鼻咽癌差异表达miRNAs筛选研究

 【目的】应用miRNAs芯片筛选原代培养的鼻咽癌细胞差异表达miRNAs，并寻找差异表达miRNAs调控的靶基因，为进一步研究其在鼻咽癌发病机制中的作用打下基础。【方法】运用miRNAs芯片技术筛选原代培养的鼻咽癌细胞差异表达miRNAs，同时运用miRNAs特异的引物组对差异表达的miRNAs进行荧光定量RTPCR验证。运用靶基因预测软件并结合全基因组表达谱芯片结果预测差异表达miRNAs可能调控的靶基因，并利用westernblot技术检测预测的靶基因在原代培养的鼻咽癌细胞中的表达。【结果】miRNAs芯片结果显示，鼻咽癌中miR-203、miR-503、miR-424、miR-141和miR-148a表达下调，而miR-25、miR-195和miR-15a表达上调，其差异表达均在2倍以上；荧光定量RTPCR结果与miRNAs芯片的结果较符合；其中miR-203的预测靶基因为CCNG1和SPARC，Western blot结果显示，其在原代培养的鼻咽癌细胞亦高表达。【结论】miR-203、miR-503、miR-424、miR-141、miR-148a、miR-25、miR-195、miR-15a在原代培养的鼻咽癌细胞与正常鼻咽上皮细胞中存在差异表达；CCNG1和SPARC可能是miR-203调控的靶基因，可能参与了鼻咽癌的发生发展。

Identification of Differentially Expressed MicroRNAs in Nasopharyngeal Carcinoma

Objective] To investigate the differentially expressed miRNAs and their target genes in primary nasopharyngeal carcinoma (NPC) cells, which are useful for further studies on functions of miRNAs in pathogenesis of nasopharyngeal carcinoma. Methods] MicroRNA microarray was used to investigate the differentially expressed miRNAs in primary NPC cells, and the discovered miRNAs were confirmed by real time RT-PCR assay. Furthermore, we predicted the possible target genes by combination anticipated algorithms and the whole expression profile of genome from primary NPC cells. Western blot was performed to detect the expression of the predicted target gene in primary NPC cells. Results] MicroRNA microarray showed that expression of miR-203, miR-503, miR-424, miR-141, and miR-148a were down-regulated in primary NPC cells by two folds, while expression of miR-25, miR-195, and miR-15a were up-regulated by more than two folds too, which was accordant with the results from real time RT-PCR. CCNG1 and SPARC, the predicted target genes of miR-203, were detected to be high expressed in primary NPC cells by Western blot assay. Conclusion] MiR-203, miR-503, miR-424, miR-141, miR-148a, miR-25, miR-195, and miR-15a were differentially expressed between NPC and NPNE cells. CCNG1 and SPARC, the putative target genes of miR-203, may play an important role in the pathogenesis and development of NPC.