ISL1-beta recombinant expression vector and application thereof in gastric cancer

Abstract

The invention discloses an ISL1-beta recombinant expression vector and application thereof in gastric cancer progression, the vector is pcDNA3.1 ISL1 beta, comprises a human ISL1-beta gene complete open reading frame (nucleotide sequence is shown as SEQ ID NO.1, 858bp), can realize high-efficiency expression under the driving of a CMV promoter, and further comprises elements such as a BGH polyA termination sequence and a Neomycin resistance gene, and the overall length of the vector is 5.4 Kb. After gastric cancer cells (SGC7901 and MGC803) are transfected by the recombinant expression vector, the expression level of ISL1-beta protein in the cells can be remarkably improved, and the effect of inhibiting the gastric cancer progress is achieved by inhibiting the proliferation of the gastric cancer cells and promoting the apoptosis of the gastric cancer cells. Meanwhile, the invention discloses a reagent and a kit containing the recombinant expression vector. The invention provides a new tool and strategy for targeted therapy of gastric cancer, and has important clinical application value.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine, specifically to an ISL1-β recombinant expression vector and its application in gastric cancer. Background Technology

[0002] Gastric cancer (GC), a common digestive tract tumor, is highly heterogeneous and often diagnosed at an advanced stage. It ranks fifth in global incidence and fourth in mortality. Similar to most malignant tumors, gastric cancer progression is caused by uncontrolled cell proliferation, invasion, and metastasis. In recent years, with the deepening of molecular biology research, molecular targeted therapy and immunotherapy are gradually becoming the focus of research in the comprehensive treatment of gastric cancer.

[0003] Insulin gene enhancer binding protein-1 (ISL1) is a pleiotropic transcription factor. Its structure includes two cysteine- and histidine-rich LIM domains that mediate protein-protein interactions; and a homeodomain that mediates protein-DNA interactions. ISL1 plays a crucial role in the development and maturation of embryonic stem cells into various tissues and organs, including the heart and neurons. In adult individuals, while ISL1 maintains high expression levels in the pancreas and motor neurons, its expression abundance is extremely low in most other tissues and organs. Recent literature reports the detection of abnormal ISL1 expression in rhabdomyosarcoma, breast cancer, and lung cancer, and ISL1 is closely related to tumorigenesis and development.

[0004] Previous studies have indicated that ISL1 can generate a second protein isoform, ISL1β, in mouse insulinomas via alternative splicing. ISL1β is preferentially expressed in insulinoma cells and is a more effective transcriptional activator of the insulin promoter, and functional differences may exist between the two isoforms of ISL1. However, the specific expression, structural variations, and functional mechanisms by which human ISL1β participates in the development and progression of gastric cancer have not been reported.

[0005] Therefore, in-depth research into the molecular mechanisms of ISL1β in gastric cancer progression and the development of new ISL1β-based targeted therapies for gastric cancer are essential to improve the efficacy and prognosis of gastric cancer patients. Summary of the Invention

[0006] To achieve the above objectives, this invention, based on previous research finding that ISL1 expression abundance is positively correlated with gastric cancer metastasis, TNM stage, and differentiation degree, and negatively correlated with patient survival rate, provides an ISL1-β recombinant expression vector and its application in gastric cancer to improve the efficacy and prognosis of gastric cancer patients. The specific technical solution is as follows:

[0007] The first objective of this invention is to provide an ISL1-β recombinant expression vector, pcDNA3.1-ISL1β, containing the complete open reading frame (ORF) of the human ISL1-β gene. The nucleotide sequence of the ORF is shown in SEQ ID NO.1, and its length is 858 bp. The recombinant expression vector uses CMV as a promoter and also contains a BGHpolyA termination sequence, a Neomycin resistance gene, a pUC origin of replication, and an SV40 origin of replication. The cloning site is BamHI / EcoRI.

[0008] Furthermore, the recombinant expression vector has the prokaryotic resistance of Ampicillin and the eukaryotic resistance of Neomycin, and the vector is 5.4 kb in length.

[0009] A second objective of this invention is to provide the application of an ISL1-β recombinant expression vector in the preparation of a drug for inhibiting the progression of gastric cancer.

