Hsa-miR-15b通过抑制DCLK1阳性肠癌干细胞干性增强直肠癌放疗敏感性研究

Radiotherapy resistance is the main reason for treatment failure during rectal cancer neoadjuvant radiotherapy. It is important to identify a set of discriminating biomarkers that can be used for characterization and prediction of response to radiotherapy in rectal cancer. Our previous results showed expression of miR-15b in pretreatment biopsy tissue samples predicts postoperative histological tumor regression grade (TRG) in LARC patients who underwent surgery after nRT(neoadjuvant radiotherapy treatment). Expression of miR-15b in post-nRT tissue samples was associated with therapeutic outcome for neoadjuvant radiotherapy. Mir-15b overexpression reverses CRC cells chemoradioresistance.DCLK1 (doublecortin and CaM kinase-like-1) is a direct target gene of miR-15b.DCLK1 positive tumor stem cells drive a tumour-initiating cell phenotype and chemoradiotherapy resistance. BMI1 is a potential downstream target of the miR-15b-DCLK1 signaling pathway. The purposes of this study is on the basis of our previous work, further to address the correlation of miR-15b expression with the response to radiotherapy and prognosis by a large-scale validation; investigate the effect of Hsa-miR-15b on DCLK1+ TICs self-renewal and tumorigenic capacity by using spheroid formation and tumorigenicity assays, the effects of miR-15b on radiosensitivity of DCLK1+ cells by using clonogenic survival; elucidate the molecular mechanism of Hsa-miR-15b by using GST pull down, Mass spectrometry, co-immunoprecipitate and identify the signal transduction pathway of Hsa-miR-15b. By these, our study provides important clues for future researches on rectal cancer prognosis and molecular therapeutic targets, which benefits the rectal cancer patients in clinical practice.

放疗抵抗是直肠癌新辅助放化疗的治疗瓶颈，寻找放疗敏感性相关标志物对于直肠癌的个体化治疗有着重要的临床意义。前期研究发现，Hsa-miR-15b的表达与直肠癌新辅助放疗敏感性正相关。Hsa-miR-15b过表达增加了肠癌细胞对放疗的敏感性，DCLK1是Hsa-miR-15b靶基因。DCLK1+细胞呈现肿瘤干细胞特性及放疗抗性。我们推测，Hsa-miR-15b可能通过调控DCLK1+肠癌干细胞干性，增加了直肠癌放疗敏感性。本研究拟通过扩大样本验证Hsa-miR-15b做为放疗敏感性预测分子标志物的可行性；通过流式分选、体内外自我更新能力、放疗实验明确Hsa-miR-15b增加放疗敏感性的作用是否与调控肿瘤干细胞相关；运用免疫共沉淀、质谱鉴定及体外结合技术深入研究Hsa-miR-15b可能参与的信号传导通路。通过本研究以期为直肠癌新辅助放疗敏感性的预测及治疗提供分子靶标，实现直肠癌治疗个体化。

直肠癌是消化道最常见的恶性肿瘤之一,其发病率在我国呈逐年上升的趋势,约占全身恶性肿瘤的15%以上。对于中低位进展期直肠癌,接受术前放疗可以使肿瘤降期明显,提高保肛率。但是,临床上发现直肠癌患者术前放疗的治疗效果个体差异较大, 放疗抵抗是新辅助放疗治疗失败的主要原因。探索放疗敏感性相关机制和分子标志物对于直肠癌的个体化治疗有着重要的临床意义。.本研究通过流式分选、体内外自我更新能力、放疗实验等探讨了Has-miR-15b增加放疗敏感性的作用以及与肿瘤干细胞干性调控之间的相关性；利用分子生物学技术深入研究Hsa-miR-15b参与的信号传导通路；探讨了Hsa-miR-15b做为放疗敏感性预测分子标志物的可行性。结果证实，Hsa-miR-15b过表达显著增强了肠癌细胞对放疗的敏感性，并可以恢复放疗抵抗肠癌细胞的放疗敏感性。Hsa-miR-15b可以抑制DCLK1(+)肿瘤干细胞干性，显著抑制其自我更新能力，增加DCLK1+细胞对放化疗的敏感性。在DCLK1+的肠癌干细胞中，WNT/β-CATENIN信号通路活化，DCLK1通过WNT/β-CATENIN信号通路调节了MYC及BMI1的表达。miR-15b通过对DCLK1的靶向调控参与BMI1信号通路调节。通过临床样本中的检测证实Hsa-miR-15b表达与放疗敏感性显著正相关，DCLK1表达与放疗敏感性显著负相关，Hsa-miR-15b和DCLK1是新辅助放化疗敏感性预测的潜在标志物。另外，在研究中发现，DCLK1与肿瘤微环境中Treg等免疫抑制性因素相关，利用CD25/CTLA4抗体清除体内Treg细胞能够增加肿瘤组织放疗疗效。.我们的研究首次证实Hsa-miR-15b在术前放疗敏感性预测及术后预后预测中的意义，可作为潜在的放疗敏感性相关的生物学标志物。明确了Hsa-miR-15b在直肠癌中的表达及在放疗敏感性中所起到的作用，深入探索了Hsa-miR-15b参与调节的信号传导通路，部分阐释了Hsa-miR-15b在直肠癌放疗敏感性中发挥作用的分子机理。