假基因PTTG3P通过结合PTBP1参与胃癌细胞Warburg效应的机制研究

Gastric cancer is one of the most prevalent malignancies in China. And Warburg effect is a core hallmark for metabolic reprogramming in gastric cancer. As a specialized form of long noncoding RNA (lncRNA), pseudogene was proved to play an important role in gastric cancer by regulating the malignant phenotypes of gastric cancer cells. We had previously discovered that a pseudogene—PTTG3P was significant upregulated in gastric cancer and played a crucial part in tumor progression. Upregulation of PTTG3P significantly enhanced glucose uptake and lactate production in gastric cancer cells. In further investigating the underlying mechanism, we identified PTBP1 as the binding target of PTTG3P, and found that PTTG3P could possibly regulate the alternative splicing of PKM gene by interacting with PTBP1. Taken together, we raised a hypothesis that PTTG3P chould regulate the alternative splicing of PKM gene by interacting with PTBP1, lead to the isoform switch from PKM1 to PKM2, facilitate the Warburg effect in gastric cancer, and consequently support gastric cancer cell growth. A series of functional and molecular biological experiments, such as protein truncated assay and RNA-scope, would be carried out to clarify: ① the regulating role of PTTG3P in faciliting the Warburg effect in gastric cancer; ② the binding site and molecular mechanism of PTTG3P-PTBP1 interaction in regulating the alternative splicing of PKM gene; ③ the clinical significance of PTTG3P, PTBP1 and PKM1/2 expression in gastric cancer. In this present study, we aim to provide theoretical basis for the screening of tumor therapeutic target via regulating glucose mechanism of gastric cancer cells.

胃癌是我国高发的肿瘤，Warburg效应是胃癌细胞代谢重编程的重要标志之一。假基因作为特殊类型的非编码RNA已被证实参与调控胃癌恶性表型。我们前期发现假基因PTTG3P在胃癌中高表达并能发挥促癌作用，且PTTG3P可促进胃癌细胞糖摄取和乳酸生成；深入研究发现PTTG3P可与PTBP1特异结合，并可影响PKM基因的可变剪接；鉴于PTBP1是重要的可变剪接调控蛋白，我们提出假设：PTTG3P可能通过与PTBP1结合，调控PTBP1对PKM基因的可变剪接，促进胃癌细胞Warburg效应并发挥促癌作用。本课题拟采用蛋白截短实验、RNA-scope等一系列功能和分子生物学实验阐明：①假基因-PTTG3P在促胃癌Warburg效应中的调控作用；②PTTG3P与PTBP1的结合位点及其调控PKM基因可变剪接的分子机制；③相关分子的表达及临床转化价值。本课题将从糖代谢角度为筛选肿瘤治疗靶标提供新的依据。

Warburg效应是胃癌细胞代谢重编程的重要标志之一。假基因作为特殊类型的非编码RNA已被证实参与调控胃癌恶性表型。项目计划研究通过过表达及敲减/沉默假基因PTTG3P，研究其通过结合PTBP1参与胃癌细胞Warburg效应的机制，但在执行过程中，及时发现了原课题假说的局限性，并调整了研究内容。项目主要完成了一下研究内容：① PTTG3P可在体内外水平促进胃癌细胞Warburg效应；② PTTG3P通过稳定PTBP1的表达促进PKM基因的可变剪接；③ HEYL在胃癌中的过表达，并与胃癌患者的预后不良密切相关；④ HEYL通过转录调控CDH11促进胃癌细胞的侵袭转移。我们的课题研究了PTTG3P、PTBP1、HEYL和CDH11等分子在胃癌中的功能和作用机制，提示这些分子有可能成为新的胃癌治疗靶点。本课题拓展了对于胃癌恶性表型及代谢重编程相关调控机制和假基因PTTG3P以及转录因子HEYL在胃癌中的作用及下游分子机制的认识；同时也为癌蛋白PTBP1和CDH11在胃癌中调控机制提供了新的研究视角和基础实验依据。本课题有助于寻找新的胃癌预后监测分子标志物和分子治疗靶标，具有良好的理论意义和临床转化价值。