Establishment of a DGKθ Endogenous Promoter Luciferase Reporter HepG2 Cell Line for Studying the Transcriptional Regulation of DGKθ Gene

Linlin Shan, Dongyang Wang, Qinwen Mao, Haibin Xia*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

DGKθ protein expression levels are closely related to the development of diseases including diabetes, cancer, and neuronal disease. To investigate the transcriptional regulation of the DGKθ gene, we used CRISPR/Cas9 to generate a DGKθ endogenous promoter luciferase reporter HepG2 cell line, in which the endogenous DGKθ promoter controls the expression of the luciferase reporter gene. To test the cell line, FXR, the transcription factor for upregulating the expression of DGKθ gene, was used to validate the cell line. Furthermore, the reported agonists for the expression of DGKθ, cAMP and GW4064, the known inhibitor for DGKθ enzyme activity, R59949, and a potential regulator for DGKθ enzyme expression, EGCG (the major catechin in green tea), were applied to the reporter cell line. The results indicated that these reagents could significantly regulate the expression of reporter luciferase. Finally, four transcription factors (E2F1, c-Myc, USF1, and Bmal1) potentially binding to the DGKθ gene’s upstream promoter region were tested. DGKθ expression was upregulated by c-Myc and downregulated by E2F1, which was also confirmed in wild-type HepG2 cells. We found that the cell line’s luciferase activity was directly correlated with DGKθ endogenous promoter activity, suggesting that it is liable and sensitive for studying DGKθ transcriptional regulation. The study provides a useful tool for high-throughput drug screening for the treatment of DGKθ-involved diseases.

Original languageEnglish (US)
Pages (from-to)1344-1355
Number of pages12
JournalApplied Biochemistry and Biotechnology
Volume187
Issue number4
DOIs
StatePublished - Apr 1 2019

Keywords

  • CRISPR/Cas9
  • DGKθ
  • EGCG
  • Green tea polyphenols
  • HepG2
  • Transcriptional regulation

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

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