Use of transcription factor sp1 in promoting expression of fbxo44 gene

Abstract

This invention relates to the field of genetic engineering technology, and particularly to the application of transcription factor SP1 in promoting FBXO44 gene expression. Addressing the problems in research on transcription factors with transcriptional regulatory functions in the promoter region of human FBXO44, this invention provides the application of transcription factor SP1 as a transcription factor in the FBXO44 promoter region. It clarifies the transcriptional regulatory role of transcription factor SP1 in FBXO44, clarifying both the direct regulatory relationship between the two and enriching the regulatory network of FBXO44, thus providing a basis for the application of FBXO44 expression regulation to the study of the regulatory mechanisms of human gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of transcription factor SP1 in promoting the expression of the FBXO44 gene. Background Technology

[0002] Gene expression in higher organisms is precisely regulated by the intracellular and extracellular environments, thus exhibiting strict temporal and spatial order. Gene expression regulation is a complex and ordered process, accomplished through multiple regulatory levels, primarily including pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational levels. Transcriptional regulation is the most critical step. The promoter is an important regulatory element at the transcriptional level, acting like a "switch." Essentially, a promoter is a DNA sequence located upstream of the 5' end of a structural gene; it does not itself regulate gene expression but interacts with numerous specific transcription factors to specifically regulate gene expression. Research on promoter function allows for a deeper understanding of the regulatory mechanisms of biological growth and development, and is of great significance for studying the expression regulation of functional genes.

[0003] FBXO44 is a member of the F-box protein family, whose members participate in various cellular regulatory mechanisms, particularly promoting protein degradation through the ubiquitin-proteasome pathway mediated by the SCF (Skp1-Cullin-F-box) complex. FBXO44 is involved in the process of tagging specific proteins for degradation, a function that can be used to develop drugs targeting protein degradation, such as PROTACs (Proteolysis Targeting Chimeras). These drugs can be designed to specifically recognize and degrade disease-related proteins. Studies have shown (Cell. 2021 Jan 21;184(2):352-369.e23.) that FBXO44 is highly expressed in various tumor cells and is positively correlated with poor prognosis. The function of FBXO44 or its complexes can activate DNA replication stress and antiviral pathways within tumor cells, thereby promoting tumor growth and enhancing immunogenicity. Another study (Nat Commun. 2023 Feb 11;14(1):778.) showed that FBXO44 was significantly upregulated in AEG (esophageal-gastric junction adenocarcinoma) and was closely associated with distant metastasis and advanced TNM stage. Higher FBXO44 expression was also associated with poorer overall survival, suggesting it may be an important therapeutic target. Furthermore, mouse models of FBXO44 overexpression showed higher tumor growth and metastasis rates, further validating its role in cancer progression. By regulating the function of FBXO44, we can control the growth and migration of cancer cells, providing new strategies for tumor immunotherapy. Therefore, identifying transcription factors that regulate the FBXO44 promoter region will provide a basis for studying the mechanisms by which FBXO44 expression regulation is applied in drug development and gene therapy. Summary of the Invention

[0004] To address the limitations in research on transcription factors with transcriptional regulatory functions in the promoter region of human FBXO44, this invention provides the application of transcription factor SP1 as a transcription factor in the FBXO44 promoter region. The transcriptional regulatory role of transcription factor SP1 in FBXO44 is clarified, defining both the direct regulatory relationship between the two and enriching the regulatory network of FBXO44, thus providing a basis for studying the expression regulation of FBXO44 in the regulatory mechanisms of human gastric cancer.

[0005] The technical solution of the present invention is as follows:

[0006] The application of transcription factor SP1 as a transcription factor in the promoter region of FBXO44, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Another object of the present invention is to protect the use of the above-mentioned transcription factor SP1 in promoting the expression of the FBXO44 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The verification process and results of this invention are as follows:

