circCTBP2通过miR-34a/Notch1信号通路增强胃癌干细胞特性促进胃癌进展的机制研究

Gastric cancer stem cells (GCSCs) contribute to growth and metastasis in gastric cancer, however, the regulating mechanisms of cancer stem cell (CSC)-like properties remain obscure. Our preliminary data found that 75 circRNAs were aberrantly expressed in GCSCs by RNA sequencing, and circCTBP2 was the most significantly upregulated circRNA, which was verified by qRT-PCR. Besides, circCTBP2 was overexpressed in GCSCs enriched by 7 gastric cell lines. For detecting the functional role of circCTBP2, circCTBP2 was overexpressed and silenced transiently in gastric cancer cell lines in vitro assays, which found that circCTBP2 promoted the CSC-like properties. Mechanistically, circCTBP2 was revealed to bind with miR-34a by bioinformatics analysis and luciferase reporter assay. In addition, Notch1, a well-known target gene of miR-34a, may mediate the function of circCTBP2 promoting the CSC-like properties. Based on these evidence, we hypothesize that circCTBP2 could promote the CSC-like properties and thus accelerate the progression of gastric cancer by upregulating Notch1 expression via competing with miR-34a. This study aims to further investigate the function and regulating mechanisms of circCTBP2. The clinical significance of circCTBP2 will also be explored. This study may provide evidence for utilizing circCTBP2 as a novel biomarker for prediction of prognosis and a potential target for CSC-targeted therapy.

胃癌干细胞（GCSC）可促进胃癌生长和转移，但GCSC特性的调控机制尚不明确。前期研究中我们通过RNA测序发现富集的GCSC中75种circRNA表达异常，结合qRT-PCR检测证实circCTBP2升高最为明显；并且，circCTBP2在7种胃癌细胞富集的GCSC中表达均升高。进一步体外实验研究发现circCTBP2能够增强GCSC特性。荧光素酶实验结果提示circCTBP2可与miR-34a结合，并且可能通过上调miR-34a靶蛋白Notch1表达增强GCSC特性。据此我们提出假设：circCTBP2可能通过miR-34a/Notch1信号通路增强GCSC特性进而促进胃癌进展。本课题拟在前期研究的基础上，在分子、细胞、裸鼠和临床标本层面进一步明确circCTBP2增强GCSC特性进而促进胃癌进展的作用和机制，并揭示其临床意义，为寻找新的预后标志物及研究靶向GCSC的治疗措施奠定基础。

肿瘤干细胞（CSCs）可为肿瘤无限生长提供驱动，并可激活促进肿瘤转移，在胃癌的进展过程中发挥极为重要的作用。环状RNA（circRNAs）在肿瘤多种生物学过程中发挥重要作用，但其在CSCs中的功能研究较少，本研究拟通过探索circRNAs在胃癌干细胞(GCSCs)的表达、功能及作用机制进一步揭示胃癌生长和转移的机制，为探索胃癌进展机制和寻找治疗靶点提供线索。为探索GCSC中circRNAs表达谱和其在胃癌进展中的功能，我们首先使用Spheroid formation富集了GCSCs，并使用高通量测序的方法检测了GCSCs和普通胃癌细胞中circRNAs表达，经筛选验证circSLC4A7是表达差异最大的circRNAs之一，我们经qRT-PCR检测证实了circSLC4A7在GCSCs、胃癌细胞和组织中高表达。CircSLC4A7具有稳定环状结构，主要定位于细胞核。我们构建了circSLC4A7稳定高表达和干扰细胞系，体外实验发现circSLC4A7高表达可显著增强GCSCs特性并促进胃癌增殖和转移，circSLC4A7干扰可明显抑制GCSCs特性并减弱胃癌增殖和转移能力。在机制探索中，我们经RNA pull-down后质谱检测发现circSLC4A7可与HSP90结合，该结果经RIP和dual RNA-FISH检测验证进一步证实，干扰HSP90表达可明显逆转circSLC4A7高表达引起的GCSCs特性的增强和转移增殖能力增加。此外，我们经多信号通路筛选发现circSLC4A7与HSP90结合后可激活NOTCH1通路进而促进胃癌进展。该研究结果初步揭示了GCSCs中circRNAs表达谱，拓展了胃癌进展机制的研究领域，为寻找新的预后标志物及研究靶向GCSC的治疗措施奠定基础。