LINC00473调控XIAP基因抑制自噬促进胃癌侵袭转移的分子机制研究

Gastric cancer is the most common malignant tumor in China. Inhibition of invasion and metastasis is important in the therapy of gastric cancer. Using lncRNAs expression profile microarray, we found that LINC00473 was highly expressed in gastric cancer compared to adjacent normal tissue. So far, its regulation mechanisms in the development of gastric cancer has not been discussed. In our pre-experiments, LINC00473 was highly expressed in not only gastric cancer tissue but also gastric cancer cell lines. Meanwhile, down-regulated expression of LINC00473 could significantly inhibit migration and invasion in gastric cancer cells. And high-throughput sequencing on transcriptome results showed that autophagy-related gene XIAP could be one target gene of LINC00473, thereby influencing autophagy in tumor cells to regulate invasion and metastasis. Bioinformatics analysis showed that LINC00473 could link to a RNA-binding protein HuR, subsequently affecting the stability of mRNAs of target gene. But the exact mechanism is not clear. We hypothesized that LINC00473 regulated the stability of target gene XIAP by combining with HuR. Then autophagy was inhibited so that invasion and metastasis of gastric cancer were promoted. In this project, we would further investigate the relationship of LINC00473 and the development of gastric cancer through clinical, cell and animal studies, which mainly focus in the biological functions of LINC00473 and its possible down-stream regulators HuR and XIAP. It would provide detail mechanisms of LINC00473 in gastric cancer, and provide novel sights of lncRNAs and possible therapy targets for gastric cancer.

胃癌是我国最常见的恶性肿瘤，如何抑制其侵袭转移是亟待解决的难题。LINC00473是本课题组通过lncRNAs表达谱芯片筛选获得的、显著高表达于胃癌组织的lncRNA，其对胃癌的调控作用迄今未见研究报道。我们发现干扰LINC00473表达可显著抑制胃癌细胞的迁移、侵袭水平；转录组高通量测序提示，自噬抑制基因XIAP可能作为其靶基因，参与抑制细胞自噬、促进侵袭转移；生物信息学分析显示LINC00473可能与HuR结合从转录后水平调控XIAP mRNA的稳定性，但确切的机制尚待研究。因此，我们提出LINC00473通过结合HuR调控靶基因XIAP的稳定性，抑制肿瘤细胞自噬，促进胃癌侵袭转移的新假设。本课题拟以临床样本验证相关分子表达规律、以细胞及动物模型验证关键分子的调控机制，初步阐明LINC00473通过XIAP调控胃癌侵袭转移的分子机制，旨在为胃癌的预后判断和治疗提供新的分子靶标。

研究背景：胃癌是我国最常见的恶性肿瘤，如何抑制其侵袭转移是亟待解决的难题。LINC00473是本课题组通过lncRNAs表达谱芯片筛选获得的，在胃癌中显著高表达。其对胃癌的调控作用未见研究报道。本研究重点探讨了LINC00473在胃癌恶性进展中的功能及其作用机制。.研究内容：应用qRT-PCR检测LINC00473在胃癌组织及癌旁组织中的表达，并分析不同表达水平与患者临床病理参数之间的相关性；通过细胞增殖实验和细胞迁移、侵袭实验验证LINC00473在体外对胃癌增殖和转移的影响；使用流式细胞术分析LINC00473对细胞周期和凋亡的影响，并进行WB实验检测周期相关蛋白的变化；体内动物实验观察裸鼠皮下成瘤能力、尾静脉注射后肺转移情况；通过ENCORI网站预测胃癌细胞中与LINC00473相互作用的miRNA，并通过实验验证miRNA的下游靶蛋白；将敲低LINC00473的胃癌细胞进行转录组测序，寻找下游靶基因，通过功能实验探究其对胃癌细胞增殖转移能力的影响，并使用拯救实验探究靶基因是否受到LINC00473调控；通过WB实验初步探究LINC00473调控靶基因的关键因子。.研究结果：（1）LINC00473在胃癌组织中高表达，并与淋巴结转移、脉管癌栓以及TNM分期显著正相关。（2）过表达LINC00473，胃癌细胞增殖与转移能力显著增强。LINC00473表达量降低的胃癌细胞周期受到抑制且细胞凋亡率增加。（3）沉默胃癌细胞LINC00473的表达会抑制其体内成瘤以及尾静脉肺转移能力；（4）MiR-16-5p是LINC00473的下游靶基因，CCND2是MiR-16-5p的下游靶蛋白。LINC00473在胃癌细胞质中通过竞争抑制miR-16-5p，降低对CCND2蛋白的抑制，促进胃癌细胞的增殖转移。（5）AQP3是LINC00473的另一下游靶基因，AQP3促进胃癌细胞增殖转移，干扰AQP3导致胃癌细胞增殖与转移能力的下降可以被过表达LINC00473部分逆转， CREB是LINC00473调控AQP3的关键因子。.科学意义：LINC00473在胃癌中促进增殖及转移，通过LINC00473/miR-16-5p/CCND2轴在胃癌发展中发挥作用，LINC00473通过CREB调节AQP3的表达，影响转移能力。LINC00473可成为一个潜在胃癌治疗的靶点。