Biotechnology Core Laboratory NIDDK, NIH, Bethesda, Maryland 20892, USA.
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biotechnol J. 2018 Feb;13(2). doi: 10.1002/biot.201700342. Epub 2017 Nov 14.
Protein expression from human embryonic kidney cells (HEK 293) is an important tool for structural and clinical studies. It is previously shown that microRNAs (small, noncoding RNAs) are effective means for improved protein expression from these cells, and by conducting a high-throughput screening of the human microRNA library, several microRNAs are identified as potential candidates for improving expression. From these, miR-22-3p is chosen for further study since it increased the expression of luciferase, two membrane proteins and a secreted fusion protein with minimal effect on the cells' growth and viability. Since each microRNA can interact with several gene targets, it is of interest to identify the repressed genes for understanding and exploring the improved expression mechanism for further implementation. Here, the authors describe a novel approach for identification of the target genes by integrating the differential gene expression analysis with information obtained from our previously conducted high-throughput siRNA screening. The identified genes were validated as being involved in improving luciferase expression by using siRNA and qRT-PCR. Repressing the target gene, HIPK1, is found to increase luciferase and GPC3 expression 3.3- and 2.2-fold, respectively.
从人胚肾细胞（HEK293）中表达蛋白质是进行结构和临床研究的重要手段。先前的研究表明，微小 RNA（小的非编码 RNA）是提高这些细胞中蛋白质表达的有效手段，通过对人微小 RNA 文库进行高通量筛选，发现几种微小 RNA 可能是提高表达的潜在候选者。其中，miR-22-3p 被选择进行进一步研究，因为它可以最小化对细胞生长和活力的影响，从而提高荧光素酶、两种膜蛋白和一种分泌融合蛋白的表达。由于每个微小 RNA 可以与几个基因靶标相互作用，因此鉴定被抑制的基因对于理解和探索提高表达的机制以进一步实施具有重要意义。在这里，作者描述了一种新的方法，通过将差异基因表达分析与之前进行的高通量 siRNA 筛选获得的信息相结合，来鉴定靶基因。通过使用 siRNA 和 qRT-PCR 验证了所鉴定的基因参与提高荧光素酶表达。抑制靶基因 HIPK1 可使荧光素酶和 GPC3 的表达分别增加 3.3 倍和 2.2 倍。
