PURα Promotes the Transcriptional Activation of PCK2 in Oesophageal Squamous Cell Carcinoma Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. RNA Extraction, Real-Time Quantitative PCR (qRT-PCR) and RNA Sequencing (RNA-seq)
2.3. ChIP-seq and ChIP-PCR Assay
2.4. Western Blot Analysis
2.5. Plasmid Construction
2.6. RNAi Interference
2.7. Transfection and Luciferase Assay
2.8. Metabolic Phenotypes
2.9. Statistical Analysis
3. Results
3.1. Motif and Function Analysis of ChIP-seq
3.2. Screening of Potential Candidate Regulated Genes for PURα
3.3. Transcriptional Activation of thePCK2 Promoter Depends on the GGN Motif bound by PURα
3.4. Identification of the Core Binding Region of the PCK2 Gene for PURα
3.5. PURα Activates the Transcription and Expression of PCK2
3.6. PURα Promotes the Mitochondrial Respiration and Glycolysis in ECSCs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence (5’→3’) |
---|---|
Human PCK2-F | GGGCACTACCTGGAACACTG |
Human PCK2-R | GCACTGTCCTCCCCCT-CTAA |
Human PURA-F | TACGGCGTGTTTATGCGAGT |
Human PURA-R | TTGCAGAAGGTGTGTCCGAA |
Human β-actin-F | CATGTACGTTGCTATCCAGGC |
Human β-actin-R | CTCCTTAATGTCAC-GCACGAT |
Primer Name | Sequence (5’→3’) |
---|---|
PCK2-C1-F | GCTCACGCCTGTAAATTCCAG |
PCK2-C1-R | TAGCTGGAATTACAGGCGC |
PCK2-C2-F | TCCAGCTACTCTGCAGGTT |
PCK2-C2-R | TCTCTCTACCCCTGGGTCT |
PCK2-C3-F | CGTTCCTAGCTTGTTTGCCAC |
PCK2-C3-R | CCAAACCGCCAAACCCATCT |
CD11c-F | TCCATCTAAGCAAAGGGCATCA |
CD11c-R | GCCAGGGGAAGGAAGAAGATT |
Antibody | Source | Identifier | Dilution Multiple |
---|---|---|---|
PCK2-Rabbit antibody | Cell Signaling Technology | Cat# 6924S | 1:1000 |
PURA-Rabbit antibody | Abcam | Cat# ab79936 | 1:1000 |
β-actin-Mouse antibody | Boao rui jing | Cat# ab1015t | 1:5000 |
Primer Name | Sequence (5’→3’) |
---|---|
PCK2-Luc-1500-227-F | CCGCTCGAGGTGTAGGCTAGAGCCTGAG |
PCK2-Luc-1500-227-R | CCGCTCGAGGTGTAGGCTAGAGCCTGAG |
PCK2-Luc-953-107-F | CCGCTCGAGCAGAAAAGGCGAGTGGATGC |
PCK2-Luc-953-107-R | CCCAAGCTTTGGCACTAGCTTTCCTCCG |
PCK2-Luc-221-107-F | CCGCTCGAGATGCCCAACCCCCTAACTTC |
PCK2-Luc-221-107-R | CCCAAGCTTTGGCACTAGCTTTCCTCCG |
PCK2-Luc-S1-MU-F | CCCTTTATTCTTAGTGTGCAGTGAGCCGAGATCG |
PCK2-Luc-S1-MU-R | GCACACTAAGAATAAAGGGTTCAAGCAATTCTCCTGTCTCA |
pCMV6-ATF4-F | CCCAAGCTTATGACCGAAATGAGCTTCCT |
pCMV6-ATF4-R | CCGCTCGAGGGGGACCCTTTTCTTCCC |
siRNA | Sequence (5’→3’) |
---|---|
siCtrl | UUCUCCGAACGUGUCACGU |
siPURα-1 | CCACCUAUCGCAACUCCAUTT |
siPURα-2 | CCAAGUUCGGACACACCUUTT |
shNC | ACAGAAGCGATTGTTGATC |
shPURα32 | CCACCAACTGACAGTTTCTCT |
shPURα34 | AGCCGCCTTACTCTCTCCATG |
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Sun, Y.; Gao, J.; Jing, Z.; Zhao, Y.; Sun, Y.; Zhao, X. PURα Promotes the Transcriptional Activation of PCK2 in Oesophageal Squamous Cell Carcinoma Cells. Genes 2020, 11, 1301. https://doi.org/10.3390/genes11111301
Sun Y, Gao J, Jing Z, Zhao Y, Sun Y, Zhao X. PURα Promotes the Transcriptional Activation of PCK2 in Oesophageal Squamous Cell Carcinoma Cells. Genes. 2020; 11(11):1301. https://doi.org/10.3390/genes11111301
Chicago/Turabian StyleSun, Yan, Jiajia Gao, Zongpan Jing, Yan Zhao, Yulin Sun, and Xiaohang Zhao. 2020. "PURα Promotes the Transcriptional Activation of PCK2 in Oesophageal Squamous Cell Carcinoma Cells" Genes 11, no. 11: 1301. https://doi.org/10.3390/genes11111301