MiR-148b参与胃癌细胞周期调控的分子机制研究

Gastric cancer is one of the most common cancers with a heavy mortality in China. It's of great significance to find attractive biomarkers for early diagnosis and new molecular targets for gastric cancer. Recently, it has been demonstrated that miRNAs which act as tumor suppressor genes or oncogenes play a significant role in tumorigenesis. In our previous work, we have already found significant down-regulation of miR-148b in gastric cancer tissues. Moreover, subsequent experiments confirmed that miR-148b inhibited cell proliferation in gastric cancer, depending on negative regulation of the expression of cholecystokinin-B receptor (CCKBR). However,we also found overexpression of miR-148b could induce the alteration of cell cycle in gastric cancer which did not related to CCKBR. Using bioinformatics methods, microarray analysis and real-time PCR, we found significant down-regulation of CDK6 on mRNA level in cells which were overexpression of miR-148b. Conclusively, we try to demonstrate that miR-148b has an effect on cell cycle depending on negative regulation of CDK6 in gastric cancer.Furthermore, we will clarify the downstream signaling pathways in this process. Maybe our research would provide potential biomarkers and therapeutic targets for eraly diagnosis and treatment against gastric cancer.

胃癌是我国最常见的恶性肿瘤之一，预后差，寻找可用于早期诊断的基因标志，发现新的分子治疗靶点，意义重大。近年来，miRNAs作为癌基因或抑癌基因参与肿瘤发生的研究颇受关注。申请者在前期工作中率先发现miR-148b在胃癌组织中显著低表达，并通过负性调控胆囊收缩素B受体（CCKBR）表达，影响胃癌细胞增殖。然而，我们在过表达miR-148b的胃癌细胞系中发现细胞周期的改变，但沉默CCKBR的表达却不能观察到类似的细胞周期变化。进一步的芯片筛查发现CDK6 mRNA的表达水平在过表达miR-148b的胃癌细胞中明显降低，real-time PCR结果也与之一致。在此基础上，本项目拟深入探寻miR-148b参与胃癌细胞周期调控的可能分子机制，试图证明miR-148b通过负性调节CDK6的表达影响细胞周期，进一步探寻二者作用后触发的下游信号通路，将可能为胃癌的早诊或治疗提供新的靶点。

近年来，miRNAs作为癌基因或抑癌基因参与肿瘤发生的研究颇受关注。申请者在前期工作中率先发现miR-148b在胃癌组织中显著低表达，并通过负性调控CCK2R表达，影响胃癌细胞增殖。然而，我们在过表达miR-148b的胃癌细胞系中发现细胞周期的改变，但沉默CCK2R的表达却不能观察到类似的细胞周期变化。进一步的芯片筛查发现CDK6 的表达水平在过表达miR-148b的胃癌细胞中明显降低。在此基础上，本项目通过构建多个相关荧光素酶报告基因，验证了miR-148b与CDK6 mRNA的3’UTR特异性结合及结合位点；采用Western Blotting和real-time PCR技术在胃癌细胞系SGC-7901、胃癌组织及癌旁组织中分别发现miR-148b与CDK6在mRNA和蛋白水平的负性相关性，从而证明胃癌细胞中CDK6是miR-148b调控的下游靶基因之一。构建相关病毒载体，CCK-8、MTT检测及生长曲线实验结果显示miR-148b 可显著抑制胃癌细胞增殖，而CDK6可明显促进其异常增殖，裸鼠成瘤实验结果也证实了上述发现。此外，CDK6过表达后，导入miR-148b表达可逆转CDK6对细胞增殖的影响。在前期工作基础中，我们发现上调miR-148b表达后，胃癌细胞MGC-803、SGC-7901的S期细胞所占比例有所增高，G2／M 期细胞的比例减低。本项目发现，在SGC-7901细胞中，过表达CDK6的实验组细胞，均出现了S期细胞所占比例有所减少，G2／M 期细胞的比例增高。这一发现提示了miR-148b通过负性调节CDK6基因参与胃癌细胞周期的调控。最后，我们在pLXSN-miR-148b、pLXSN-control中，应用芯片筛查其lncRNA以及mRNA的表达谱。经过聚类分析、Gene Ontology分析及Pathways等分析，发现与胃癌发生、发展相关的细胞周期调控、细胞侵袭、血管生成等信号通路上存在复杂的网络调控。Western Blotting和real-time PCR技术证实miR-148b过表达后，可引起细胞周期素A1 (CCNA1)的升高。上述研究成果证明了CDK6是miR-148b参与胃癌细胞周期调控的靶基因，并找出miR-148b与CDK6作用后触发的下游信号通路，将丰富胃癌发生的理论研究，为胃癌的早期诊断或阻断治疗提供新的靶点。