[0010] Furthermore, the drug, by transfecting gastric cancer cells, efficiently expresses the ISL1-β protein, thereby inhibiting the proliferation of gastric cancer cells and promoting apoptosis.

[0011] Furthermore, the gastric cancer cells are SGC7901 cells or MGC803 cells.

[0012] A third objective of this invention is to provide a reagent for inhibiting the progression of gastric cancer, the reagent comprising an ISL1-β recombinant expression vector.

[0013] A fourth object of the present invention is to provide a kit comprising an ISL1-β recombinant expression vector and primers, antibodies or reagents for detecting ISL1-β gene or protein expression.

[0014] Furthermore, the primer sequences for detecting the ISL1-β gene include:

[0015] Forward primer 5'-TCTGTTCGTAGGAATGCCCC-3'

[0016] Reverse primer 5'-GATCCCGTACAACCAAGGGA-3'.

[0017] Compared with existing technologies, the beneficial effects of this invention are as follows: The recombinant vector pcDNA3.1 ISL1β described in this invention, through efficient expression of ISL1-β protein, acts on gastric cancer cells and significantly inhibits gastric cancer progression. Experiments have shown that after transfection of MGC803 and SGC7901 gastric cancer cells with this vector, cell proliferation can be significantly inhibited, and the CCK-8 assay shows a significant decrease in cell proliferation; secondly, colony formation assays confirm that this vector has the ability to inhibit the colony formation of gastric cancer cells; finally, flow cytometry experiments show that ISL1-β protein can promote apoptosis of gastric cancer cells, demonstrating an inhibitory effect on gastric cancer progression. Attached Figure Description

[0018] Figure 1 The relative expression levels of ISL1-β compared to ISL1 in MGC803 and SGC7901 cells;

[0019] Figure 2 Western blot was used to detect the protein expression level of ISL1β in gastric cancer cells MGC803 and SGC7901 transfected with the corresponding plasmids, where:

[0020] A represents the protein expression level of ISL1β in MGC803.

[0021] B. Protein expression level of ISL1β in SGC7901

[0022] Figure 3 ISL1β inhibits the proliferation of gastric cancer cells, including:

[0023] A. CCK-8 cell proliferation-toxicity assay to detect cell viability of gastric cancer cells MGC803 and SGC7901 transfected with plasmid (NC) and overexpression group of ISL1β (OE-ISL1β) (left), statistical analysis (right), (means ±s, n = 3, *P < 0.05 vs. NC);

[0024] B. Colony formation assay to detect the colony formation ability of MGC803 and SGC7901 cells after transient transfection with control plasmid and overexpression of ISL1β plasmid.

[0025] Figure 4 Results of ISL1β promoting apoptosis in gastric cancer cells, including:

[0026] A. Scatter plot (left) and statistical analysis plot (right) of apoptosis in the SGC7901 gastric cancer cell line NC group and OE-ISL1β group detected by flow cytometry.

[0027] B. Flow cytometry analysis of apoptosis in the NC and OE-ISL1β groups of MGC803 gastric cancer cell lines (left) and statistical analysis (right), (means ±s, n = 3, ***P < 0.001 vs. NC).

[0028] Figure 5 This is a map of the ISL1β primer sequence and the ISL1-β overexpression plasmid. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0030] Example 1: Construction and identification of the ISL1-β recombinant expression vector pcDNA3.1-ISL1β

[0031] I. Construction of the recombinant expression vector pcDNA3.1-ISL1β

[0032] Using cDNA containing the complete open reading frame (ORF, 858 bp, coding sequence in SEQ ID NO.1) of human ISL1-β as a template, the target fragment was obtained by PCR amplification. The ISL1-β ORF was inserted into the multiple cloning site (MCS) of the eukaryotic expression vector pcDNA3.1 using restriction endonuclease double digestion or homologous recombination. The vector's built-in CMV promoter drives efficient ISL1-β expression, the BGH polyA termination sequence ensures transcription termination, the Neomycin resistance gene is used for eukaryotic cell selection, and the pUC and SV40 replication origins support vector replication in *E. coli* and eukaryotic cells, respectively. The recombinant vector pcDNA3.1-ISL1β was finally constructed. The specific construction method is as follows:

