Type 2 diabetes accounts for 90% of all kinds of diabetes caused by a declining response to insulin, the victims suffer from huge side effects. And the number of cases is increasing rapidly. Moreover, it is gradually spreading among younger crowds. Therefore, we find it helpful to many people to design a drug used to prevent symptoms of type 2 diabetes.
Forkhead box protein (Fox) is a family of transcription factors. The DNA binding region of this family of proteins has a conserved winglike helix structure. There are currently 17 subfamilies in this family, of which the FoxO subfamily is the most well studied. There are four subtypes: FoxO1, FoxO3, FoxO4, and FoxO6 in mammals. FoxO1 has four domains, which are DNA-binding domain, nuclear localization domain, nuclear export sequence, and transcriptional activation domain. It binds with IRE sequence and plays a role in regulating downstream genes. FoxO1 is mainly expressed in insulin-responsive tissues, The main role of FoxO1 is to regulate downstream target genes, such as PEPCK, G6Pase, PGC1-α, and PDK-4 to promote gluconeogenesis and can regulate cell proliferation, gluconeogenesis, and energy metabolism. Thus, we intend to reduce the expression of the two enzymes by regulating the expression of FoxO1.
In order to construct a FoxO1 expression plasmid that can duplicate both in E.coli and HepG2 cells, we designed the DNA sequences of hFoxO1 to be inserted into the XhoI and KpnI sites of the pcDNA3.1 vector (Fig.1), and transfect the HepG2 cells with the recombinant plasmid and set up our experiment platform.
Fig. 1 The map of recombinant plasmid pcDNA3.1-hFoxO1
To build the plasmid, we use PCR to amplify the hFoxO1 gene from template DNA (HepG2 cell genome), and extract the target fragment (Fig.2). At the same time, we did the plasmid extraction to obtain the plasmid pcDNA3.1. The second step was double-enzyme digestion with XhoI and KpnI. The goal of digestion was to get the linearized pcDNA3.1 vector and inserted DNA fragments of hFoxO1. The third step was to ligate the inserts and linearized vector and transfer the ligation product into DH5α competent.
Fig.2 Agarose gel electrophoresis of PCR product. We send the constructed recombinant plasmid to a sequencing company for sanger sequencing. The returned sequencing comparison results showed that the plasmid was successfully constructed (Fig.3). Then we extract the recombinant plasmid from E.coli DH5α and transfect it into HepG2 cells to express hFoxO1 proteins.
Fig.3 Agarose gel electrophoresis diagram of the clone. In order to verify if hFoxO1 protein was successfully expressed in HepG2 cells which were transfected with the correct recombinant plasmid after 12 hours, we did Western blot (Fig.4). The result shows that the protein expression level of hFoxO1 increased accompanied by the increase of plasmid concentration, indicating that hFoxO1 was expressed successfully in the cell.
Fig.4 Protein determination of HepG2 cells after transfection. To confirm the activity of hFoxO1 protein to activate downstream gene expression, we chose two kinds of luciferase for detection. What’s more, we also chose AS1842856, which is a small component that can inhibit the activity of hFoxO1, as control group.
Fig.5 hFoxO1 activity verification through luciferase activity detection
As shown in Fig.5, after the addition of different concentrations of positive control (AS1842856), we could see a gradient of luciferin activity and concentration dependence, 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.
We have already collected the data and figures from our experiments. hFoxO1 plays a role in regulating downstream genes, especially in gluconeogenesis. And we find out that within a certain range, we could see a gradient of luciferin activity and concentration dependence.
After expressing hFoxO1 protein in HepG2 cells, we can easily detect the downstream gene expression through a fluorescence reporting system. By the way, when we compared these data with the positive control group, we find that the activity of the hFoxO1 is easily regulated by special small components.
Because of the great effect of the hFoxO1, we believe that if we can regulate the expression of hFoxO1 in the future and promote it in the market, it will become a great power to fight against type 2 diabetes, and it may be applied to clinical disease treatment, improve the quality of life of patients, and even reduce the number of diabetes patients.
