Regulation and control of cinnamyl aldehyde on hyperuricemia related gene promoter region G quadruplex

Abstract

The invention belongs to the technical field of biology, and particularly relates to regulation and control of cinnamyl aldehyde on hyperuricemia related gene promoter region G quadruplex. According to the application disclosed by the invention, firstly, the capacity of cinnamyl aldehyde for regulating and controlling a G quadruplex in a hyperuricemia related gene promoter region is explored by utilizing circular dichroism, and the influence of cinnamyl aldehyde on regulation and control of a nucleic acid structure is further evaluated by utilizing a fluorospectro photometer, so that support is provided for a molecular mechanism of cinnamyl aldehyde for relieving hyperuricemia on a gene level. In conclusion, the invention discloses a brand new mechanism that cinnamyl aldehyde regulates and controls uric acid metabolism related genes through a targeted G quadruplex structure, a precise action target and a potential drug development direction are provided for treatment of hyperuricemia, and meanwhile, an established small molecule and G quadruplex interaction verification method provides technical reference for similar research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the regulation of G quadruplexes in promoter regions of hyperuricemia-related genes by cinnamaldehyde. Background Art

[0002] Recent studies have found that cinnamaldehyde, the main active ingredient in cinnamon, shows potential application value in the treatment of hyperuricemia. Studies have shown that cinnamaldehyde has anti-inflammatory, antioxidant, and uric acid-lowering effects. On the one hand, cinnamaldehyde can inhibit the activity of xanthine oxidase in the liver, reducing uric acid production. Xanthine oxidase is a key enzyme in uric acid production in the body, catalyzing the conversion of hypoxanthine and xanthine to uric acid. Inhibiting this enzyme can effectively lower uric acid levels.

[0003] On the other hand, cinnamaldehyde can also play a therapeutic role in hyperuricemia and protect liver and kidney tissue by regulating kidney-related uric acid transporters, such as upregulating the renal transporters OAT1 and ABCG2 and downregulating GLUT9 and URAT1 levels, promoting uric acid excretion and inhibiting renal tubular uric acid reabsorption. Furthermore, cinnamaldehyde can synergistically inhibit NLRP3 inflammasome activation and suppress the secretion of downstream inflammatory interleukin-1β (IL-1β) and interleukin-18 (IL-18), alleviating renal inflammatory responses and enhancing antioxidant levels.

[0004] Among them, cinnamaldehyde plays an important role in regulating the expression of uric acid transporter 1 (URAT1) and glucose transporter 9 (GLUT9), key pathogenic genes of hyperuricemia. URAT1 is primarily responsible for uric acid reabsorption, while GLUT9 is involved in uric acid transport. When both are overexpressed, uric acid accumulates in the body, causing hyperuricemia. Cinnamaldehyde may regulate gene expression through different pathways: on the one hand, the active ingredient may act directly on gene regulatory regions, inhibiting their transcription and translation processes; on the other hand, it may indirectly regulate gene expression by affecting intracellular signaling pathways. Therefore, further clarifying the pathways by which active ingredients regulate gene expression may help alleviate the symptoms of patients with hyperuricemia and reduce the frequency of symptom attacks.

[0005] This study first used circular dichroism spectroscopy to explore the ability of cinnamaldehyde to regulate G-quadruplexes in the promoter region of hyperuricemia-related genes, and further used a fluorescence spectrophotometer to evaluate the effect of cinnamaldehyde on nucleic acid structure regulation, in order to provide support for the molecular mechanism of cinnamaldehyde in alleviating hyperuricemia at the genetic level. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to propose a method for regulating the G-quadruplex in the promoter region of hyperuricemia-related genes by cinnamaldehyde.

[0007] To achieve the above objectives, on the one hand, the present invention proposes the use of a DNA sequence as shown in any one of SEQ ID NOs. 1 to 7 or SEQ ID NOs. 9 to 12 as a binding target on the URAT1 promoter in regulating URAT1 gene expression by cinnamaldehyde.

[0008] On the other hand, the present invention proposes the use of the DNA sequence shown in SEQ ID NO. 15 as a binding target on the GLUT9 promoter in regulating GLUT9 gene expression by cinnamaldehyde.