[0033] 1. Primer Design and Target Gene Amplification: Based on the coding region sequence of the human ISL1-β gene (SEQ ID NO.1, full length 858 bp), the DNA fragment was obtained using whole-gene synthesis or RT-PCR technology. To adapt to the pcDNA3.1(+) vector, a BamHI restriction site (GGATCC) and a Kozak sequence (GCCACC) were introduced at the 5' end of the fragment, and an EcoRI restriction site (GAATTC) and protective bases were introduced at the 3' end. The synthesized / amplified products were sequenced to ensure complete identity with the SEQ ID NO.1 sequence.

[0034] 2. Vector digestion: Take appropriate amounts of the empty vector pcDNA3.1(+) and the ISL1-β target fragment prepared in step 1, respectively. Add BamHI and EcoRI restriction endonucleases and their corresponding buffers to a sterile system, and digest at 37°C for 2-3 hours. Separate the digestion products by 1% agarose gel electrophoresis. Extract and recover approximately 5.4 kb of the linearized vector backbone and approximately 870 bp of the target gene fragment with sticky ends, respectively. Use a DNA purification kit to recover and determine the concentration for later use.

[0035] 3. Ligation and Transformation: The purified and recovered linearized pcDNA3.1(+) vector and the enzyme-digested ISL1-β fragment were mixed at a molar ratio of 1:3, T4 DNA ligase and buffer were added, and ligation was carried out overnight at 16℃ to construct the recombinant plasmid pcDNA3.1-ISL1β. Subsequently, the ligation product was transformed into E. coli DH5α competent cells, and after heat shock treatment at 42℃ and recovery culture, it was plated on LB agar plates containing 100 μg / mL ampicillin and cultured upside down overnight at 37℃ for antibiotic resistance selection.

[0036] 4. Screening and Identification of Positive Clones: Single colonies were randomly selected from the plates, expanded, and plasmids were extracted for double identification. First, double digestion with BamHI and EcoRI was performed. Electrophoresis showed that clones yielding approximately 5.4 kb of vector and approximately 858 bp of target bands were considered positive. Subsequently, Sanger sequencing was performed on the positive clones using universal primers (T7 Promoter and BGH Reverse). The sequencing results were completely identical to SEQ ID NO.1 and showed no mutations, confirming the successful construction of the recombinant expression vector.

[0037] 5. Endotoxin Removal and Plasmid Preparation: Positive clones verified by sequencing were cultured in large quantities, and high-purity recombinant plasmids were extracted using an endotoxin-removed plasmid extraction kit. After the plasmid concentration and purity (A260 / A280≈1.8) were verified to be acceptable, the plasmids were stored at -20℃ for subsequent transfection and functional experiments targeting SGC7901 and MGC803 gastric cancer cells.

[0038] Table 1. Specific primer sequences for ISL1α, ISL1β, and the internal reference gene GAPDH

[0039]

[0040] Example 2: Application of recombinant expression vector pcDNA3.1-ISL1β in gastric cancer

[0041] Gastric cancer drug screening was conducted using MGC803 and SGC7901 cells. Combining the characteristics of "low differentiation (high invasion / drug resistance) vs. moderate differentiation (high sensitivity)" of these two cells, a process of "in vitro cell-level screening → functional verification → preliminary mechanism exploration" was used to rapidly evaluate the antitumor activity, specificity, and applicable scenarios of the drugs. The specific methods are as follows:

[0042] I. Pre-experimental preparation: Cell and drug pretreatment

[0043] 1. Cell Culture

[0044] SGC7901 and MGC803 cell lines were placed in a constant temperature incubator at 37°C and 5% CO2 and cultured using DMEM complete medium containing 10% fetal bovine serum and 100 U / ml penicillin and streptomycin. When the cells reached 80%-90% confluence, they were passaged or seeded by trypsin digestion.

