Application of transcription factor SP1 in promotion of FBXO44 gene expression

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a transcription factor SP1 in promotion of FBXO44 gene expression. The invention provides application of a transcription factor SP1 as a transcription factor of a promoter region of FBXO44 in order to solve the problems existing in research of a transcription factor with a transcription regulation effect in the promoter region of human FBXO44. The transcription regulation effect of the transcription factor SP1 on the FBXO44 is defined, the direct regulation relation between the transcription factor SP1 and the FBXO44 is defined, the regulation network of the FBXO44 is enriched, and a basis is provided for application of expression regulation of the FBXO44 to research of a human gastric cancer regulation mechanism.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to application of transcription factor SP1 in promoting FBXO44 gene expression. Background Art

[0002] The expression of genes in higher organisms is finely regulated by the internal and external environment of the cell, and therefore has strict temporal and spatial order. The regulation of gene expression is a complex and orderly process, which is completed by multiple stages of regulation levels, which mainly include five levels: pre-transcription, transcription, post-transcription, translation, and post-translation. Among them, the regulation of the transcription level is the most critical link. The promoter is an important regulatory element at the transcription level, playing a role similar to a "switch". The promoter is essentially a DNA sequence located in the 5' upstream region of the structural gene. It does not have the function of regulating genes itself, but interacts with many specific transcription factors to specifically regulate the expression of genes. The study of promoter function enables us to have a deeper understanding of the regulatory mechanism of biological growth and development, which is of great significance for studying the expression regulation of functional genes.

[0003] FBXO44 is a member of the F-box protein family, which is involved in a variety of cellular regulatory mechanisms, especially promoting protein degradation through the ubiquitin-proteasome pathway mediated by the SCF (Skp1-Cullin-F-box) complex. FBXO44 is involved in the process of marking specific proteins for degradation, and this function can be used to develop drugs that target 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. 2021Jan 21; 184(2): 352-369.e23.) that FBXO44 is highly expressed in a variety of tumor cells and is positively correlated with poor prognosis. The function of FBXO44 or its complex can activate DNA replication pressure and antiviral pathways in tumor cells, thereby promoting tumor growth and improving immunogenicity. Another study showed that (Nat Commun. 2023Feb 11; 14(1): 778.) FBXO44 was found to be significantly upregulated in AEG (esophageal gastric junction adenocarcinoma) and was closely associated with distant metastasis and advanced TNM stage of the tumor. Higher FBXO44 expression was also associated with poor overall survival, indicating that it may be an important therapeutic target. In addition, experiments on mouse models with overexpression of FBXO44 showed higher tumor growth and metastasis rates, further verifying its role in cancer progression. We can control the growth and migration of cancer cells by regulating the function of FBXO44, which provides new strategies for tumor treatment. Therefore, identifying transcription factors that have transcriptional regulatory effects on the promoter region of FBXO44 will provide a basis for the study of the mechanism of FBXO44 expression regulation in drug development and gene therapy. Summary of the invention

[0004] In view of the problems existing in the research of transcription factors with transcriptional regulatory effects in the promoter region of human FBXO44, the present invention provides the application of transcription factor SP1 as a transcription factor in the promoter region of FBXO44. The transcriptional regulatory effect of transcription factor SP1 on FBXO44 is clarified, which not only clarifies the direct regulatory relationship between the two but also enriches the regulatory network of FBXO44, providing a basis for the study of the expression regulation of FBXO44 in the regulatory mechanism of human gastric cancer.

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

[0006] The transcription factor SP1 is used as the transcription factor of the FBXO44 promoter region, and the nucleotide sequence of the FBXO44 promoter region 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 FBXO44 gene.

