长链非编码RNAlnc-CC3作为细胞内β-catenin“调节器”增强宫颈癌侵袭转移的分子机制研究

Patients with recurrent and metastasis cervical neoplasm always present poor quality of life and high death rate. Long noncoding RNA as a focus area in cancer research is closely related to tumor metastasis. We cloned a novel lncRNA, named lnc-CC3, can increase metastasis in cervical cancer by increasing Slug expression. Subsequent studies showed that lnc-CC3 can combine with β-catenin and enhance the abundance of β-catenin in nucleus, thus increasing the expression of tumor metastasis related genes like Slug. The analysis of RNA pull down assay suggested that lnc-CC3 may weaken the intercellular adhesion and β-catenin degradation by affecting the interaction of β-catenin with related proteins. Published reports showed that β-catenin is a promoter of Slug and play an important role in tumor metastasis. In this project, we will employ CO-IP, IF, CRISPR-Cas9 and other methods to reveal the cause of abnormal β-catenin localization and repressed degradation. And the metastasis ability change of lnc-CC3-mutant cervical cancer cell will be tested in vitro and vivo. The clarification of lnc-CC3 as an intracellular β-catenin modulator in increasing cervical cancer metastasis can provide new insights for tumor clinical decision-making and treatment.

宫颈癌的复发转移严重影响患者生存时间及质量。长链非编码RNA与肿瘤转移密切相关，是肿瘤分子机制研究的焦点领域。Lnc-CC3是申请者克隆的新lncRNA，最初发现其可提高Slug表达而增强宫颈癌的侵袭转移。后续研究显示lnc-CC3可与Slug的转录促进子β-catenin结合，并提高核内β-catenin的丰度，增强相关基因的表达。综合RNA pull down结果，推测lnc-CC3与β-catenin胞内定位及降解抑制的相关调控有关。因此本课题拟利用免疫共沉淀、免疫荧光、CRISPR-Cas9等技术揭示lnc-CC3导致细胞内β-catenin定位异常及降解抑制的原因，并通过体内外实验检测lnc-CC3相关蛋白结合序列缺失后宫颈癌侵袭转移能力的改变，阐明lnc-CC3作为β-catenin胞内“调节器”增强宫颈癌侵袭转移的分子机制，为肿瘤临床决策与基因治疗提供理论参考。

宫颈癌的复发转移严重影响患者生存时间及质量。长链非编码RNA与肿瘤转移密切相关，是肿瘤分子机制研究的焦点领域。Lnc-CC3是申请者克隆的新lncRNA，最初发现其可提高Slug表达而增强宫颈癌的侵袭转移，本课题针对lnc-CC3增加宫颈癌侵袭转移能力的分子机制进行了深入探索。通过RNA pull down和RNA免疫共沉淀实验确定了lnc-CC3与β-catenin蛋白的相互作用，以β-catenin蛋白为基点开展相关研究发现过表达lnc-CC3后β-catenin蛋白及其结合蛋白GSK3β在细胞核内发生了累积并存在共定位，同时细胞质内β-catenin蛋白与α-catenin、E-cadherin等膜定位蛋白的结合发生减少。综上所述，lnc-CC3可以减少β-catenin与细胞膜上相关蛋白的结合而削弱细胞的粘附能力，并通过增加β-catenin和GSK3β蛋白的入核从而引起宫颈癌细胞侵袭转移能力的增强。我们分析实验结果后推测lnc-CC3并非通过阻止GSK3β在细胞质内对β-catenin降解的降解，而是协助β-catenin和GSK3β入核并导致蛋白累积，该RNA/蛋白复合体在细胞核内发挥增强宫颈癌侵袭转移能力的作用值得进一步研究。此外，本课题利用CRISPR-KRAB-dCas9质粒敲降lnc-CC3表达并筛选得到两个有效敲降靶点，未来可用于lnc-CC3相关的基因治疗。整个课题的研究内容可以为lnc-CC3的靶向干预以及β-catenin、GSK3β蛋白定位异常引起的疾病基因治疗提供理论参考，并作为分子靶标用于相关疾病的基因分子诊断。

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