微小RNA-301a(miR-301a)在胃癌中的生物学功能及其调控机制的研究

Gastric cancer is featured as high incidence and poor prognosis. The mechanism of its generation and progression is largely unknown. Recently, miRNAs are found to be important in human carcinogenesis including gastric cancer. In our previously study, we found that miR-301a was highly expressed in human gastric cancer tissues which indicate its importance in gastric cancer. Hence, explore the role and mechanism of miR-301a in gastric cancer is necessary. We will first examine the biological functions of miR-301a in gastric cancer. The expression pattern and clinic-pathological correlations of miR-301a in gastric cancer tissues will be investigated. To this end, we will modulate the expression of miR-301a in gastric cancer cells via transfection of its mimics or inhibitors and analyze the effects on cell growth and metastasis. Second, we will use the predicting soft ware to find the possible target genes regulated by miR-301a. Luciferase-reporter analysis will be used to identify these target genes. The correlations of the proteins coded by target genes with miR-301a in gastric cancer cells and paired gastric cancer/normal tissues will be analyzed. Third, we will investigate the transcriptional factors that regulate the expression of miR-301a. Possible transcriptional factors will be predicted via software. The verification of transcriptional factors will be performed by using CHIP and EMSA analyses. This project explores the network centered of miR-301a and provide experimental basis for clinical application of miR-301a as a target for therapy.

胃癌发病率高，预后差，发生发展机制不清。微小RNA(microRNA, miRNA)与人类癌症的发生发展有着密切的联系，在诸多恶性肿瘤中都发现有miRNA表达异常。本课题组前期研究表明miR-301a在胃癌中呈高表达，且与胃癌的发生发展密切相关，但其作用及其调控机制还不清楚。本研究拟采用通过上调或下调miR-301a在胃癌细胞中的表达，观察其对细胞生物学行为的作用，研究其在胃癌发生发展中的调控机制。通过预测miR-301a的可能靶基因，再通过蛋白水平和构建荧光素酶报告基因载体进行验证，进一步分析其在胃癌中的作用通路。进一步由EMSA和CHIP技术寻找调控miR-301a异常表达的转录因子。本项目不仅有助于阐明miR-301a作用于肿瘤的分子机理，也为临床采用针对miR-301a的靶向调节治疗提供新的思路。

目的：我们通过微阵列技术筛选出一系列胃癌与正常对照差异表达的miRNA，而miR-301a是其中差异最显著的之一。本课题主要目的在于探讨miR-301a在胃癌组织中的表达和功能，并探索其调控机制。材料与方法：（1）应用qRT-PCR对前期microRNA微阵列筛选出的miR-301a在九株胃癌细胞株与永生化胃黏膜细胞GES-1和正常胃黏膜组织中的表达水平进行验证，检测其在51对配对的胃癌肿瘤组织与癌旁组织中的表达水平，并进一步分析胃癌组织中miR-301a表达水平与胃癌临床病理特征之间的关系；（2）采用miR-301a mimics与miR-301a逆转录病毒表达载体上调胃癌细胞株SGC-7901 中miR-301a的表达水平，以及miR-301a inhibitors下调该细胞株miR-301a的表达水平，观察其对胃癌细胞生物学行为的影响；（3）生物信息学预测靶基因，然后结合3’UTR 双荧光素酶报告系统检测, qRT-PCR和免疫印迹的方法，筛选并验证miR-301a作用的靶基因，初步明确miR-301a的发挥生物学功能的分子机制。结果：qRT-PCR检测结果显示，miR-301a在胃癌细胞株中的表达水平明显高于其在GES-1和正常胃黏膜组织中的表达水平，与microRNA微阵列中的检测结果趋势相一致。miR-301a在51例胃癌组织中的平均表达水平显著高于其配对癌旁组织，且胃癌组织miR-301a表达水平越高者，肿瘤组织分化程度越差。上调miR-301a表达水平能有效增强胃癌细胞株SGC-7901体外增殖、克隆形成、迁移侵袭及体内成瘤能力，抑制凋亡，下调miR-301a除了逆转上述生物学行为，还诱导细胞周期发生G2/M期阻滞。生物信息学分析提示转录因子RUNX3的mRNA的3’UTR含有miR-301a直接作用的种子序列，双荧光素酶报告系统检测进一步证实了该靶序列，qRT-PCR及Immunoblot证实miR-301a对RUNX3蛋白表达的调控发生在转录后水平。结论：miR-301a在胃癌细胞和组织中处于高表达状态，其表达水平与肿瘤组织分化负相关；上调miR-301a表达水平能增强胃癌细胞的恶性表型，反之亦然；miR-301a通过对其靶基因RUNX3的调控参与了胃癌的发生发展过程。