Dataset Information

Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen.

ABSTRACT: 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.

SUBMITTER: Inwood S

PROVIDER: S-EPMC5823020 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen.

Inwood Sarah S Buehler Eugen E Betenbaugh Michael M Lal Madhu M Shiloach Joseph J

Biotechnology journal 20171114 2

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 ...[more]

PMID: 28987030

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Project description:BackgroundPituitary tumor transforming gene (PTTG), also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293) cells that do not express PTTG and analyzed the downstream genes using microarray analysis.ResultsA total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA) and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad) or adenovirus vector expressing GFP (AdGFP). Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of < or = 0.05 and a fold change of > or =2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family.ConclusionPTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study demonstrated that PTTG may induce apoptosis by down-regulation of oncogenes such as v-Jun and v-maf and up-regulation of the histone family of genes.

Dataset Information

Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen.

Publications

Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen.

Similar Datasets

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