[0009] 1. Different fragments of the human FBXO44 gene promoter region were isolated and cloned, resulting in one FBXO44-WT plasmid and two FBXO44 deletion vectors. Using the JASPAR online prediction website (https: / / jaspar.elixir.no), it was discovered that the binding site of transcription factor SP1 to the FBXO44 gene is located proximal to the promoter. Therefore, human genomic DNA was extracted from human embryonic kidney cells (HEK293) as a template, and three promoter fragments of different lengths were amplified by PCR: approximately 1991–0 bp from the human FBXO44 gene promoter region, and -1991–-563 bp and -563–0 bp (counting backwards from the proximal promoter). Their nucleotide sequences are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. These fragments were then fused into the dual-luciferase reporter vector pGL3-Basic to construct three recombinant dual-luciferase reporter plasmids. Then, the plasmids were transfected into AGS cells along with the pcDNA-SP1 overexpression plasmid. After 24 hours, the intracellular dual-luciferase activity was measured using a dual-luciferase reporter system. Results are as follows: Figure 4 As shown, compared with the control group FBXO44-WT plasmid, the luciferase activity of plasmids containing -563~0bp was significantly increased, while there was no significant difference in -1991~-563bp. This experiment can determine the approximate binding region of transcription factor SP1 to the promoter of the FBXO44 gene and provides a basis for subsequent detection of the binding site of transcription factor SP1 to FBXO44.

[0010] 2. The FBXO44-WT plasmid was co-transfected into AGS cells with the overexpression vectors pcDNA-SP1 and pRL-TK-intronless plasmid, respectively. Cell lysates were collected after 24 hours, and dual-luciferase activity was measured. The results are shown in Figure 5. Compared with the control group NC (0 μg), luciferase activity was significantly increased after SP1 overexpression. Furthermore, after inhibiting SP1 transcription factor expression through siRNA knockdown and drug inhibitors, and then co-transfecting AGS cells with the FXBO44 wild-type vector and pRL-TK-intronless plasmid, the measured dual-luciferase activity was significantly reduced. Figure 6 , 7 As shown. The results of the rescue experiment for SP1 are as follows. Figure 8 As shown, this also indicates that transcription factor SP1 can promote the transcription of the FBXO44 gene, thus clarifying the transcriptional regulatory role of transcription factor SP1 in FBXO44.

[0011] 3. Using the JASPAR online prediction website (https: / / jaspar.elixir.no), three potential SP1 binding sites were identified in the promoter region of the FBXO44 gene: -563bp to -554bp, -129bp to -120bp, and -10bp to -1bp. Site-directed mutagenesis was used to truncate these three predicted binding sites, constructing three SP1 transcription binding site site-directed mutagenesis vectors (mut1, mut2, and mut3). The nucleotide sequence of mut1 is shown in SEQ ID NO.5; the nucleotide sequence of mut2 is shown in SEQ ID NO.6; and the nucleotide sequence of mut3 is shown in SEQ ID NO.7.

[0012] The constructed FBXO44-WT plasmid and three mutant plasmids (mut1, mut2, and mut3) were then co-transfected into AGS cells with the pcDNA-SP1 overexpression plasmid and the pRL-TK-intronless plasmid, respectively. Cell lysates were collected after 24 hours, and dual-luciferase activity was measured. The results, shown in Figure 9, indicate that the activities of mut1, mut2, and mut3 were significantly reduced compared to the FBXO44-WT plasmid. Furthermore... Figure 4 The results showed that SP1 can target the FBXO44 promoter region through the above three binding sites, thereby promoting the transcription of FBXO44.

[0013] In summary, the binding site is located at -563 to 0 bp upstream of the promoter of the FBXO44 gene; and the nucleotide sequence of the binding site mut1 is GGGGAGGGG; the nucleotide sequence of mut2 is CCCTCCCC; and the nucleotide sequence of mut3 is GGGGCGGCG.

[0014] The present invention has the following beneficial effects:

[0015] 1. This invention identifies the promoter region of the FBXO44 gene, providing a new promoter resource for gene therapy and the development of novel tumor-targeted drugs, with advantages such as high sensitivity and fast speed.

[0016] This invention clarifies the transcriptional regulatory role of transcription factor SP1 in FBXO44, bringing a deeper understanding to the expression regulation of FBXO44. It has good application prospects for drug development and gene therapy mechanism research. Attached Figure Description

[0017] Figure 1 The structural map of the pGL3-Basic vector containing the human FBXO44-WT promoter of 1991 bp;

[0018] Figure 2 The structural map of the internal reference pRL-TK-intronless plasmid;

[0019] Figure 3 Electrophoretic gel images of the FBXO44 WT promoter and three promoter fragments of different lengths: -1991 to -563 bp and -563 to 0 bp (counting backwards from the proximal promoter as 0).