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

[0009] 1. Different fragments of the promoter region of the human FBXO44 gene were isolated and cloned, and one FBXO44-WT promoter plasmid and two FBXO44 deletion fragment vectors were constructed. First, the JASPAR online prediction website (https: / / jaspar.elixir.no) was used to find that the binding sites of the transcription factor SP1 to the FBXO44 gene were all located proximal to the promoter, so we extracted human genomic DNA from human embryonic kidney cells (HEK293) as a template, and amplified the promoter region of the human FBXO44 gene by PCR, about 1991 to 0 bp, -1991 to -563 bp, -563 to 0 bp (counting backward from the proximal promoter as 0), a total of three promoter fragments of different lengths, whose nucleotide sequences are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively; these fragments were fused into the dual-luciferase reporter vector pGL3-Basic to construct three recombinant dual-luciferase reporter plasmids. Then, the pcDNA-SP1 overexpression plasmid was transfected into AGS cells, and the dual-luciferase activity in the cells was measured by dual-luciferase reporter system analysis 24 hours later. Figure 4 As shown, compared with the control group FBXO44-WT promoter plasmid, the luciferase activity of the plasmid containing -563 to 0 bp increased significantly, while there was no significant difference in -1991 to -563 bp. This experiment can determine the approximate binding region of the transcription factor SP1 to the promoter of the FBXO44 gene, and provide a basis for subsequent detection of the binding site of the transcription factor SP1 to FBXO44.

[0010] 2. The FBXO44-WT promoter plasmid was co-transfected with the overexpression vector pcDNA-SP1 and pRL-TK-intronless plasmid into AGS cells, and the cell lysate was collected after 24 hours to measure the dual luciferase activity. Figure 5 As shown in the figure, compared with the control group NC (0 μg), the luciferase activity was significantly increased after overexpression of SP1. We also inhibited the expression of SP1 transcription factor by si-RNA knockdown and drug inhibitors, and then co-transfected FXBO44 wild-type vector and pRL-TK-intronless plasmid into AGS cells. The measured dual luciferase activity was significantly reduced. The results are shown in the figure. Figure 6 , 7 The results of the rescue experiment on SP1 are shown in Figure 8As shown, it also shows that transcription factor SP1 can promote the transcription of FBXO44 gene, thus clarifying the transcriptional regulatory effect of transcription factor SP1 on FBXO44.

[0011] 3. Using the JASPAR online prediction website (https: / / jaspar.elixir.no), we found that there were three potential SP1 binding sites in the promoter region of the FBXO44 gene at -563bp~-554bp, -129bp~-120bp, and -10bp~-1bp. By using site-directed mutagenesis technology, the three predicted binding sites were truncated and mutated, and three SP1 transcription binding site site-directed mutagenesis vectors (mut1, mut2, and mut3) were constructed respectively; the nucleotide sequence of mut1 is shown in SEQ ID NO.5; the nucleotide sequence of mut2 is shown in SEQ ID NO.6; the nucleotide sequence of mut3 is shown in SEQ ID NO.7.

[0012] Then the constructed FBXO44-WT promoter plasmid and three mutant plasmids (mut1, mut2 and mut3) were co-transfected with pcDNA-SP1 overexpression plasmid and pRL-TK-intronless plasmid into AGS cells, and the cell lysate was collected after 24 hours to measure the dual luciferase activity. Fig. 9 As shown, the activities of mut1, mut2, and mut3 were significantly reduced compared with those of the FBXO44-WT promoter plasmid. Figure 4 The results showed that SP1 could target the promoter region of FBXO44 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 CCCTCCCCC; and the nucleotide sequence of mut3 is GGGGCGGCG.

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

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

[0016] The present invention clarifies the transcriptional regulatory effect of transcription factor SP1 on FBXO44, provides a deeper understanding of the expression regulation of FBXO44, and has good application prospects in drug development and research on the mechanism of gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0019] Figure 3 The electrophoresis gel identification diagram of FBXO44 WT promoter and three promoter fragments of different lengths, -1991 to -563 bp, -563 to 0 bp (counting backwards from the proximal promoter as 0);

[0020] Figure 4 The relative luciferase activity test results of AGS cells co-transfected with FBXO44-WT promoter plasmid, overexpression vector pcDNA-SP1 and pRL-TK-intronless internal reference plasmid for 24 hours, **P<0.01; ****P<0.0001;

[0021] Figure 5 After knocking down SP1 using si-RNA, the cells were transfected with FBXO44 WT vector and pRL-TK-intronless internal reference plasmid. The relative luciferase activity was detected after 24 h. ****P<0.0001;

