U2AF65/circATP2B1靶向调控miR-326基因簇影响胃癌细胞“Warburg效应”的机制研究

"Warburg effect" is one of the most important factors of gastric cancer cells over proliferation. Our preliminary data showed that circular RNA hsa_circ_000826 (also called circATP2B1) was highly expressed in gastric cancer tissues, it promoted aerobic glycolysis in gastric cancer cells. While The mechanism of the process is unknown. Bioinformatic analysis predicts that splicing factor U2AF65 (U2 snRNP auxiliary factor 65kDa subunit) obtains the ability to bind to circATP2B1 flanking sequences and may take part in the cyclization and intracellular transportation. Gene Ontology analysis shows that the potential downstream genes miR-326 gene cluster (miR-326-3p/miR-330-5p) functionally focuses on cell growth and metabolic processes. Therefore we propose the hypothesis that”U2AF65 promotes the cyclization and enrichment of circATP2B1 in cytoplasma, circATP2B1 binds to miR-326-3p/miR-330-5p therefore induced aerobic glycolysis”. The study will utilize RNA pull down, eukaryotic expression technique, real-time PCR, RNA interruption, luciferase reporter assay, nude mice xenograft assay and so on to illustrate the accumulation mechanism and role of circATP2B1 in glycolysis of gastric cancer, it might help to provide novel targets for reversing cancer metabolic reprogramming.

“Warburg效应”即有氧糖酵解，是胃癌过度增殖的重要因素。申请者前期研究发现环状RNA circATP2B1在胃癌组织中明显高表达，能显著促进胃癌细胞有氧糖酵解，但其富集原因及作用机制尚不清楚。生物信息学预测剪接因子U2AF65能与circATP2B1侧翼序列结合，可能参与其RNA生成及细胞内转运。富集分析提示circATP2B1的潜在下游基因簇miR-326-3p/miR-330-5p功能集中于细胞生长代谢过程。因此，申请者提出“U2AF65促进circATP2B1生成并将其转运至细胞质富集，从而结合miR-326基因簇促进胃癌细胞Warburg效应”的假说。本研究拟通过RNA pull down、真核表达、荧光定量PCR、RNA干扰、荧光素酶报告基因、裸鼠移植瘤实验等方法，探讨circATP2B1在胃癌中富集及促进糖酵解的部分机制，为逆转肿瘤代谢重编程提供新的靶点。

“Warburg效应”即有氧糖酵解，是胃癌过度增殖的重要因素。环状RNA是重要的遗传物质。本课题本研究采用RNA 免疫共沉淀、真核表达、荧光定量PCR、RNA干扰、荧光素酶报告基因、裸鼠移植瘤实验的方法，探讨circATP2B1在胃癌中富集及促进糖酵解的部分机制。.本研究发现，胃癌组织中circATP2B1较正常胃组织升高。过表达U2AF65的MKN45及SGC7901细胞中circATP2B1的环状分子表达上升。提示U2AF65能促进circATP2B1成环。我们发现circATP2B1 过表达能引起胃癌细胞系有氧糖酵解的程度增加，miR-326-3p/miR-330-5p含量显著下降。光素酶实验证实circATP2B1与miR-326-3p/miR-330-5p直接作用。miR-326-3p/miR-330-5p过表达能下调PKM2含量。裸鼠移植瘤实验发现circATP2B1沉默及miR-326-3p/miR-330-5p过表达均能抑制移植瘤生长，二者共同作用抑制作用相协同。.我们的研究能得出如下结论；生理状态下胃腺体细胞中circATP2B1含量较低。在胃癌细胞中由于剪接因子U2AF65表达升高，从而促使circATP2B1成环，进而与抑癌基因miR-326-3p/miR-330-5p形成RNA沉默复合体，下调miR-326-3p/miR-330-5p表达含量。下调的miR-326-3p/miR-330-5p减轻了对于PKM2的抑制，加速了有氧糖酵解的进程，为癌细胞增殖提供了能量基础。通过本项目的系统研究，能够加深对胃癌发病机理的理解，为胃癌预防和治疗的新靶点提供理论依据和实验基础。

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