[0020] Figure 4 The results show the relative luciferase activity of AGS cells 24 h after co-transfection with FBXO44-WT plasmid, overexpression vector pcDNA-SP1, and pRL-TK-intronless internal control plasmid. **P<0.01; ****P<0.0001;

[0021] Figure 5 To determine the relative luciferase activity of cells after 24 hours of si-RNA knockdown of SP1 followed by transfection with FBXO44 WT vector and pRL-TK-intronless internal control plasmid, ****P<0.0001;

[0022] Figure 6 AGS cells were treated with Plicamycin for 24 h and then transfected with FBXO44 WT vector and pRL-TK-intronless internal control plasmid. Relative luciferase activity was measured 24 h later. ****P<0.0001;

[0023] Figure 7 To salvage the relative decrease in luciferase caused by SP1 deficiency by using si-RNA to knock down SP1 and then co-transfecting AGS cells with FBXO44 WT vector, overexpression vector pcDNA-SP1 and pRL-TK-intronless internal control plasmid, **P<0.01;

[0024] Figure 8 The relative luciferase activity of AGS cells transfected with the three potential SP1 binding site mutant vectors mut1, mut2, and mut3, the FBXO44-WT vector, and the pRL-TK-intronless internal control plasmid for 24 hours was measured. ****P<0.0001;

[0025] Figure 9The relative luciferase activity was measured 24 h after co-transfecting AGS cells with the FBXO44-WT plasmid and three fluorescent vectors with promoters of different lengths (-1991~-563bp and -563~0bp) with the overexpression vector pcDNA-SP1 and the pRL-TK-intronless internal control plasmid, respectively. *** P<0.001;

[0026] Figure 10 To predict the top three binding sites of transcription factor SP1 on the FBXO44 promoter using the JASPAR database. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Isolation, Cloning, and Identification of the FBXO44 Gene Promoter Fragment

[0029] 1. Human genomic DNA extraction and detection

[0030] Human genomic DNA extraction was performed using an EZBioscience DNA extraction kit. The specific steps are as follows:

[0031] (1) Discard the culture medium and wash the cells with PBS (add PBS along the side wall, gently shake, and then aspirate the PBS along the side wall).

[0032] (2) Add 500 μL of lysis buffer and pipette repeatedly 30 times, blowing and scraping at the same time to fully lyse the cells. Then place the plate on a shaker at room temperature and shake for 5 min (120~180 rpm) to fully lyse the cells.

[0033] (3) Transfer the supernatant of the cell lysis product or the tissue lysis product after centrifugation (avoiding aspirating the precipitate) to the DNA spin column, centrifuge at 4000 g for 1 min, and discard the liquid.

[0034] (4) Add 500 μL Wash Buffer 1 to the centrifuge column and centrifuge at 12000 g for 1 min. Carefully remove the centrifuge column, discard the waste liquid, and use absorbent paper to clean the remaining liquid at the collection tube opening.

[0035] (5) Add 500 μL Wash Buffer 2 to the centrifuge column and centrifuge at 12000 g for 2 min. Discard the waste liquid and use absorbent paper to clean the remaining liquid at the collection tube opening.

[0036] (6) Put the centrifuge column back into the collection tube, centrifuge at 12000 g for 1 min to remove the residual waste liquid, then discard the collection tube, transfer the centrifuge column to an RNase-free 1.5 ml EP tube, open the cap and let it air dry for 2 min.

[0037] (7) Add 20~100 μL of elution buffer preheated at 70°C to the membrane in the center of the centrifuge column and incubate at 70°C for 1 min.

[0038] (8) Centrifuge at 12000 g for 1 minute, discard the centrifuge column, and use a NanoDrop 2000 DNA / RNA concentration meter to measure the DNA concentration and OD value, and store at -80℃ for later use.

[0039] (9) Take 3 μL of DNA, add 4 μL of loading buffer, mix well, and then spot onto a 1% agarose gel (EB staining solution). At the same time, spot 10 μL of standard molecular weight DL 5000 DNA Marker as a reference. Electrophoresis is performed at 15 V / cm. The results are then observed and photographed in a gel imaging system.

