Li Shiquan, Yan Guoqiang, Liu Wei, Li Chenyao, Wang Xu
Department of Colorectal and Anal Surgery, The First Hospital of Jilin University, Changchun, Jilin, China.
Department of Spinal Surgery, The First Hospital of Jilin University, Changchun, Jilin, China.
Mol Carcinog. 2020 Dec;59(12):1323-1342. doi: 10.1002/mc.23259. Epub 2020 Oct 30.
This study aimed to investigate the role of circ0106714-miR-942-5p-discs large homolog 2 (DLG2), a novel interactome, in colorectal cancer (CRC). Circ0106714 was found to be the most significantly downregulated circular RNA in CRC using a bioinformatics method, and we researched whether the ability of circ0106714 to sponge miR-942-5p and release DLG2 could affect CRC development via Hippo-YES-associated protein (YAP) signaling. We first employed qRT-PCR and immunoblotting to detect messenger RNA (mRNA) and protein expression, respectively. Live imaging of mice tumor xenografts was then conducted to study the effect of circ0106714 on tumor progression in vivo. Reporter gene assays were subsequently conducted to verify the predicted targeting relationship between circ0106714, miR-942-5p, and DLG2 mRNA in SW480 and HCT116 cell lines. As well as using flow cytometry for both apoptosis and cell cycle profile analyses, CCK-8 and clone foci formation assays were performed to assess cell survival. Wound healing assay and transwell invasion assay were later carried out to evaluate the migration and invasion of the cell lines. Findings revealed that circ0106714 and DLG2 were significantly downregulated, while miR-942-5p was significantly upregulated in human CRC tissues and cell lines. However, circ0106714 upregulation significantly suppressed tumor progression in vivo and inhibited the malignancy phenotypes of tumor cells in vitro by targeting miR-942-5p. Also discovered in this research was that miR-942-5p could directly target DLG2 mRNA, thus enhancing the malignancy phenotypes of CRC cells. We even found that DLG2 overexpression resulted in enhanced phosphorylation of YAP, a critical downstream effector of DLG2. This downstream effector was demonstrated to have a tumor-suppressive capacity in CRC cell lines. In sum, circ0106714 could suppress CRC by sponging miR-942-5p and releasing DLG2, thus promoting YAP phosphorylation.
本研究旨在探究一种新型相互作用组circ0106714-miR-942-5p-盘状大同源蛋白2（DLG2）在结直肠癌（CRC）中的作用。利用生物信息学方法发现，circ0106714是CRC中下调最显著的环状RNA，我们研究了circ0106714通过海绵吸附miR-942-5p并释放DLG2的能力是否会通过Hippo-YES相关蛋白（YAP）信号通路影响CRC的发展。我们首先采用qRT-PCR和免疫印迹法分别检测信使核糖核酸（mRNA）和蛋白质表达。随后对小鼠肿瘤异种移植进行活体成像，以研究circ0106714对体内肿瘤进展的影响。接着进行报告基因检测，以验证circ0106714、miR-942-5p和DLG2 mRNA在SW480和HCT116细胞系中的预测靶向关系。除了使用流式细胞术进行凋亡和细胞周期分析外，还进行了CCK-8和克隆集落形成试验以评估细胞存活率。随后进行伤口愈合试验和Transwell侵袭试验以评估细胞系的迁移和侵袭能力。研究结果显示，在人CRC组织和细胞系中，circ0106714和DLG2显著下调，而miR-942-5p显著上调。然而，circ0106714的上调显著抑制了体内肿瘤进展，并通过靶向miR-942-5p抑制了体外肿瘤细胞的恶性表型。本研究还发现，miR-942-5p可直接靶向DLG2 mRNA，从而增强CRC细胞的恶性表型。我们甚至发现，DLG2的过表达导致YAP磷酸化增强，YAP是DLG2的关键下游效应因子。在CRC细胞系中证实该下游效应因子具有肿瘤抑制能力。总之，circ0106714可通过海绵吸附miR-942-5p并释放DLG2来抑制CRC，从而促进YAP磷酸化。