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

[0023] Figure 7 To rescue the decrease of relative luciferase caused by SP1 deficiency after knocking down SP1 with si-RNA, AGS cells were co-transfected with FBXO44 WT vector, overexpression vector pcDNA-SP1 and pRL-TK-intronless internal reference plasmid, **P<0.01;

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

[0025] Fig. 9The relative luciferase activity test results of AGS cells after 24 hours of co-transfection of FBXO44-WT promoter plasmid and three promoter fluorescence vectors with different lengths of -1991 to -563 bp and -563 to 0 bp with overexpression vector pcDNA-SP1 and pRL-TK-intronless internal reference plasmid, ***P<0.001;

[0026] Fig.10 The top three binding sites of transcription factor SP1 on the FBXO44 promoter were predicted using the JASPAR database. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 was extracted using the 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, shake gently, and then aspirate the PBS along the side wall).

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

[0033] (3) Transfer the supernatant of the cell lysate or the above-mentioned tissue lysate after centrifugation (avoid aspirating the precipitate) to a 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 12,000 g for 1 min. Carefully remove the centrifuge column, discard the waste liquid, and use absorbent paper to absorb the remaining liquid at the collection tube.

[0035] (5) Add 500 μL Wash Buffer 2 to the centrifuge column and centrifuge at 12,000 g for 2 min. Pour away the waste liquid and use absorbent paper to absorb the remaining liquid at the collection tube.

[0036] (6) Place the spin column back into the collection tube and centrifuge at 12,000 g for 1 min to remove any residual waste liquid. Discard the collection tube and transfer the spin column to an RNase-free 1.5 ml EP tube. Open the lid 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 min, discard the centrifuge column, and use NanoDrop 2000 DNA / RNA concentration meter to measure the DNA concentration and OD value, and store at -80°C for future use.

[0039] (9) Take 3 μL of DNA, add 4 μL of loading buffer, mix well, and then spot on 1% agarose gel (EB staining solution), and spot 10 μL of standard molecular weight DL 5000 DNA marker as a reference. Perform electrophoresis at 15 V / cm, then observe the results in a gel imaging system and take photos for preservation.

[0040] 2. Construction of dual luciferase vector containing FBXO44 gene promoter deletion fragment

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

[0042] According to the sequence information of the human FBXO44 gene in the NCBI database GenBank (Gene ID: 93611), the transcription direction was observed to be positive, and then the position of the translation start site ATG was found. The NCBI database search tool was used to extend 2000bp forward to obtain the promoter sequence of FBXO44. The binding of SP1 transcription factor to the FBXO44 promoter was analyzed using the JASPAR online prediction data website ( Fig.10 ), and determine the scope of deletion and mutation vector primer design based on the predicted information.

[0043] Primer design for promoter deletion fragment: Based on the information predicted by bioinformatics, the scope of primer design for the deletion fragment is comprehensively considered.

[0044] According to the homologous recombination method, 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), FBXO44-R-2 (nucleotide sequence as shown in SEQ ID NO.11) and one upstream and downstream primer V-FOR (nucleotide sequence as shown in SEQ ID NO.12) and V-REV (nucleotide sequence as shown in SEQ ID NO.13) of the pGL3-Basic vector were designed. Since the homologous recombination method is used to connect the fragment and the vector, an additional 15bp homology arm will be designed in the primer to allow the target fragment and the vector to recombine at the corresponding homologous sequence, thereby achieving seamless insertion of the fragment.

[0045] (3) PCR amplification of the promoter deletion fragment

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

[0047] PCR reaction system (50 μL):

[0048] Table 1 PCR amplification system

[0049]

[0050] Reaction procedure: pre-denaturation at 98°C for 30 sec; denaturation at 98°C for 10 sec, annealing at 72°C for 5 sec, extension at 72°C for 4-5 sec / kb, 35 cycles; final cycle at 72°C for 1 min, and storage at 4°C.