[0040] 2. Construction of a dual-luciferase vector with a deleted fragment from the FBXO44 gene promoter

[0041] (1) Bioinformatics analysis of the FBXO44 gene promoter

[0042] Based on the sequence information of the human FBXO44 gene (Gene ID: 93611) in the NCBI GenBank database, its transcription direction was observed to be positive. The translation start site (ATG) was then located, and the NCBI database search tool was used to extend forward 2000 bp to obtain the FBXO44 promoter sequence. The binding of the SP1 transcription factor to the FBXO44 promoter was analyzed using the JASPAR online prediction database. Based on the prediction information, the scope of primer design for deletion and mutant vectors was determined.

[0043] Promoter deletion primer design: Based on bioinformatics predictions, the scope of primer design for deletion fragments is comprehensively considered.

[0044] Based on homologous recombination, two upstream primers, FBXO44-FOR-1 (nucleotide sequence as shown in SEQ ID NO. 8) and FBXO44-FOR-2 (nucleotide sequence as shown in SEQ ID NO. 9), two downstream primers, FBXO44-R-1 (nucleotide sequence as shown in SEQ ID NO. 10) and FBXO44-R-2 (nucleotide sequence as shown in SEQ ID NO. 11), and one upstream and one downstream primer for the pGL3-Basic vector, V-FOR (nucleotide sequence as shown in SEQ ID NO. 12) and V-REV (nucleotide sequence as shown in SEQ ID NO. 13), were designed. Because homologous recombination is used to connect the fragment and the vector, an additional 15bp homologous arm is designed in the primers to allow recombination of the target fragment and the vector at the corresponding homologous sequences, thereby achieving seamless insertion of the fragment.

[0045] (3) PCR amplification promoter deletion fragment

[0046] Using the primers designed above, and with human genomic DNA as a template, three fragments of the FBXO44 promoter were amplified by PCR (product lengths were 1991bp, 1428bp, and 563bp, respectively).

[0047] PCR reaction system (50 μL):

[0048] Table 1 PCR amplification system

[0049]

[0050] Reaction program: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, 72℃ annealing for 5 seconds, 72℃ extension for 4-5 seconds / kb, 35 cycles; 72℃ for the final 1 minute, store at 4℃.

[0051] Take 50 μL of PCR amplification product, add 5 μL of Loading Buffer, mix well, and spot onto a 1% agarose gel. Use 10 μL of DNA standard molecular weight DL 5000 Marker as a reference, and perform electrophoresis at 15 V / cm. After electrophoresis, observe the results in a gel imaging system and take pictures for storage.

[0052] The target bands on the agarose gel were extracted and recovered using the GENEray agarose gel extraction kit, following the instructions in the kit's manual.

[0053] (4) Homologous recombination

[0054] The purified PCR products were analyzed using a NanoDrop 2000 DNA / RNA concentration analyzer to determine the DNA concentration and OD value, and to calculate the amount of each reagent used in the homologous recombination system. The PCR enzyme and homologous recombination enzyme were purchased from Vazyme.

[0055] Table 2 Homologous recombination reaction system

[0056]

[0057] Calculation of carrier and fragment usage:

[0058] The optimal amount of cloning vector used in the ClonExpress II recombination reaction system is 0.03 pmol, and the optimal amount of insert fragment is 0.06 pmol (vector to insert molar ratio of 1:2). The DNA mass corresponding to these molar amounts can be roughly calculated using the following formula:

[0059] The optimal amount of cloning vector used = [0.02 × number of base pairs in the cloning vector] ng (0.03 pmol)

[0060] The optimal amount of insert used = [0.04 × number of base pairs in the insert] ng (0.06 pmol)

[0061] Therefore, the volumes (X, Y) of the inserted fragment and the linearized vector in the homologous recombination system can be directly calculated using the above formula.

[0062] The reaction system was reacted at 37°C for 30 min, then cooled to 4°C and stored on ice.

[0063] (5) Transformation, sequencing and plasmid extraction

[0064] ① Under aseptic conditions, add 10 μL of the above ligation product to 50 μL of DH5α competent cells (TransGen), mix well, incubate on ice for 30 min, heat shock at 42°C for 90 sec, immediately place on ice for 3 min, then spread evenly on a plate (containing 100 mg / L Amp), add 500 µL of antibiotic-free LB medium to the competent bacteria, and incubate at 37°C with shaking for 45 min (220 rpm). Spread the transformed bacteria evenly on LB plates containing antibiotics, and incubate upside down in a 37°C incubator overnight.

