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目的对人miR-34a(hsa-miR-34a)下游靶基因进行预测,并构建含自噬相关基因靶结合序列的GFP报告基因载体。方法使用TargetScan Release 5.1和RNA22软件分析hsa-miR-34a可能的靶基因,并利用Gominer软件对靶基因功能进行分析。根据预测结果,合成含自噬相关基因hsa-miR-34a靶序列的寡核苷酸,以pEGFPC2为载体构建系列质粒,并检测各载体的真核表达情况。结果预测结果显示hsa-miR-34a共有2904个下游靶基因,功能涵盖生物过程、细胞组分、分子功能三大类,其中与自噬作用直接相关的靶基因有beclin 1和sestrin 2。构建含beclin 1和sestrin 2作用位点的GFP载体共5个,质粒转染Hela细胞后在荧光显微镜下可见绿色荧光。结论成功构建hsa-miR-34a下游自噬相关基因靶位点序列GFP报告基因载体,为进一步研究hsa-miR-34a对自噬的调控作用奠定基础。
Objective To predict the downstream targets of human miR-34a (hsa-miR-34a) and construct GFP reporter vectors containing the target-binding sequences of autophagy-related genes. Methods TargetScan Release 5.1 and RNA22 software were used to analyze the possible target genes of hsa-miR-34a, and the function of target genes was analyzed by Gominer software. According to the results of the prediction, oligonucleotides containing the autophagy-related gene hsa-miR-34a were synthesized, and a series of plasmids were constructed by using pEGFPC2 as a vector, and the eukaryotic expression of each vector was detected. Results The results showed that there were 2904 downstream target genes in hsa-miR-34a. The function of them included three types of biological processes, cell components and molecular functions. The target genes directly related to autophagy were beclin 1 and sestrin 2. A total of 5 GFP vectors containing the sites of beclin 1 and sestrin 2 were constructed. The plasmids transfected into Hela cells showed green fluorescence under a fluorescence microscope. Conclusion The successful construction of GFP reporter gene vector downstream of autophagy-related gene hsa-miR-34a lay the foundation for the further study on the regulation of autophagy by hsa-miR-34a.