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

[0052] The target bands shown on the agarose gel ( Figure 3 ), and the agarose gel recovery kit of GENEray was used for gel excision and recovery. The specific operation steps were carried out according to the instructions of the kit.

[0053] (4) Homologous recombination

[0054] The recovered and purified PCR products were used to measure the DNA concentration and OD value using a NanoDrop 2000 DNA / RNA concentration meter to calculate the dosage of each reagent in the homologous recombination system. Both PCR enzyme and homologous recombination enzyme were purchased from Vazyme.

[0055] Table 2 Homologous recombination reaction system

[0056]

[0057] Calculation of vector and fragment usage:

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

[0059] The most suitable amount of cloning vector to use = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0060] The most suitable amount of insert fragment = [0.04 × number of base pairs of insert fragment] 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 incubated at 37°C for 30 min, cooled to 4°C and stored on ice.

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

[0064] ① Take 10 μL of the above ligation product under sterile conditions and add it to 50 μL of DH5α competent cells (TransGen), mix well and place it in an ice bath for 30 minutes, heat shock at 42℃ for 90 seconds, immediately place it on ice for 3 minutes, then evenly spread it on a plate (containing 100 mg / L Amp), add 500 μL of LB medium without antibiotics to the competent bacteria, and shake and culture at 37℃ for 45 minutes (220rpm). Evenly spread the transformed bacteria on the LB plate containing antibiotics, place it flat in a 37℃ constant temperature incubator and invert it for overnight culture.

[0065] ② Use a small gun tip to pick up a single colony with normal morphology on the plate, place it in a 1.5 mL centrifuge tube containing 400 μL LB liquid culture medium (containing 100 mg / L Amp), and culture it in a constant temperature shaker at 37°C for 24 hours.

[0066] ③ Take part of the bacterial solution for PCR testing, and send the positive bacterial solution containing the recombinant plasmid to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0067] ④ The bacterial solution with correct sequencing was expanded again, and 120 μL of bacterial solution was added to a 50 mL centrifuge tube containing 30 mL of liquid culture medium (containing 100 mg / LAmp), and cultured in a 37°C constant temperature shaker for 18-24 hours. Plasmid extraction was performed according to the instructions of TIANprep Mini Plasmid Kit (TIANGEN BIOTECH), and the extracted plasmid was measured by NanoDrop 2000 DNA / RNA concentration meter to determine the DNA concentration and OD value, and stored at -20°C for future use.

[0068] (6) Cell transfection

[0069] Cells were seeded into 6-well plates and transfected when the cells grew to 80% after about 24 hours of culture. 1 μg of FBXO44 WT promoter plasmid ( Figure 1 ) and 1 μg pRL-TK ( Figure 2 ) plasmid was diluted in 250 μL Opti-MEM, and 4 μL Lipofectamine 2000 was diluted in 250 μL Opti-MEM. After 5 minutes, the two parts were mixed, and after standing at room temperature for 20 minutes, they were evenly and slowly added to the cell culture plate. After 24 hours of transfection, the cells were lysed by diluting to 1× with 5× Cell Lysis Buffer and collecting the cell lysate. The cells were stood at room temperature or shaken for 5 minutes, and the cell lysate was pipetted and aspirated into a 1.5 ml centrifuge tube, and centrifuged at 11200 rpm (12,000×g) for 2 minutes at room temperature. The supernatant was used for subsequent detection.

[0070] (7) Dual luciferase activity assay

[0071] The relative fluorescence activity of the fluorescent carrier was detected in a multifunctional microplate reader using the Dual-Reporter Assay System: 20 μL of cell lysate was added to the microplate, and 100 μL of Luciferase Substrate was first added to read the activity value A of the firefly luciferase in the sample, and 100 μL of 100 μl of freshly prepared Renilla substrate was added to read the activity value B of the sea renilla luciferase in the sample. The A / B ratio represents the activity of the fluorescent carrier, and the results were analyzed by one-way ANOVA using Prism9 software. The difference was significant when p<0.05, and extremely significant when p<0.01.

[0072] 3. Results and analysis

[0073] The three recombinant dual-luciferase reporter plasmids released bands of the expected size ( Figure 3 ), indicating that the vector was successfully constructed.