[0045] 2. Cell transfection

[0046] (1) 10cm×10cm culture dishes: When the cell confluence reaches 70-80%, replace the culture medium with 15mL DMEM medium containing serum-free and penicillin-streptomycin antibiotics 2 hours in advance. Then, perform transient plasmid transfection using Lipofectamine 3000. The transfection system for each culture dish is as follows: 3μg plasmid is added to 15μl of infection enhancement solution p3000 and diluted with 375μL Opti-MEM; 15μl of Lipo3000 is diluted with 375μL Opti-MEM. After standing for 10 minutes, gently mix the diluted DNA solution and the diluted Lipo3000 solution, and after standing for another 15 minutes, evenly drop the mixture into the culture medium. After 6 hours, replace the original culture medium with complete culture medium containing nutrient serum and antibiotics to continue cell culture. After 48 hours, samples can be collected to detect the corresponding mRNA and protein expression.

[0047] 3. Total RNA extraction and real-time quantitative PCR

[0048] (1) 48 h after cell transfection, the original culture medium was discarded, the cells were washed twice with cold PBS solution, 1 ml of Trizol was added, the cells were mixed by pipetting for 1 minute and collected in 1.5 ml EP tubes and allowed to stand for 5 minutes.

[0049] (2) Add 200 μl of chloroform to the centrifuge tube, invert and mix until the solution turns milky white, then let stand for 5 minutes. Centrifuge at 4 ºC and 16000 g for 15 minutes.

[0050] (3) Take the supernatant and add an equal volume of isopropanol into a new EP tube. After standing for 10 minutes, perform the operation at 4 ºC, 16000 g, for 15 minutes.

[0051] (4) Discard the supernatant and mix with 1 ml of 75% ethanol to suspend the precipitate. Centrifuge at 16000 g for 10 minutes at 4 ºC.

[0052] (5) Repeat step 4.

[0053] (6) Thoroughly remove the supernatant, open the lid and dry until the liquid has completely evaporated.

[0054] (7) Add an appropriate amount of sterile, enzyme-free water and determine its concentration and purity.

[0055] (8) Generate the first strand of mRNA cDNA using the cDNA reverse transcription kit.

[0056] (9) After adding the FastStart Universal SYBR Green Master (ROX) reaction system, perform RT-PCR on a real-time quantitative PCR instrument. The reaction conditions are: 50 ºC for 2 minutes, 95 ºC for 10 minutes, 95 ºC for 15 seconds, 60 ºC for 1 minute, for 40 cycles. The reaction system is as follows:

[0057] Reagents Volume

[0058] ROX 12.5 μl

[0059] Forward Primer (10 μM) 0.25 μl

[0060] Reverse Primer (10 μM) 0.25 μl

[0061] mRNA and first-strand cDNA (25 ng) 2.5 μl

[0062] 9.5 μl of distilled water

[0063] (10) Statistical Analysis: Each sample was tested in triplicate. The Ct values ​​of each replicate were measured and averaged. The 2-ΔΔCt value was then calculated to compare gene expression differences between the control and treatment groups. All experiments were repeated three times. GraphPad Prism software was used for data analysis and bar chart plotting. Independent samples t-tests were performed using SPSS 23.0 software. The calculation formulas are as follows:

[0064] △Ct=Ct(target gene in control group / experimental group)-Ct(internal reference gene in control group / experimental group)

[0065] △△Ct=△Ct(experimental group)-△Ct(control group)

[0066] The relative expression levels of two isoforms of ISL1 (ISL1β and ISL1α) in two gastric cancer cell lines (MGC803 and SGC7901) were experimentally detected and compared. The results are as follows: Figure 1 As shown, the mRNA expression level of ISL1α was significantly higher than that of ISL1β in MGC803 and SGC7901 cells (the expression level of ISL1β was close to 0 in MGC803 cells).

[0067] The significantly higher mRNA expression levels of ISL1α in MGC803 and SGC7901 cells compared to ISL1β indicate that ISL1β is expressed at low or almost no levels in these two cell types. In untreated gastric cancer cells, ISL1 is primarily expressed in the α isoform, with extremely low β isoform expression. This provides an experimental basis for further investigation into the function of ISL1β through recombinant vector overexpression—that is, the endogenous ISL1β content in gastric cancer cells is insufficient to exert a significant effect, necessitating exogenous introduction to observe its impact on the biological behavior of gastric cancer cells.