[0009] Furthermore, the present invention proposes the use of a DNA sequence as shown in any one of SEQ ID NOs. 1 to 7 or SEQ ID NOs. 9 to 12 as a binding target of cinnamaldehyde in regulating URAT1 gene expression by cinnamaldehyde.

[0010] Finally, the present invention proposes the use of the DNA sequence shown in SEQ ID NO. 15 as a binding target of cinnamaldehyde in regulating GLUT9 gene expression by cinnamaldehyde.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. This study demonstrates for the first time that cinnamaldehyde can interact with the G-quadruplex structure in the promoter regions of key hyperuricemia pathogenic genes, URAT1 and GLUT9, to alter higher-order nucleic acid conformations and thereby regulate gene expression. This discovery expands research on the molecular mechanisms of cinnamaldehyde's treatment of hyperuricemia and provides a new perspective on the pathogenesis and treatment strategies of hyperuricemia.

[0013] 2. The present invention precisely locates specific nucleic acid sequences in the promoter regions of the URAT1 and GLUT9 genes that have the potential to form G-quadruplexes. Compared with traditional treatment methods, the regulatory effect of cinnamaldehyde based on G-quadruplexes can more accurately act on key gene expression links, reduce interference with other non-related metabolic pathways, and is expected to improve the specificity and effectiveness of treatment and reduce the risk of side effects.

[0014] 3. This study demonstrates the ability of cinnamaldehyde to regulate the G-quadruplex structure of hyperuricemia-related genes, laying a solid experimental foundation for the development of novel hyperuricemia therapeutics based on cinnamaldehyde. Further efforts targeting this mechanism of action could be made to optimize the structure of cinnamaldehyde or screen for compounds with similar regulatory effects, accelerating the development of safe and effective anti-hyperuricemia drugs.

[0015] 4. This study uses a combination of circular dichroism spectroscopy and fluorescence experiments to establish an experimental system that effectively validates the interaction of small molecules with G-quadruplexes and their ability to regulate their conformation. This method, characterized by its ease of use and reliable results, is not only applicable to the study of cinnamaldehyde but also provides a valuable technical tool for studying the interactions of other small molecules with G-quadruplexes, thereby promoting methodological development in the field of gene regulation related to hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Circular dichroism spectrum of URAT1 448 (SEQ ID NO. 1) bound to cinnamaldehyde.

[0017] Figure 2 Circular dichroism spectrum of URAT1 1221 (SEQ ID NO. 2) bound to cinnamaldehyde.

[0018] Figure 3 Circular dichroism spectrum of URAT1 1226 (SEQ ID NO. 3) bound to cinnamaldehyde.

[0019] Figure 4 Circular dichroism spectrum of URAT1 1263 (SEQ ID NO. 4) bound to cinnamaldehyde.

[0020] Figure 5 Circular dichroism spectrum of URAT1 1342 (SEQ ID NO. 5) bound to cinnamaldehyde.

[0021] Figure 6 Circular dichroism spectrum of URAT1 1345 (SEQ ID NO. 6) bound to cinnamaldehyde.

[0022] Figure 7 Circular dichroism spectrum of URAT1 1350 (SEQ ID NO. 7) bound to cinnamaldehyde.

[0023] Figure 8 Circular dichroism spectrum of URAT1 1439 (SEQ ID NO. 8) bound to cinnamaldehyde.

[0024] Figure 9 Circular dichroism spectrum of URAT1 1497 (SEQ ID NO. 9) bound to cinnamaldehyde.

[0025] Figure 10 Circular dichroism spectrum of URAT1 1499 (SEQ ID NO. 10) bound to cinnamaldehyde.

[0026] Figure 11Circular dichroism spectrum of URAT1 1603 (SEQ ID NO. 11) bound to cinnamaldehyde.

[0027] Figure 12 Circular dichroism spectrum of URAT1 1638 (SEQ ID NO. 12) bound to cinnamaldehyde.

[0028] Figure 13 Circular dichroism spectrum of URAT1 1758 (SEQ ID NO. 13) bound to cinnamaldehyde.

[0029] Figure 14 Circular dichroism spectrum of GLUT9 972 (SEQ ID NO. 14) bound to cinnamaldehyde.

