FoxO1 is a member of the Forkhead Box protein family. It has an important role in gluconeogenesis, cell proliferation, and energy metabolism. To obtain the target hFoxO1 gene, PCR and agarose electrophoresis were performed.
Fig.1 Agarose gel electrophoresis of PCR product. In order to obtain the target fragment, we used PrimeStar high-fidelity polymerase to amplify hFoxO1 DNA products. Then we obtained the target DNA fragments (Fig.1). Afterward, we cloned the corresponding plasmids and used DNA sanger sequencing to verify the construction.
Under the premise of having extracted the target DNA fragment, we selected the appropriate endonuclease and digested both the DNA fragments and plasmid carrier simultaneously. The correct fragments are recovered by electrophoresis. In addition, the T4 DNA ligase, viscous terminal carrier, target fragment, and buffer were all added proportionately for conversion.
Fig.2 Agarose gel electrophoresis diagram of the clone. In Fig.2A, we can see that there are obvious bands in the range of 1900-2000bp in 1-6 lines, proving that our recombinant cloning products were constructed successfully. In Fig.2B, we can see that there is no difference in the result of template and construction, which represents the success of construction. This meant that we can carry out subsequent cell transfection and characterization qualitative detection.
In order to construct a transcriptional activation platform of hFoxO1, we transfected HepG2 cells with hFoxO1 plasmids, which were verified by Western Blot.
Fig.3 Protein determination of HepG2 cells after transfection. After transfected HepG2 cells with different concentrations of hFoxO1 plasmids, the protein expression level of hFoxO1 increased accompanied by the increase of plasmid concentration, indicating that hFoxO1 was expressed successfully in the cell (Fig.3A). By comparing the expression level of internal reference proteins, we could obtain hFoxO1 plasmids with the appropriate concentration. We can construct a transcriptional activation platform and perform tests on it.
To test if the hFoxO1 transcriptional activation platform we constructed worked well, we tested it with three plasmids: pcDNA3.1-hFoxO1, pGL3-3IRE, pRL-sv40 (Fig.4A, 4B, 4C), a transcriptional regulation-hFoxO1, and a positive control-AS1842856
Fig.4 Plasmid profiles and test results In Fig.4D, “+” represents the presence of the corresponding plasmid (or transcriptional regulation) in the sample, and “-” represents the negative control. The results indicated that luciferase activity was only affected by pcDNA3.1 plasmid and hFoxO1 transcription regulator. Fig.4D also showed that hFoxO1 had a good luciferase activation effect. After the addition of different concentrations of positive control (AS1842856), we could see a gradient of luciferin activity and a concentration dependence (Fig.4E), proving that positive control has an inhibitory effect on hFoxO1 transcriptional activation. This means the hFoxO1 transcription activation platform constructed by us is successful and can be used for follow-up experiments.
After the feasibility of the hFoxO1 transcription activation platform was confirmed, the original positive control (AS1842856) was replaced by different small molecular compounds from the database to examine their effects on gluconeogenesis (Table1). Compound 335 was found to have a significant role in reducing glucose concentrations in HepG2 cells (Fig.5A).
| Compound | Inhibitory rate (%) |
|---|---|
| AS1842856 | 50.13 |
| 177 | 49.54 |
| 222 | 63.2 |
| 240 | 38.12 |
| 340 | 31.91 |
| 355 | 97.17 |
| 389 | 45.31 |
| 391 | 40.28 |
| 392 | 77.57 |
| 396 | 50.06 |
| 460 | 25.27 |
| 548 | 57.52 |
| 550 | 41.47 |
In Fig.5A, in the third column of the histogram, the luciferase activity level was still low in the presence of the hFoxO1 transcriptional activator under the action of compound 355, so it could be concluded that compound 355 has an obvious inhibitory effect on hFoxO1 transcriptional activation.
Fig.5 Plasmid profiles and test results Compound 355 was also used to inhibit both glucose concentration in HepG2 cells and mRNA abundance of key enzymes of gluconeogenesis. In Fig.5B, we can find that with the gradual increase of the concentration of compound 355, the glucose level showed a counter gradient change and was concentration-dependent, indicating that compound 355 could significantly inhibit gluconeogenesis in hepatocytes.
G6pase is an enzyme that releases glucose into the blood by hydrolyzing glucose-6-phosphate in liver tissue. PEPCK is a gluconeogenic enzyme that allows hepatic parenchymal cells to produce glucose from pyruvate derived from amino acid metabolism. In Fig. 5C and 5D, when the volume of compound 355 increases gradually, the mRNA levels of G6pase and PEPCK gradually decreased in a counter gradient and were also concentration-dependent, indicating that compound 355 could significantly reduce the mRNA levels of key enzymes in gluconeogenesis