[0065] ② Use a small pipette tip to pick up a single colony with normal morphology from the plate and place it in a 1.5 mL centrifuge tube containing 400 μL LB liquid medium (containing 100 mg / L Amp), and incubate at 37℃ in a shaker for 24 h.

[0066] ③ Take a portion of the bacterial culture for PCR testing. The bacterial culture containing the recombinant plasmid that is positive is sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0067] ④ Re-expand the culture of the correctly sequenced bacterial culture. Add 120 μL of bacterial culture to a 50 mL centrifuge tube containing 30 mL of liquid culture medium (containing 100 mg / L Amp) and incubate at 37 °C in a shaker for 18-24 h. Extract plasmids according to the TIANprep Mini Plasmid Kit (TIANGEN BIOTECH) instructions. Measure the DNA concentration and OD value of the extracted plasmids using a NanoDrop 2000 DNA / RNA analyzer and store at -20 °C for later use.

[0068] (6) Cell transfection

[0069] Cells were seeded into 6-well plates and transfected after approximately 24 hours of culture when the cells reached 80% confluency. 1 μg of promoter plasmid and 1 μg of pRL-TK were added to each well. Figure 2 The plasmid was diluted in 250 μL of Opti-MEM, and 4 μL of Lipofectamine 2000 was diluted in 250 μL of Opti-MEM. After 5 min, the two portions were mixed and incubated at room temperature for 20 min before being slowly and evenly added to the cell culture plate. 24 h after transfection, the cells were diluted to 1 × 10⁻⁶ with 5 × Cell Lysis Buffer, and the cell lysis buffer was collected. The cells were incubated at room temperature or shaken for 5 min to lyse. The cell lysis products were pipetted and aspirated into 1.5 ml centrifuge tubes and centrifuged at 11200 rpm (12,000 × g) at room temperature for 2 min. The supernatant was used for subsequent detection.

[0070] (7) Dual-luciferase activity assay

[0071] The relative fluorescence activity of fluorescent carriers was detected using a Dual-Reporter Assay System in a multifunctional microplate reader: 20 μL of cell lysis buffer was added to the microplate. First, 100 μL of Luciferase Substrate was added to read the activity value A of firefly luciferase in the sample. Then, 100 μL of freshly prepared Renilla substrate was added to read the activity value B of Renilla luciferase in the sample. The A / B ratio represents the activity of the fluorescent carrier. One-way ANOVA was performed on the results using Prism9 software. A p < 0.05 was considered statistically significant, and a p < 0.01 was considered highly statistically significant.

[0072] 3. Results and Analysis

[0073] Three recombinant dual-luciferase reporter vector plasmids released bands of the expected size. Figure 3 This indicates that the carrier has been successfully constructed.

[0074] Plasmids with three different promoter regions were co-transfected into AGS cells along with the overexpression vectors pcDNA-SP1 and pRL-TK-intronless internal control plasmids. Cell lysates were collected 24 hours later, and dual-luciferase activity was measured. Results are as follows: Figure 4 As shown, compared with the FBXO44 WT vector, the fluorescence activity of the vector containing the -1991 to -563 bp promoter region was significantly reduced, while the fluorescence activity of the vector containing the -563 to 0 promoter sequence was not different, indicating that the -563 to 0 sequence is more important in the entire promoter region.

[0075] Example 2: Transcriptional Regulation of FBXO44 by Transcription Factor SP1

[0076] 1. Co-transfection of FBXO44-WT plasmid with SP1 overexpression vector

[0077] AGS cells were seeded into 6-well plates and transfected after approximately 24 hours of culture when the cells reached 80% confluency. 1 μg of FBXO44-WT plasmid, 1 μg of SP1 overexpression plasmid, and 1 μg of pRL-TK plasmid were added to each well in 250 μL of opti-MEM, and 6 μL of Lipofectamine 2000 was added to the same well in 250 μL of opti-MEM. After 5 minutes, the two solutions were mixed and incubated at room temperature for 20 minutes before being slowly and evenly added to the cell culture plate. Cells were lysed 24 hours after transfection, and the cell lysate was collected to measure dual-luciferase activity.

