Establishment of a DGKθ Endogenous Promoter Luciferase Reporter HepG2 Cell Line for Studying the Transcriptional Regulation of DGKθ Gene
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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.
KeywordsCRISPR/Cas9 DGKθ Transcriptional regulation Green tea polyphenols EGCG HepG2
This work was supported by the Fundamental Research Funds for Free Exploration Funds of Graduate Programs, Shaanxi Normal University (No. 2016TS058), and research grants to H.X. from the National Natural Science Foundation of China (No. 81471772 and No. 81773265) and Key Research and Development Plan of Shaanxi Province (No. 2018SF-106).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflicts of interest.
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