[0074] The plasmids of three different promoter regions were co-transfected with the overexpression vector pcDNA-SP1 and the pRL-TK-intronless internal reference plasmid into AGS cells. After 24 hours, the cell lysate was collected and the dual luciferase activity was measured. Figure 4 As shown, compared with the FBXO44 WT vector, the fluorescence activity of the vector containing the -1991 to -563bp promoter region was significantly reduced, while there was no difference in the fluorescence activity of the promoter sequence containing -563 to 0, indicating that the sequence of -563 to 0 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 promoter plasmid and SP1 overexpression vector

[0077] AGS cells were seeded into 6-well plates and transfected when the cells grew to 80% after about 24 hours of culture. 1 μg FBXO44-WT promoter plasmid, 1 μg SP1 overexpression plasmid and 1 μg pRL-TK plasmid diluted in 250 μL opti-MEM were added to each well, and 6 μL Lipofectamine 2000 was diluted in 250 μL opti-MEM. After 5 minutes, the two parts were mixed, and after standing at room temperature for 20 minutes, they were evenly and slowly added to the cell culture plate. After 24 hours of transfection, the cells were lysed and the cell lysate was collected to measure the dual luciferase activity ( Figure 5 ).

[0078] 2. Knockdown of SP1 and transfection of FBXO44-WT promoter plasmid

[0079] The cells were seeded into 6-well plates and transfected when the cells grew to 60% after about 24 hours of culture. First, 5 μL si-RNA-SP1 diluted in 250 μL opti-MEM was added to each well, and 6 μL Lipofectamine 2000 was diluted in 250 μL opti-MEM. After 5 minutes, the two parts were mixed, and they were allowed to stand at room temperature for 20 minutes before being evenly and slowly added to the cell culture plate. After 12 hours of transfection, 1 μg FBXO44-WT promoter plasmid and 1 μg pRL-TK plasmid were added to each well and diluted in 250 μL opti-MEM, and 4 μL Lipofectamine 2000 was diluted in 250 μL opti-MEM. After 5 minutes, the two parts were mixed, and they were allowed to stand at room temperature for 20 minutes before being evenly and slowly added to the cell culture plate. After 24 hours, the cells were lysed and the cell lysate was collected to measure the dual luciferase activity ( Figure 6 ).

[0080] 3. Treatment with drug inhibitor Plicamycin

[0081] Plicamycin can compete with SP1 binding sites for DNA binding and inhibit SP1-mediated gene transcription.

[0082] The cells were inoculated into 6-well plates. When the cells grew to 80% after about 24 hours of culture, the medium was changed. Plicamycin was prepared with gradient concentrations of 0nM, 25nM, 50nM, 100nM, and 200nM in the antibiotic-free medium. After culturing the cells for 24 hours, 1μg FBXO44-WT promoter plasmid and 1μg pRL-TK plasmid were transfected into each well using Lipofectamine 2000. After 24 hours, the cells were lysed and the cell lysate was collected to measure the dual luciferase activity ( Figure 7 ).

[0083] 4. SP1 rescue experiment

[0084] The cells were seeded into a 6-well plate and transfected when the cells grew to 80% after about 24 hours of culture. One well was left empty as a control and only transfection reagent was added. The remaining 3 wells were transfected with si-SP1. After 12 hours of transfection, one well was only transfected with transfection reagent as a control, and the remaining 2 wells were transfected with 1μg, 2μg of SP1 overexpression vector, 1μg FBXO44-WT promoter plasmid and 1μg pRL-TK plasmid respectively. After 24 hours of transfection, the cells were lysed and the cell lysate was collected to measure the dual luciferase activity ( Figure 8 ).