[0078] 2. After knocking down SP1, transfect the FBXO44-WT plasmid.

[0079] Cells were seeded into 6-well plates and transfected after approximately 24 hours of culture when the cells reached 60% confluency. First, 5 μL of si-RNA-SP1 diluted in 250 μL opti-MEM and 6 μL of Lipofectamine 2000 diluted in 250 μL opti-MEM were added to each well. After 5 minutes, the two solutions were mixed and allowed to stand at room temperature for 20 minutes before being slowly and evenly added to the cell culture plate. 12 hours after transfection, 1 μg of FBXO44-WT plasmid and 1 μg of pRL-TK plasmid diluted in 250 μL opti-MEM and 4 μL of Lipofectamine 2000 diluted in 250 μL opti-MEM were added to each well. After 5 minutes, the two solutions were mixed and allowed to stand at room temperature for 20 minutes before being slowly and evenly added to the cell culture plate. After 24 hours, cells were lysed, and the cell lysates were collected to determine dual-luciferase activity.

[0080] 3. Treatment with the drug inhibitor Plicamycin

[0081] Plicamycin can competitively bind to DNA at the SP1 binding site, thereby inhibiting SP1-mediated gene transcription.

[0082] Cells were seeded into 6-well plates and cultured for about 24 hours until the cells reached 80% confluence. The medium was then changed. Plicamycin was prepared in gradient concentrations of 0 nM, 25 nM, 50 nM, 100 nM, and 200 nM with antibiotic-free medium. After culturing the cells for 24 hours, each well was transfected with 1 μg of FBXO44-WT plasmid and 1 μg of pRL-TK plasmid using Lipofectamine 2000. After 24 hours, the cells were lysed and the cell lysate was collected to measure dual-luciferase activity.

[0083] 4. SP1 rescue experiment

[0084] Cells were seeded into 6-well plates and transfected when the cells reached 80% confluency after approximately 24 hours of culture. One well was left as a control, containing only the transfection reagent. The remaining three wells were transfected with si-SP1. After 12 hours of transfection, one well was left with only the transfection reagent, and the remaining two wells were transfected with 1 μg and 2 μg of the SP1 overexpression vector, 1 μg of FBXO44-WT plasmid, and 1 μg of pRL-TK plasmid, respectively. Cells were lysed 24 hours after transfection, and the cell lysates were collected to measure dual-luciferase activity.

[0085] 5. Dual-luciferase activity assay

[0086] Discard the cell culture medium, wash twice with PBS, add an appropriate amount of 1× Cell Lysis Buffer, incubate at room temperature for 5 min for lysis, pipette and aspirate the cell lysis products into a 1.5 ml centrifuge tube, centrifuge at 11,200 rpm (12,000 × g) at room temperature for 2 min, add 20 μL of the supernatant cell lysis buffer to an ELISA plate, then add 100 µl of Luciferase Substrate equilibrated to room temperature to the ELISA plate, mix quickly and immediately.

[0087] The activity (A) of the Firefly luciferase reporter gene was detected using a microplate reader. 100 µl of freshly prepared Renilla substrate working solution was added to the above reaction solution, quickly mixed, and immediately the Renilla luciferase reporter gene activity (B) was detected using a microplate reader. The A / B ratio represents the activity of the fluorescent carrier. One-way ANOVA was performed on the results using Prism software; p < 0.05 was considered statistically significant, and p < 0.01 was considered highly statistically significant.

[0088] 3. Results and Analysis

[0089] To investigate the effect of SP1 on the transcriptional activity of FBXO44, we co-transfected the FBXO44-WT plasmid obtained in Example 1 with the overexpression vectors pcDNA-SP1 and pRL-TK plasmid for 24 hours and then measured the dual-luciferase activity. The results are as follows: Figure 5 As shown, compared with the control group NC (0 μg), the concentration gradient of SP1 increased, and the luciferase concentration also showed an increasing trend. Furthermore, the effects of directly knocking down SP1 expression and inhibiting SP1 transcription with the drug inhibitor Plicamycin on FBXO44 expression were also verified. Figure 6 , 7 As shown, different methods of inhibiting SP1 expression also resulted in a significant decrease in luciferase. To verify that SP1 specifically regulates FBXO44, we first knocked down SP1 expression with siRNA and then reintroduced it. The results are as follows. Figure 8 As shown, luciferase expression was successfully restored after inoculation. These experiments collectively demonstrate that transcription factor SP1 promotes the transcription of the FBXO44 gene, clarifying that SP1 is a transcriptional regulator of the FBXO44 gene.

