lncRNA SNHG6通过促进前列腺肿瘤干细胞自我更新调控激素难治性前列腺癌耐药及其机制研究

Prostate cancer being resistant to castration (CRPC) is a major cause of treatment failure, and the prostate cancer stem cells play a major role in the development of the CRPC progress. Previous reports demonstrate that lncRNA SNHG6 promotes various cancer diseases development, however the role and the mechanism of lncRNA SNHG6 in prostate cancer is widely ambiguous. We found that the quantity of the prostate cancer stem cells and the formed holoclones of PC3 cells decreased obviously after knockdown the lncRNA SNHG6 in PC3 line cells, at the same time, the proliferation of the PC3 cells was decreased while the apoptosis inhibited, and the docetaxel-resistant was reduced. Therefore, we speculate that lncRNA SNHG6 regulates the CRPC progress and the chemotherapy of the docetaxel through promoting the self-renewal of the prostate cancer stem cells. This study will further demonstrate lncRNA SNHG6 influences the CRPC progress and docetaxel-resistant through combining ANXA2 then regulating downstream target gene B and the PI3K/AKT pathway signal using lncRNA pull down IP and Western bolts assay, and provides potential targets for treatment.

前列腺癌进展为去势抵抗型(CRPC)是导致治疗失败的主要原因，前列腺肿瘤干细胞在CRPC进展及治疗过程中起到重要作用。研究发现：lncRNA SNHG6促进多种肿瘤的进展，但在前列腺癌进展中的作用及机制尚不明确。我们发现：在CRPC细胞系PC3中沉默lncRNA SNHG6后，肿瘤干细胞数量明显减少，克隆成球能力明显降低，细胞增殖能力明显下降，凋亡数量明显升高，对化疗药物多西他赛的耐药性明显降低。故而我们推测：lncRNA SNHG6可能通过促进前列腺肿瘤干细胞自我更新，进而影响CRPC进展及多西他赛耐药。本课题进一步通过lncRNA pulldown IP及Western blots实验，发现lncRNA SNHG6可能通过结合蛋白ANXA2调控下游靶基因B及PI3K/AKT信号通路影响CRPC进展及多西他赛耐药，为新药研发提供理论依据。

课题组通过流式分选了CD44+/CD133+前列腺肿瘤干细胞和CD44-/CD133-对照细胞；通过RNA测序筛选了差异表达基因lncRNA SNHG6并验证了lncRNA SNHG6在前列腺癌样本中高表达，且与前列腺癌样本的Gleason评分正相关，生物信息学分析提示lncRNA SNHG6的高表达与前列腺癌的不良预后相关。为了研究lncRNA SNHG6对前列腺癌的影响，课题组构建了敲减/过表达lncRNA SNHG6的前列腺癌细胞系（DU145和LNCaP）稳转株，通过CCK-8实验，流式细胞周期实验以及Transwell细胞迁移和侵袭实验，从细胞水平验证了lncRNA SNHG6可以增强前列腺癌细胞增殖，迁移和侵袭活性。通过裸小鼠皮下荷瘤和转移模型从动物水平验证lncRNA SNHG6可以促进前列腺癌生长和转移。课题组进一步检测了lncRNA SNHG6在细胞中的定位以探索其影响PCa进展的内在机制，发现lncRNA SNHG6主要定位于前列腺癌细胞系的细胞质中，提示其可能发挥ceRNA的作用。课题组通过检索TargetScan和starbase2.0数据库，发现miR-26a-5p与lncRNA SNHG6及POLR3G mRNA的3’UTR片段存在结合位点，而POLR3G的表达被发现有助于维持前列腺癌细胞的干性。为此，我们构建了相应的野生型和突变型质粒，通过双荧光素酶报告基因实验验证了miR-26a-5p可以分别与lncRNA SNHG6和POLR3G mRNA的3’UTR结合，通过Realtime PCR实验验证了lncRNA SNHG6的表达与miR-26a-5p的表达呈负相关，而POLR3G mRNA的表达亦与miR-26a-5p呈负相关，提示lncRNA SNHG6可能通过miR-26a-5p/POLR3G轴促进前列腺癌进展。我们在敲减了lncRNA SNHG6的前列腺细胞系中转染了miR-26a-5p inhibitor或POLR3G的过表达质粒以开展挽救实验，并验证了lncRNA SNHG6可以通过miR-26a-5p/POLR3G维持前列腺癌细胞干性，从而促进前列腺癌的进展。相关研究成果已经编撰成文并投稿。