[0068] 4. Western blot (WB) of proteins

[0069] (1) 48 h post-transfection, total protein extraction: Collect cells, centrifuge at 111 g for 5 minutes, discard the supernatant, resuspend cells in 1 ml cold PBS, centrifuge again to precipitate cells, and discard the supernatant. Add 100 µl of high-efficiency RIPA lysis buffer, 1 µl of PMSF and 1 µl of protease inhibitor solution to each tube, and mix by pipetting. Lyse on ice for 30 minutes. Centrifuge at 16000 g for 15 minutes at 4 ºC, and collect the supernatant as the total protein solution.

[0070] (2) Determination of total protein concentration: The BCA protein assay kit was used for sample pretreatment, and then the OD value was measured at a wavelength of 562 nm using an ELISA reader. The accurate concentration of the measured protein solution was calculated by comparing the measured absorbance value with a pre-established standard curve.

[0071] (3) Gel preparation: Install and fix the glass plate in the vertical electrophoresis plate, prepare the separating gel, pour the gel and press the gel with double distilled water in time. After the separating gel solidifies, use filter paper to absorb the double distilled water; pour the compression gel again, insert the comb in time and wait for it to solidify for later use.

[0072] (4) Sample processing: Mix the total protein solution of the sample with 5× SDS-PAGE protein loading buffer at a ratio of 4:1, and boil at 100 °C for 5 minutes to denature the protein.

[0073] (5) Sample loading: Place the electrophoresis plate into the electrophoresis tank and add enough electrophoresis buffer. Add 16 μg of protein sample and 3 μl of protein pre-stained marker per well.

[0074] (6) Electrophoresis: First, use 80 V constant voltage for 30 minutes for electrophoresis, then adjust the voltage to 120 V and electrophoresis for 60 minutes until the bromophenol blue reaches the bottom of the separating gel, then stop the electrophoresis.

[0075] (7) Electrotransfer: Prepare the PVDF membrane by soaking it in methanol solution for 10 minutes. Place a sponge pad and four filter papers on the top and bottom of the transfer clamp, and wet the system with transfer buffer. Then place the PVDF membrane on the white side of the transfer clamp, place the electrophoretically deposited gel on the black side, and fix the device. Place the transfer clamp in the electrotransfer tank, inject the electrotransfer buffer, place an ice box, and place the electrotransfer tank on ice. Electrotransfer at 200 mA for 2 hours.

[0076] (8) Sealing: Place the PVDF membrane in 5% skim milk and seal at room temperature for 1 hour.

[0077] (9) Primary antibody incubation: Remove 5% of the skim milk, wash the membrane three times with 1×TBST solution for 5 minutes each time. Then add the primary antibody and incubate overnight at 4 ℃.

[0078] (10) Secondary antibody incubation: The primary antibody was recovered into a centrifuge tube and returned to 4 °C ice. The membrane was then washed three times with 1×TBST solution for 10 minutes each time. The corresponding species of secondary antibody was then added and the membrane was gently incubated on a shaker at room temperature for 1 hour.

[0079] (11) Chemiluminescence: Remove the secondary antibody, wash the membrane 6 times with 1×TBST solution, 5 minutes each time. Mix chemiluminescent agent A and B in a 1:1 ratio, then immerse the membrane in the mixture and place it in a dark room for exposure.

[0080] The results are as follows Figure 2 As shown, in the WT and pcDNA3.1 groups, the ISL1β band in MGC803 / SGC7901 cells was extremely weak (low background expression, consistent with previous RT-qPCR results); in the pcDNA3.1-ISL1β group, the ISL1β band was significantly brighter, indicating that the ISL1β protein was successfully overexpressed in both cell types.

[0081] Changes in ISL1α expression: There was no significant difference in the band brightness of ISL1α among the groups, indicating that overexpression of ISL1β does not affect the protein expression of ISL1α.

[0082] Internal control validation: The α-tubulin bands in each group showed consistent brightness, indicating that the protein loading was uniform and the experimental results were reliable.