[0090] Example 3: Determination of the binding site between transcription factor SP1 and FBXO44

[0091] 1. Construction of SP1 transcription binding site-directed mutagenesis vector

[0092] (1) Bioinformatics Analysis

[0093] Using the Jaspar online prediction website, three potential SP1 binding sites were found in the promoter region of the FBXO44 gene: -563bp to -554bp, -129bp to -120bp, and -10bp to -1bp. Therefore, we speculate that the transcription factor SP1 may target the promoter region of the FBXO44 gene by binding to one or more of these transcription binding sites.

[0094] (2) Construction of mutant vectors

[0095] Using the FBXO44-WT plasmid constructed in the first step as a template, nine primers were designed based on the principle of homologous recombination. The predicted three SP1 binding sites were truncated and mutated. The primer sequences used are shown in Table 4.

[0096] Table 3. List of primer nucleotide sequences

[0097]

[0098] Taking mut1 as an example, the specific steps are as follows: The mut1 (upper) fragment is amplified by conventional PCR using FBX-FOR and MUT-1-REV as primers, and then the mut1 (lower) fragment is amplified by PCR using V-MUT-FOR and FBX-REV as primers. ApexHF HS DNA polymerase premixed solution-CL from AG is used here, which has good effects on short fragments and can effectively avoid non-specific amplification.

[0099] Table 4 PCR reaction system (50 μL)

[0100]

[0101] Reaction program: 94℃ pre-denaturation for 1 min; 98℃ denaturation for 10 sec, 60℃ annealing for 5 sec, 68℃ extension for 30 sec / kb, 35 cycles; store at 4℃.

[0102] Take 50 μL of PCR amplification product, add 5 μL of Loading Buffer, mix well, and spot onto a 1% agarose gel. Use 10 μL of DNA standard molecular weight DL 5000 Marker as a reference, and perform electrophoresis at 15 V / cm. After electrophoresis, observe the results in a gel imaging system and take pictures for storage.

[0103] The target bands on the agarose gel were recovered by cutting the gel using the GENEray agarose gel recovery kit. The specific operation steps were performed according to the kit's instructions.

[0104] (3) Homologous recombination

[0105] The purified PCR products were analyzed using a NanoDrop 2000 DNA / RNA concentration meter to determine the DNA concentration and OD value, and the volume of each component in the homologous recombination system was calculated. Subsequently, fragments mut1 (top) and mut1 (bottom) were combined with the vector for homologous recombination.

[0106] Table 5 Homologous recombination reaction system

[0107]

[0108] Calculation of carrier and fragment usage:

[0109] The optimal amount of cloning vector used in the ClonExpress II recombination reaction system is 0.03 pmol, and the optimal amount of insert fragment is 0.06 pmol (vector to insert molar ratio of 1:2). The DNA mass corresponding to these molar amounts can be roughly calculated using the following formula:

[0110] The optimal amount of cloning vector used = [0.02 × number of base pairs in the cloning vector] ng (0.03 pmol)

[0111] The optimal amount of insert used = [0.04 × number of base pairs in the insert] ng (0.06 pmol)

[0112] Therefore, the volumes (X, Y, Z) of the inserted fragment and the linearized vector in the homologous recombination system can be directly calculated using the above formula. The homologous recombination reaction conditions are: 37℃ for 30 min, followed by a 4℃ hold.

[0113] (6) Transformation, sequencing and plasmid extraction

[0114] ① Under aseptic conditions, add 10 μL of the above ligation product to 50 μL of DH5α competent cells (TransGen), mix well, incubate on ice for 30 min, heat shock at 42°C for 90 sec, immediately place on ice for 3 min, then spread evenly on a plate (containing 100 mg / L Amp), add 500 µL of antibiotic-free LB medium to the competent bacteria, and incubate at 37°C with shaking for 45 min (220 rpm). Spread the transformed bacteria evenly on LB plates containing antibiotics, and incubate upside down in a 37°C incubator overnight.

