Department of Gastrointestinal Colorectal and Anal surgery, China-Japan Union Hospital of Jilin University, NO.126 Xiantai Street, Changchun, Jilin 130033, China.
Department of Neurology, The First Hospital of Jilin University, Jilin 130021, China.
Dig Liver Dis. 2021 Apr;53(4):509-516. doi: 10.1016/j.dld.2020.12.115. Epub 2021 Jan 18.
lncRNA cancer susceptibility 9 (CASC9) is a pivotal modulator in various cancers, such as colorectal cancer, breast cancer and esophageal cancer. However, its exact role in gastric cancer (GC) has not been systematically studied. Here, using a combination of molecular and cell biology approaches, we found that CASC9 also acts as a factor promoting the progression of GC. First, mRNA and protein expression levels were quantified by real-time quantitative reverse transcription PCR (qRT-PCR) and western blot, respectively. Second, CCK-8 assay, colony formation assay and cell cycle analysis were performed to compare the cell growth abilities when CASC9 was knocked down. Third, the proliferative cells were determined by labeling Edu and the regulatory effect of CASC9 on miR-370 was detected by RNA-protein pull-down and luciferase reporter assays. Finally, in vivo mice model was established to verify the role of CASC9 in promoting GC progression. Our results showed that CASC9 was up-regulated significantly in both GC tissues and cell lines. Conversely, CASC9 knockdown inhibited GC growth in vitro. Further analysis indicated that CASC9 directly targeted miR-370 and negatively regulated miR-370 expression in GC. Besides, EGFR (epidermal growth factor receptor) was identified as a direct target gene of miR-370. Taken together our results support a model in which CASC9 promotes GC progression through miR-370/EGFR/ERK/AKT pathway. Finally, in vivo CASC9 knockdown resulted in impaired GC growth. In sum, this study firstly demonstrates that lncRNA CASC9 acts as an oncogene through altering EGFR expression level via negatively regulating miR-370 expression.
长链非编码 RNA 癌症易感性 9(CASC9)是多种癌症(如结直肠癌、乳腺癌和食管癌)中的关键调节因子。然而,其在胃癌(GC)中的确切作用尚未得到系统研究。在这里,我们采用分子和细胞生物学方法的组合,发现 CASC9 也作为促进 GC 进展的因素发挥作用。首先,通过实时定量逆转录 PCR(qRT-PCR)和 Western blot 分别定量 mRNA 和蛋白质表达水平。其次,进行 CCK-8 测定、集落形成测定和细胞周期分析,以比较 CASC9 敲低时的细胞生长能力。第三,通过标记 Edu 测定增殖细胞,并通过 RNA-蛋白下拉和荧光素酶报告基因测定检测 CASC9 对 miR-370 的调节作用。最后,建立体内小鼠模型以验证 CASC9 在促进 GC 进展中的作用。我们的结果表明,CASC9 在 GC 组织和细胞系中均显著上调。相反,CASC9 敲低抑制了体外 GC 的生长。进一步分析表明,CASC9 直接靶向 miR-370,并负调控 GC 中 miR-370 的表达。此外,表皮生长因子受体(EGFR)被鉴定为 miR-370 的直接靶基因。总之,我们的研究结果支持 CASC9 通过 miR-370/EGFR/ERK/AKT 通路促进 GC 进展的模型。最后,体内 CASC9 敲低导致 GC 生长受损。总之,本研究首次证明长链非编码 RNA CASC9 通过负调控 miR-370 的表达来改变 EGFR 表达水平,从而作为癌基因发挥作用。