[0085] 5. Dual luciferase activity assay

[0086] Aspirate the cell culture medium, wash twice with PBS, add an appropriate amount of 1× Cell Lysis Buffer, let stand at room temperature for 5 minutes, pipette and pipette the cell lysate into a 1.5 ml centrifuge tube, centrifuge at 11,200 rpm (12,000×g) for 2 minutes at room temperature, take 20 μL of the supernatant cell lysate and add it to the ELISA plate, then add 100 μL of Luciferase Substrate balanced to room temperature to the ELISA plate, mix quickly and immediately

[0087] The Firefly luciferase reporter gene activity A was detected in an ELISA instrument. 100 μl of freshly prepared Renilla substrate working solution was added to the above reaction solution, mixed quickly, and then the Renilla luciferase reporter gene activity, i.e., the activity value B of Renilla luciferase, was detected in an ELISA instrument immediately. The A / B ratio represents the activity of the fluorescent carrier. The results were analyzed by one-way ANOVA using Prism software. The difference was significant when p<0.05, and extremely significant when p<0.01.

[0088] 3. Results and analysis

[0089] In order to detect the effect of SP1 on the transcriptional activity of FBXO44, we co-transfected the FBXO44-WT plasmid obtained in Example 1 with the overexpression vector pcDNA-SP1 and pRL-TK plasmid and measured the dual luciferase activity for 24 hours. Figure 5 As shown in the figure, compared with the control group NC (0 μg), the SP1 concentration gradient increased and the luciferase also showed a gradient increase trend. In addition, the effects of directly knocking down the expression of SP1 and inhibiting SP1 transcription with the drug inhibitor Plicamycin on the expression of FBXO44 were also verified. Figure 6 , 7 As shown in the figure, after inhibiting SP1 expression by different methods, luciferase also showed a significant decrease. In order to verify that SP1 specifically regulates FBXO44, we first knocked it down by si-RNA and then restored the expression of SP1. The results are shown in the figure. Figure 8 As shown, after complementation, luciferase successfully restored high luciferase expression. The above experiments together proved that transcription factor SP1 promoted the transcription of FBXO44 gene, and confirmed that SP1 is a transcriptional regulatory factor of gene FBXO44.

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

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

[0092] (1) Bioinformatics analysis

[0093] Using the Jaspar online prediction website, we found that there were three potential SP1 binding sites at -563bp to -554bp, -129bp to -120bp, and -10bp to -1bp in the promoter region of the FBXO44 gene ( Fig.10 ). Therefore, we speculated whether the transcription factor SP1 targets the promoter region of the FBXO44 gene by binding to one or more of the above transcription binding sites.

[0094] (2) Construction of mutation vector

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

[0096] Table 3 List of nucleotide sequences of primers

[0097]

[0098] Taking mut1 as an example, the specific steps are as follows: mut1 (upper) fragment is amplified by conventional PCR using FBX-FOR and MUT-1-REV as primers, and mut1 (lower) fragment is amplified by PCR using V-MUT-FOR and FBX-REV as primers. Here, AG's ApexHF HSDNA polymerase premix-CL is used, which has a good effect on short fragments and can effectively avoid nonspecific amplification.

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

[0100]

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

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

[0103] The target bands on the agarose gel were excised and recovered using the agarose gel recovery kit from GENEray, and the specific operation steps were carried out according to the instructions of the kit.

[0104] (3) Homologous recombination

[0105] The recovered and purified PCR products were measured using a NanoDrop 2000 DNA / RNA concentration meter to determine the DNA concentration and OD value, and the volumes of each component of the homologous recombination system were calculated. Subsequently, the fragments mut1 (upper) and mut1 (lower) were homologously recombined together with the vector.

[0106] Table 5 Homologous recombination reaction system

[0107]

[0108] Calculation of vector and fragment usage:

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

[0110] The most suitable amount of cloning vector to use = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0111] The most suitable amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0112] Therefore, the volume (X, Y, Z) of the inserted fragment and the linearized vector in the homologous recombination system can be directly calculated by the above formula. The homologous recombination reaction conditions are: 37°C for 30 minutes and 4°C for maintenance.

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

[0114] ① Take 10 μL of the above ligation product under sterile conditions and add it to 50 μL of DH5α competent cells (TransGen), mix well and place it in an ice bath for 30 minutes, heat shock at 42℃ for 90 seconds, immediately place it on ice for 3 minutes, then evenly spread it on a plate (containing 100 mg / L Amp), add 500 μL of LB medium without antibiotics to the competent bacteria, and shake and culture at 37℃ for 45 minutes (220rpm). Evenly spread the transformed bacteria on the LB plate containing antibiotics, place it flat in a 37℃ constant temperature incubator and invert it for overnight culture.