[0115] ② Use a small pipette tip to pick up a single colony with normal morphology from the plate and place it in a 1.5 mL centrifuge tube containing 400 μL LB liquid medium (containing 100 mg / L Amp), and incubate at 37℃ in a shaker for 24 h.

[0116] ③ Take a portion of the bacterial culture for PCR testing. The bacterial culture containing the recombinant plasmid that is positive is sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0117] ④ Re-expand the culture of the correctly sequenced bacterial culture. Add 120 μL of bacterial culture to a 50 mL centrifuge tube containing 30 mL of liquid culture medium (containing 100 mg / L Amp) and incubate at 37 °C in a shaker for 18-24 h. Extract plasmids according to the TIANprep Mini Plasmid Kit (TIANGEN BIOTECH) instructions. Measure the DNA concentration and OD value of the extracted plasmids using a NanoDrop 2000 DNA / RNA analyzer and store at -20 °C for later use.

[0118] 2. Expression experiments of FBXO44-WT plasmid and mut (1, 2, 3) vector

[0119] AGS cells were seeded into 6-well plates and transfected after approximately 24 hours of culture when the cells reached 80% confluency. Control group 1 was transfected with 1 μg of FBXO44-WT plasmid and 1 μg of pRL-TK plasmid; control group 2 was transfected with 1 μg of FBXO44-WT plasmid, 1 μg of SP1 overexpression vector plasmid, and 1 μg of pRL-TK plasmid. Experimental groups were transfected with 1 μg of MUT (-1, -2, -3) vector plasmid, 1 μg of SP1 overexpression vector plasmid, and 1 μg of pRL-TK plasmid, respectively. Cells were lysed 24 hours after transfection, and the cell lysates were collected to measure dual-luciferase activity.

[0120] 3. Dual-luciferase activity assay

[0121] Discard the cell culture medium, wash twice with PBS, add an appropriate amount of 1× Cell Lysis Buffer, incubate at room temperature for 5 min for lysis, pipette and aspirate the cell lysis products into a 1.5 ml centrifuge tube, centrifuge at 11,200 rpm (12,000 × g) at room temperature for 2 min, add 20 μL of the supernatant cell lysis buffer to an ELISA plate, then add 100 µl of Luciferase Substrate equilibrated to room temperature to the ELISA plate, mix quickly and immediately.

[0122] The activity (A) of the Firefly luciferase reporter gene was detected using a microplate reader. 100 µl of freshly prepared Renilla substrate working solution was added to the above reaction solution, and after rapid mixing, the Renilla luciferase reporter gene activity (B) was immediately detected using a microplate reader. The A / B ratio represents the activity of the fluorescent carrier. One-way ANOVA was performed on the results using Prism9 software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly statistically significant.

[0123] 3. Results and Analysis

[0124] AGS cells were transfected with the FBXO44-WT plasmid and three mutant vector plasmids, respectively. Cell lysates were collected 24 hours later, and dual-luciferase activity was measured. Results are as follows: Figure 9 As shown, compared with the control group (Full Length) and the control group (Full Length+SP1), the fluorescence activity of the three mutant vector plasmids transfected was significantly reduced.

[0125] The above results preliminarily indicate that SP1 can target the FBXO44 promoter region by binding to the above three sites, thereby promoting the transcription of FBXO44. The binding sites are located upstream of the FBXO44 gene promoter at -563bp to -554bp, -129bp to -120bp, and -10bp to -1bp.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of transcription factor SP1 in promoting FBXO44 gene expression, characterized in that, The nucleotide sequence of the transcription factor SP1 is shown in SEQ ID NO.

1. The transcription factor SP1 targets the FBXO44 promoter region through three binding sites mut1, mut2, and mut3 to promote the transcription of the FBXO44 gene. The binding site is located at -563 to 0 bp upstream of the promoter of the FBXO44 gene; the nucleotide sequence of the binding site mut1 is GGGGAGGGG, the nucleotide sequence of mut2 is CCCTCCCC, and the nucleotide sequence of mut3 is GGGGCGGCG.

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