[0030] Figure 15 Circular dichroism spectrum of GLUT9 1625 (SEQ ID NO. 15) bound to cinnamaldehyde.

[0031] Figure 16 The fluorescence intensity changes of the system when cinnamaldehyde binds to different sequences. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] Example 1. Mining of G-quadruplex-forming potential sequences in the URAT1 and GLUT9 promoter regions

[0034] URAT1 is a recombinant protein on the cell membrane, belonging to a subfamily of otochrome acetylcholine (OAT). It plays a crucial role in regulating uric acid. The functional structure of URAT1 is quite complex, and it plays a crucial role in uric acid metabolism.

[0035] GLUT9 is a glucose facilitator transporter present on the basolateral membrane of proximal tubular epithelial cells. It is a new type of high-capacity, low-affinity uric acid transporter that plays an important role in maintaining uric acid levels in the body.

[0036] Interventional regulation of promoter regions can affect the expression of downstream genes. Therefore, to investigate whether G-quadruplex elements exist in the promoter regions of the hyperuricemia-related genes URAT1 and GLUT9, we used the G-quadruplex prediction website G4 Hunter (https: / / bioinformatics.ibp.cz / # / analyse / quadruplex) to predict the G-quadruplex structure of the promoter sequences before the transcription start site (TSS) of URAT1 and GLUT9. Fifteen nucleic acid sequences with scores greater than 0.9 were obtained, as shown in SEQ ID NOs. 1 to 15, indicating that these 15 sequences may have the potential to form high-order G-quadruplex conformations (Table 1).

[0037] Table 1 Summary of nucleic acid sequences

[0038]

[0039]

[0040] Example 2. Verification of the interaction between nucleic acid sequences and cinnamaldehyde using circular dichroism spectroscopy

[0041] Furthermore, circular dichroism spectroscopy was used to verify the ability of cinnamaldehyde to interact with the above-mentioned specific nucleic acid sequences.

[0042] The specific procedure was as follows: 20 μmol / L cinnamaldehyde and 10 μmol / L of the specific nucleic acid sequence were mixed in 20 mmol / L Tris-HCl (containing 30 mmol / L KCl) buffer (pH 7.4) to prepare a total volume of 200 μL of the test solution. The solution was scanned at 1 nm / s over a range of 220 to 320 nm using a JASCO J-1500 circular dichroism spectrometer at 20°C. Before the sample measurement, the background signal of the buffer solution was scanned separately and automatically subtracted during the subsequent sample measurement.

[0043] G-quadruplexes of different conformations have characteristic manifestations in the circular dichroism spectrum: the parallel type has a strong positive peak at about 260nm, the antiparallel type has a strong positive peak at about 290nm and a negative peak near 265nm, and the mixed type may show the peak characteristics of the former two at the same time.

[0044] Depend on Figures 1 to 15 It can be seen that all nucleic acid sequences present typical circular dichroism peaks of G-quadruplex, and different sequences have different types of G-quadruplex conformations, including parallel, antiparallel and mixed types, indicating that the alternative sequences all have G-quadruplex structures.

[0045] like Figures 1 to 7As shown in Figures 9-12 and 15, the circular dichroism spectra of SEQ ID NOs. 1-7, 9-12, and 15 mixed with cinnamaldehyde solution exhibit distinct spectral characteristics compared to the nucleic acid sequence alone. Specifically, the ellipticity of the circular dichroism spectra varies to varying degrees. This is because the intensity of the characteristic circular dichroism peak is related to the number of bases in the DNA helix. Cinnamaldehyde molecules embed themselves into the higher-order conformations formed by the corresponding folded nucleic acid sequences, altering the number of bases initially forming the conformation. This in turn causes changes in the intensity of the characteristic circular dichroism peaks, leading to varying degrees of downward or upward movement, or shifting and repositioning of the characteristic peaks of the G-quadruplex. This suggests that cinnamaldehyde has a certain influence on the order and compactness of the G-quadruplex structure formed by the nucleic acid sequences.

[0046] In summary, based on the changes in the position and intensity of the characteristic peaks of the circular dichroism spectrum, it is shown that cinnamaldehyde can cause changes in the higher-order conformation of nucleic acids by interacting with the G-quadruplex in the promoter regions of URAT1 and GLUT9.