[0083] Key findings: The pcDNA3.1-ISL1β recombinant vector can effectively overexpress ISL1β protein in gastric cancer cells (MGC803 and SGC7901 cells), providing a reliable cell model for subsequent research on the function of ISL1β.

[0084] 5. Quality Control: Before the experiment, cell morphology was observed under a microscope (MGC803 cells were spindle-shaped and loosely adhered to the cell wall; SGC7901 cells were polygonal and tightly adhered to the cell wall) to rule out contamination; then, the cell proliferation curve was detected using a CCK-8 cell proliferation assay. The specific procedures are as follows:

[0085] (1) Stable cell lines NC-MGC803, OE-ISL1β-MGC803, NC-SGC7901 and OE-ISL1β-SGC7901 were seeded in 96-well plates.

[0086] (2) Absorbance measurement: 10 μL of CCK-8 solution was injected into each sample well and incubated in a cell culture incubator for 1 hour. Then, the absorbance of the cells was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance measured 24 hours after cell seeding with CCK-8 reagent solution was the absorbance value at 0 hours. The absorbance was then measured every 24 hours for 4 consecutive days, for a total of 96 hours.

[0087] The results are as follows Figure 3 As shown in Figure A, the CCK-8 experiment revealed that for SGC7901 cells, the absorbance values ​​(OD450nm) of the experimental group at 24h, 48h, 72h, and 96h were significantly lower than those of the control group, and the difference between the two groups gradually increased with the extension of culture time (e.g., at 96h, the absorbance value of the experimental group was only 60%-70% of that of the control group). For MGC803 cells, the absorbance values ​​of the experimental group at each time point were also significantly lower than those of the control group, and the proliferation inhibition trend was consistent with that of SGC7901 cells. Therefore, it can be concluded that, for both SGC7901 and MGC803 cells, the rate of absorbance increase in the OE-ISL1β group was significantly slower than that in the NC group (lower curve slope); indicating that overexpression of ISL1β can inhibit the short-term proliferation ability of gastric cancer cells (P<0.05, * in the figure indicates significant difference).

[0088] 6. Cloning experiments

[0089] (1) MGC803 cells and SGC7901 cells that have been stably overexpressed with ISL1β and their controls or transfected with the corresponding plasmids were digested with 0.25% trypsin, centrifuged at 1000 rpm for 4 min, and collected in centrifuge tubes. Cells were counted from the cell suspensions resuspended in complete culture medium to obtain the corresponding cell concentrations.

[0090] (2) Add 2 ml of complete culture medium to each well of a 6-well plate, then add the cell suspension mentioned above (800 cells per well for MGC803 cells; and 1000 cells per well for SGC7901 cells). Gently pipette to mix the cells so that they are evenly distributed at the bottom of the plate. Incubate the cells in a 37ºC, 5% CO2 incubator for about 2 weeks, until white cell clumps are visible to the naked eye, then terminate the cell culture.

[0091] See results Figure 3 B: For SGC7901 cells (1000 cells per well): After 2 weeks of culture, the number of visible white clonal clusters in the experimental group was only 40%-50% of that in the control group, and the clonal volume was smaller. For MGC803 cells (800 cells per well): The number of clones formed in the experimental group was approximately 30%-40% of that in the control group, and the clonal density was significantly reduced. Therefore, it can be seen that the number of clones in the OE-ISL1β group was much less than that in the NC group in both SGC7901 and MGC803 cells; this indicates that overexpression of ISL1β can significantly weaken the long-term proliferation / clonal formation ability of gastric cancer cells (i.e., long-term proliferative potential), and its inhibitory effect on gastric cancer cell proliferation was verified at the cell population level.

[0092] The results of CCK-8 and plate colony experiments show that ISL1β plays a "tumor-suppressing role" in gastric cancer cells—overexpression of ISL1β can simultaneously inhibit the short-term proliferation rate and long-term colony formation ability of cells.

[0093] 7. Flow cytometry detection of apoptosis

[0094] (1) Collect cells, centrifuge at 111 g for 5 minutes, discard the supernatant, add 1 ml of cold PBS, resuspend the cells, centrifuge again to precipitate the cells, and discard the supernatant.

[0095] (2) Add 400 µl of 1×Buffer to the test tube and mix well by blowing.