[0115] ② Use a small gun tip to pick up a single colony with normal morphology on the plate, place it in a 1.5 mL centrifuge tube containing 400 μL LB liquid culture medium (containing 100 mg / L Amp), and culture it in a constant temperature shaker at 37°C for 24 hours.

[0116] ③ Take part of the bacterial solution for PCR testing, and send the positive bacterial solution containing the recombinant plasmid to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0117] ④ The bacterial solution with correct sequencing was expanded again, and 120 μL of bacterial solution was added to a 50 mL centrifuge tube containing 30 mL of liquid culture medium (containing 100 mg / LAmp), and cultured in a 37°C constant temperature shaker for 18-24 hours. Plasmid extraction was performed according to the instructions of TIANprep Mini Plasmid Kit (TIANGEN BIOTECH), and the extracted plasmid was measured by NanoDrop 2000 DNA / RNA concentration meter to determine the DNA concentration and OD value, and stored at -20°C for future use.

[0118] 2. FBXO44-WT promoter plasmid and mut (1, 2, 3) vector expression experiment

[0119] AGS cells were seeded into 6-well plates and transfected when the cells grew to 80% after about 24 hours of culture. Control group 1 was transferred with 1μg FBXO44-WT promoter plasmid and 1μg pRL-TK plasmid, and control group 2 was transferred with 1μg FBXO44-WT promoter plasmid, 1μg SP1 overexpression vector plasmid and 1μg pRL-TK plasmid. The experimental groups were transferred with 1μg MUT (-1, -2, -3) vector plasmid, 1μg SP1 overexpression vector plasmid and 1μg pRL-TK plasmid, respectively. After 24 hours of transfection, the cells were lysed and the cell lysate was collected to measure the dual luciferase activity ( Fig. 9 ).

[0120] 3. Dual luciferase activity assay

[0121] Aspirate the cell culture medium, wash twice with PBS, add an appropriate amount of 1× Cell Lysis Buffer, let stand at room temperature for 5 minutes, pipette and pipette the cell lysate into a 1.5 ml centrifuge tube, centrifuge at 11,200 rpm (12,000×g) for 2 minutes at room temperature, take 20 μL of the supernatant cell lysate and add it to the ELISA plate, then add 100 μL of Luciferase Substrate balanced to room temperature to the ELISA plate, mix quickly and immediately

[0122] The Firefly luciferase reporter gene activity A was detected in a microplate reader. 100 μl of freshly prepared Renilla substrate working solution was added to the above reaction solution, mixed quickly, and then the Renilla luciferase reporter gene activity, i.e., the activity value B of Renilla luciferase, was detected in a microplate reader immediately. The A / B ratio represents the activity of the fluorescent carrier. The results were analyzed by one-way ANOVA using Prism9 software. The difference was significant when p<0.05, and extremely significant when p<0.01.

[0123] 3. Results and analysis

[0124] The FBXO44-WT promoter plasmid and the three mutant vector plasmids were transfected into AGS cells, and the cell lysates were collected after 24 hours to measure the dual luciferase activity. Fig. 9 As shown, compared with the control group (Full Length) and the control group (Full Length + SP1), the fluorescence activity of the cells transfected with the three mutant vector plasmids was significantly reduced.

[0125] The above results preliminarily indicate that SP1 can target the promoter region of FBXO44 by binding to the above three sites, thereby promoting the transcription of FBXO44. The binding sites are located at -563bp to -554bp, -129bp to -120bp, and -10bp to -1bp upstream of the promoter of the FBXO44 gene; and the nucleotide sequence of the binding site is shown in SEQ ID NO.18.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of transcription factor SP1 as a transcription factor in the promoter region of FBXO44, characterized in that: The nucleotide sequence of the FBXO44 promoter region is shown in SEQ ID NO.

1.

2. The use 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.

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