WNT/β-catenin信号通路对Linc-ROR的调控及其在结直肠癌发生中的作用机制研究

Linc-ROR has been confirmed to be related to the development and metastasis of different cancers. The correlation between Linc-ROR and P53 as well as its function as ceRNA to adsorb miR-145 may be responsible for such phenomenon. However, the upstream of Linc-ROR remains unclear. In our previous studies, we found that the levels of Linc-ROR were evidently up regulated in human colorectal cancer samples. With further bioinformatics analysis and luciferase experiment, we found that TCF7L2, a key transcription factor in WNT/β-catenin pathway, might regulate Linc-ROR expression. As so, in the present study, we are going to measure Linc-ROR level in normal colorectal mucosa,colorectal adenoma and colorectal cancer; find out the relationship among Linc-ROR, P53 and WNT/β-catenin by luciferase and siRNA technology; confirm the regulation of miR-145/143 by Linc-ROR through RIP, luciferase and anti-AGO2 RIP; verify the influence of Linc-ROR to colorectal cancer cell proliferation by overexpression Linc-ROR or knockdown Linc-ROR in vitro and in vivo. In order to confirm the molecular function of Linc-ROR and provide a new theoretical basis for how Linc-ROR regulate colorectal cancer development.

Linc-ROR已被证实与肿瘤的发生和转移相关，其机制可能与P53的相互调控及吸附miR-145有关，但导致其表达增高的上游调控机制尚未明确。我们前期发现，Linc-ROR在结直肠癌中表达明显增高。生物信息学分析及luciferase实验提示，WNT/β-catenin信号通路成员TCF7L2可能作为转录因子调控Linc-ROR的表达。本研究检测人正常结肠粘膜、结肠腺瘤和腺癌中Linc-ROR水平；luciferase转染等验证WNT/β-catenin 和P53对Linc-ROR的调节及三者间关系；RNA免疫沉淀和anti-AGO2 RIP实验验证Linc-ROR与miR-145/143的结合作用；构建Linc-ROR过表达及敲减模型，通过体内外实验验证Linc-ROR水平对结直肠癌细胞的影响。为明确Linc-ROR功能，探讨结直肠癌发生中非编码RNA调控机制提供新的理论依据。

Linc-ROR于诱导多功能干细胞（iPSCs）研究中被首次发现，已被证实与多种肿瘤的发生和转移相关，机制可能与P53的相互调控有关，但导致其表达增高的上游调控机制尚未明确。我们前期发现，Linc-ROR在结肠癌中表达明显增高。生物信息学分析及荧光素酶（Luciferase）实验提示，WNT/β-catenin信号通路成员TCF7L2可能作为转录因子调控Linc-ROR的表达。因此研究WNT/β-catenin和P53对Linc-ROR的调节及三者之间作用关系，对于揭示Linc-ROR在结肠癌发生中的分子机制具有重要意义。.本研究实时荧光定量qRT-PCR结果发现结肠腺癌样本组织中Linc-ROR表达水平显著高于结肠腺瘤及正常粘膜组织。通过构建包含预测结合位点序列的荧光素酶Luciferase质粒，检测发现TCF7L2可与Linc-ROR启动子结合，转染敲减TCF7L2后HCT-116结肠癌细胞Linc-ROR表达量显著减低，P53表达量增高，表明WNT/β-catenin信号通路能够激活Linc-ROR表达，同时调节P53的表达。其次，成功构建了Luciferase质粒稳转癌细胞，以及三种结肠癌细胞株的Linc-ROR稳定过表达及敲减模型。通过体内外实验我们观察到，与阴性对照组相比，转染过表达质粒HCT-116结肠癌细胞的增殖、迁移及侵袭能力显著增强，且在DNA损伤的情况下（Dox处理），过表达Linc-ROR显著抑制P53的表达；转染shLinc-ROR敲减质粒 HCT-116细胞的增殖、迁移及侵袭能力明显减弱，在DNA损伤的情况下（Dox处理），敲减Linc-ROR能够增加P53的表达。.本研究证实了Linc-ROR在结肠癌中表达上调，受到WNT/β-catenin通路转录调节，并进一步通过抑制P53的表达在结肠腺癌发生过程中发挥重要调控作用。为探讨结肠癌发生中非编码RNA调控机制提供新的理论依据，P53相关的Linc-ROR可能成为结肠癌诊断的重要新型标志和治疗的潜在靶点。