Rorβ通过下调Wnt通路活性抑制结直肠癌干细胞自我更新的作用机制研究

Colorectal cancer stem cells (CCSC) remaining is one of the main causes for metastasis, recurrence, and treatment resistance in colorectal cancer. Therefore, the removal of CCSCs is the focus of the colorectal cancer treatment. However, there is no targeted drug to inhibit or remove colorectal cancer stem cells. To clarify the molecular mechanism of CCSCs self-renewal, and identify the key molecular targets is the basis and premise in the colorectal cancer treatment by targeting CCSCs. Our previous studies have found that Rorβ was expressed in colorectal cancer cells, but at a lower level in colorectal cancer tissue than in matched para-carcinoma tissues. Overexpression of Rorβ could inhibit the colospheres formation ability, the tumorigenesis in vivo and the expression of downstream molecules of Wnt, indicating that Rorβ may inhibit the self-renew of CCSCs via regulating the activity of Wnt pathway as a CSCs-targeted therapy in colorectal cancer. However, the molecular mechanisms of Rorβ regulating CCSCs remain unclear. This study focuses on previous basis to identify the effector proteins of RORβ through LC-MS/MS; to investigate the effects of RORβ and its key targets on the biological function of CCSCs. This study provides a strategy for drug design to eradicate CCSCs in future.

结直肠癌干细胞的残存是结直肠癌转移、复发和耐药的主要原因。因此清除结直肠癌干细胞是结直肠癌治疗的重点。然目前尚无靶向抑制或清除大肠癌干细胞药物。阐明结直肠癌干细胞自我更新的分子机制，鉴定其关键分子靶点是靶向结直肠癌干细胞治疗的基础和前提。我们先前研究发现Rorβ在结直肠癌细胞和组织中均存在表达，在原代结直肠癌组织中表达明显下调，过表达Rorβ能够显著抑制结直肠癌细胞的克隆球成球能力、体外致瘤能力和Wnt通路下游靶分子表达。提示Rorβ可能通过调控Wnt通路活性抑制结直肠癌干细胞自我更新，是结直肠癌干细胞的潜在治疗靶点。然Rorβ调控结直肠癌干细胞的作用机制尚不明了。本研究拟在先前研究基础上，通过亲和串联质谱方法筛选和鉴定Rorβ的作用蛋白，探讨Rorβ及其关键靶分子对结直肠癌干细胞自我更新的生物学功能的影响和信号通路的鉴定。为今后研制靶向清除结直肠癌干细胞的药物打下坚实的基础。

结直肠癌干细胞的残存是结直肠癌转移、复发和耐药的主要原因。因此清除结直肠癌干细胞是结直肠癌治疗的重点。我们首先通过免疫组化和mRNA荧光原位杂交验证大肠癌病理组织切片中维甲酸相关孤儿受体β（Retinoic acid-related orphan receptor beta,Rorβ）的表达，并采用Real-time qPCR检测大肠癌细胞和临床样本中Rorβ的表达情况。接着，我们采用亲和串联质谱方法从大肠癌细胞中筛选Rorβ相互作用靶分子，通过免疫共沉淀方法进行验证；并对其相互作用结构域进行鉴定。通过构建和生产可诱导的Rorβ及其关键靶分子过表达/沉默的慢病毒载体，感染结直肠癌细胞系，验证Rorβ及其关键靶分子对结直肠癌干细胞自我更新能力的影响。最后构建Rorβ及其关键靶分子可诱导表达/沉默细胞，结合高通量cDNA和蛋白质芯片筛选方法，鉴定Rorβ和其关键作用靶分子Wnt通路的调控。我们首次发现Rorβ除表达于神经体统外，还表达于大肠上皮和大肠癌组织中，且在原代结直肠癌组织中呈表达下调趋势。异位表达Rorβ后明显抑制结直肠癌干细胞体外成球能力，致瘤性及CD44+CD24+细胞数量。通过基因芯片检测HMG盒转录因子1(High Mobility Group-box transcription factor1,HBP1)是Rorβ下游靶分子，EMSA提示Rorβ确实能结合到HBP1上游启动子区序列，促进了后者对HBP1的转录，加强了HBP1对TCF4与DNA结合的抑制效应，从而通过激活经典Wnt通路促进了结直肠癌干细胞的自我更新。上述结果说明Rorβ通过HBP1抑制Wnt 通路活性抑制结直肠癌干细胞自我更新。为今后研制靶向清除结直肠癌干细胞的药物打下坚实的基础。