[0096] (3) Divide the cell suspension into four equal parts, each containing 100 µl of cell suspension, and set them as blank tubes, single-stain tubes, and detection tubes respectively.

[0097] (4) Mix the cells in the corresponding groups of SGC7901 and MGC803 thoroughly, wrap them in tin foil to protect them from light, and incubate them at room temperature for about 15 minutes.

[0098] (5) Add 400 µl of 1×Buffer to each test tube.

[0099] (6) Detection on the machine: After adjusting the voltage compensation, apoptosis flow cytometry data were obtained. The detected apoptosis rate was the value of the Q2+Q3 region in the cell flow cytometry apoptosis scatter plot, and the apoptosis rate of gastric cancer cells was obtained (means ±s, n = 3, ***P < 0.001 vs. NC).

[0100] (7) Each experiment was repeated 3 times. The data were analyzed and bar charts were drawn using GraphPad Prism software. Independent samples t-test analysis was performed using SPSS 23.0 statistical software.

[0101] Table 2. Experimental results of ISL1β promoting apoptosis in gastric cancer cells.

[0102] SGC7901 (Q2+ Q3) MGC803 (Q2+ Q3) Control group (NC) 9.89% 5.4% Experimental group (OE-ISL1β) 16.3% 12.45%

[0103] The results are as follows Figure 4 The experimental results showed that ISL1β promoted apoptosis in gastric cancer cells, specifically SGC7901 cells ( Figure 4 A) A significant increase in apoptosis rate: The apoptosis rate in the control group (NC) was approximately 9.89% (Q2=7.73%, Q3=1.98%; Q2+Q3=9.89%), while the apoptosis rate in the experimental group (OE-ISL1β) significantly increased to 16.3% (Q2=7.72%, Q3=8.54%; Q2+Q3=16.3%), and the difference was statistically significant (*P<0.05).

[0104] MGC803 cells ( Figure 4 B): The apoptosis rate in the control group was only 5.4% (Q2=4.72%, Q3=0.68%; Q2+Q3=5.4%), while the apoptosis rate in the experimental group increased significantly to 12.45% (Q2=5.33%, Q3=7.12%; Q2+Q3=12.45%), and the difference was statistically significant (***P<0.001).

[0105] Both sets of results showed that ISL1β overexpression can significantly increase the apoptosis rate of gastric cancer cells, and the pro-apoptotic effect on MGC803 cells is more obvious, confirming that ISL1β can play a role in inhibiting the progression of gastric cancer by inducing apoptosis in gastric cancer cells.

[0106] In summary, overexpression of ISL1β significantly promotes apoptosis in gastric cancer cells, and combined with the proliferation inhibition results, this further demonstrates that ISL1β plays an anti-cancer role in gastric cancer. Therefore, ISL1β promoters can be used as effective components for the treatment of gastric cancer.

[0107] Sequence list information:

[0108] DTD Version: V1_3

[0109] Filename: An ISL1-β Recombinant Expression Vector and Its Application in Gastric Cancer.xml

[0110] Software Name: WIPO Sequence

[0111] Software version: 2.1.0

[0112] Generation Date: 2025-12-31

[0113] Basic Information:

[0114] Current application / applicant file name: 12530000431201824Q

[0115] Applicant's Name or Organization: Yunnan Cancer Hospital (The Third Affiliated Hospital of Kunming Medical University)

[0116] Applicant's name or organization name / Language: zh

[0117] Applicant's Name or Title / Latin Name: Yunnan Cancer Hospital / The Third Affiliated Hospital of Kunming Medical University

[0118] Invention Title: An ISL1-β Recombinant Expression Vector and Its Application in Gastric Cancer (zh)

[0119] Total sequence count: 1

[0120] sequence:

[0121] Serial Number (ID): 1

[0122] Length: 858

[0123] Molecular type: AA

[0124] Feature location / qualifier:

[0125] - source, 1..858

[0126] > mol_type, protein

[0127] > organism, synthetic construct

[0128] Residue:

[0129] ATGGAACCTA TAAAGATTGT CCCTTGGTTG TACGGGATCA AATGCGCCAA GTGCAGCATC 60

[0130] GGCTTCAGCA AGAACGACTT CGTGATGCGT GCCCGCTCCA AGGTGTATCA CATCGAGTGT 120

[0131] TTCCGCTGTG TGGCCTGCAG CCGCCAGCTC ATCCCTGGGG ACGAATTTGC GCTTCGGGAG 180

[0132] GACGGTCTCT TCTGCCGAGC AGACCACGAT GTGGTGGAGA GGGCCAGTCT AGGCGCTGGC 240

[0133] GACCCGCTCA GTCCCCTGCA TCCAGCGCGG CCACTGCAAA TGGCAGCGGA GCCCATCTCC 300

[0134] GCCAGGCAGC CAGCCCTGCG GCCCCACGTC CACAAGCAGC CGGAGAAGAC CACCCGCGTG 360

[0135] CGGACTGTGC TGAACGAGAA GCAGCTGCAC ACCTTGCGGA CCTGCTACGC CGCAAACCCG 420

[0136] CGGCCAGATG CGCTCATGAA GGAGCAACTG GTAGAGATGA CGGGCCTCAG TCCCCGTGTG 480

[0137] ATCCGGGTCT GGTTTCAAAA CAAGCGGTGC AAGGACAAGA AGCGAAGCAT CATGATGAAG 540

[0138] CAACTCCAGC AGCAGCAGCC CAATGACAAA ACTAATATCC AGGGGATGAC AGGAACTCCC 600

[0139] ATGGTGGCTG CCAGTCCAGA GAGACACGAC GGTGGCTTAC AGGCTAACCC AGTGGAAGTA 660

[0140] CAAAGTTACC AGCCACCTTG GAAAGTACTG AGCGACTTCG CCTTGCAGAG TGACATAGAT 720

[0141] CAGCCTGCTT TTCAGCAACT GGTCAATTTT TCAGAAGGAG GACCGGGCTC TAATTCCACT 780

[0142] GGCAGTGAAG TAGCATCAAT GTCCTCTCAA CTTCCAGATA CACCTAACAG CATGGTAGCC 840

[0143] AGTCCTATTG AGGCATGA 858

[0144] END

Claims

1. An ISL1-β recombinant expression vector, characterized in that, The vector is pcDNA3.1 ISL1β, which contains the complete open reading frame of the human ISL1-β gene. The nucleotide sequence of the open reading frame is shown in SEQ ID NO.1, and its length is 858 bp.

2. The ISL1-β recombinant expression vector according to claim 1, characterized in that, The recombinant expression vector also includes a CMV promoter, a BGH polyA termination sequence, a Neomycin resistance gene, a pUC origin of replication, and an SV40 origin of replication. The human ISL1-β gene is efficiently expressed under the drive of the CMV promoter.

3. The ISL1-β recombinant expression vector according to claim 1, characterized in that, The cloning site of the vector is BamHⅠ / EcoRⅠ, the vector is 5.4Kb in length, the prokaryotic resistance is Ampicillin, and the eukaryotic resistance is Neomycin.

4. The use of the ISL1-β recombinant expression vector according to any one of claims 1-3 in the preparation of a product for inhibiting gastric cancer.

5. The application according to claim 4, characterized in that, The product, through transfection of gastric cancer cells, efficiently expresses the ISL1-β protein, thereby inhibiting the proliferation of gastric cancer cells and / or promoting apoptosis of gastric cancer cells.

6. The application according to claim 5, characterized in that, The gastric cancer cells include either SGC7901 cells or MGC803 cells.

7. A reagent for inhibiting the progression of gastric cancer, characterized in that, It includes the ISL1-β recombinant expression vector according to any one of claims 1-3.

8. A reagent kit, characterized in that, It comprises the ISL1-β recombinant expression vector according to any one of claims 1-3, and primers, antibodies or reagents for detecting ISL1-β gene or protein expression.

9. The reagent kit according to claim 8, characterized in that, The primer sequences for detecting the ISL1-β gene are: forward primer 5'-TCTGTTCGTAGGAATGCCCC-3', and reverse primer 5'-GATCCCGTACAACCAAGGGA-3'.

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