of STMN1P2 increased the migration of MDA- MB-231 cells (P<0.05). Our results showed that STMN1P2 might not be related to cell apoptosis and cell cycle. RNA pull-down results showed that heterogeneous nuclear ribonucleoprotein U (hnRNPU) might be the binding protein of STMN1P2, and theexpression of hnRNPU might be regulated by STMN1P2 (P<0.05). The results of the rescue experiment showed that downregulating hnRNPU expression reversed the effect of STMN1P2 on promoting cell migration (P<0.05). Conclusion: STMN1P2 may promote breast cancer cell migration via binding and interacting with hnRNPU.