[0047] Example 3. Verification of the interaction between nucleic acid sequences and cinnamaldehyde using fluorescence experiments

[0048] This study, combined with a small molecule dye, uses a fluorescence spectrophotometer to verify the ability of cinnamaldehyde to regulate the conformational structure of 15 nucleic acid sequences (SEQ ID NOs. 1-15) in the promoter region of hyperuricemia-related genes after they fold into G-quadruplexes. The method first induces the nucleic acid sequences to fold into G-quadruplex structures and enhances the fluorescence signal by binding to thioflavin T (ThT). If the fluorescence signal changes upon subsequent addition of an active ingredient, this indicates that the active ingredient interacts with the G-quadruplex, affecting its structural stability and causing the higher-order nucleic acid topology to become more stable or disintegrate, thereby demonstrating the potential of cinnamaldehyde to regulate gene expression.

[0049] The specific experimental operation is as follows: centrifuge the EP tube containing the freeze-dried powder of nucleic acid sequence at 10000-12000 r / min for 10-15 minutes, add ultrapure water to dilute to a concentration of 100μmol / L nucleic acid mother solution and mix well, then heat at 95℃ for 10 minutes in a PCR instrument for denaturation, and then cool to 4℃ for use. + The nucleic acid sequence was diluted to 1 μmol / L in a buffer solution (pH 7.4, containing 30 mmol / L KCl) and thoroughly mixed with a 5 μmol / L ThT solution. Subsequently, a cinnamaldehyde solution with a final concentration of 2 μmol / L was added, vortexed and immediately collected using a fluorescence spectrophotometer at an excitation wavelength of 443 nm and an emission wavelength of 492 nm. In the control sample, the cinnamaldehyde solution was replaced with an equal volume of Tris-K+ Buffer solution.

[0050] like Figure 16 As shown, when cinnamaldehyde is not introduced into the system, ThT can bind to 15 nucleic acid sequences (SEQ ID NOs. 1-15), resulting in increased autofluorescence, indicating that all 15 sequences can form G-quadruplex higher-order conformations. Similar to the circular dichroism spectroscopy results, after the addition of cinnamaldehyde solution, the fluorescence intensity of the solution systems of SEQ ID NOs. 1-7, SEQ ID NOs. 9-12, and SEQ ID NO. 15 increased or decreased to varying degrees compared to the control group, indicating that the addition of cinnamaldehyde can affect the conformational stability of the higher-order G-quadruplex structure, confirming the interaction ability of the cinnamaldehyde small molecule with SEQ ID NOs. 1-7, SEQ ID NOs. 9-12, and SEQ ID NO. 15.

[0051] In summary, the fluorescence spectrophotometer was used to verify that 15 nucleic acid sequences had the potential to form G-quadruplexes, and cinnamaldehyde had the ability to regulate the higher-order conformation of specific sequences in the promoter region of key pathogenic genes of hyperuricemia.

[0052] In summary, the combination of fluorescence experiments and circular dichroism spectroscopy experimental results revealed that cinnamaldehyde interacts with specific nucleic acid sequences in the promoter region of key pathogenic genes of hyperuricemia, and regulates the expression of genes GLUT9 and URAT1 by causing changes in higher-order nucleic acid conformations such as G-quadruplexes, thereby potentially reducing the risk of hyperuricemia to a certain extent.

Claims

1. Use of a DNA sequence as shown in any one of SEQ ID NOs. 1 to 7 or SEQ ID NOs. 9 to 12 as a binding target on the URAT1 promoter in regulating URAT1 gene expression by cinnamaldehyde.

2. Application of the DNA sequence shown in SEQ ID NO.15 as a binding target on the GLUT9 promoter in regulating GLUT9 gene expression by cinnamaldehyde.

3. Use of the DNA sequence shown in any one of SEQ ID NOs. 1 to 7 or SEQ ID NOs. 9 to 12 as a binding target for cinnamaldehyde in regulating URAT1 gene expression by cinnamaldehyde.

4. Use of the DNA sequence shown in SEQ ID NO. 15 as a binding target for cinnamaldehyde in regulating GLUT9 gene expression.

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