Application of GCKR and its inhibitors in the preparation of drugs for treating pancreatic cancer

Abstract

This invention provides the application of GCKR and its inhibitors in the preparation of drugs for treating pancreatic cancer, belonging to the field of biomedical technology. The application of GCKR and its inhibitors in the preparation of drugs for treating pancreatic cancer reveals for the first time that GCKR is a key metabolic switch driving liver metastasis in pancreatic cancer, and that high expression of the GCKR gene can promote the growth and liver metastasis of pancreatic cancer in mice. The proliferation, invasion, and migration abilities of pancreatic cancer cells in the GCKR low-expression group were significantly lower than those in the normal control group. Using a pancreatic cancer orthotopic xenograft model constructed in highly immunodeficient mice, the invention demonstrated in vivo that knocking out GCKR can inhibit the growth of pancreatic cancer, indicating that GCKR inhibitors block the progression of pancreatic cancer by downregulating GCKR; conversely, overexpression plasmids that promote GCKR expression can upregulate GCKR expression and promote pancreatic cancer progression. This provides a new application of GCKR and offers new ideas for the treatment of pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of GCKR and its inhibitors in the preparation of drugs for treating pancreatic cancer. Background Technology

[0002] Metabolic reprogramming plays a crucial role in driving tumorigenesis and development. Previous studies have shown that pancreatic cancer cells enhance their invasive and migratory abilities through glycolytic reprogramming. However, the key genes driving this glycolytic process and promoting pancreatic cancer progression remain to be explored.

[0003] GCKR (glucokinase regulator) is a protein that regulates glucokinase. Current research mainly focuses on the association between GCKR and metabolic diseases (such as alcoholic fatty liver disease and diabetes). Studies have demonstrated that CRISPR-Cas9 gene knockout technology can target and suppress gene expression. Furthermore, epigenetic editing techniques targeting specific enhancer regions can regulate gene expression by affecting the transcription initiation complex without altering the structure and sequence of the target gene.

[0004] Currently, there are no studies reporting the biological function and clinical significance of GCKR in pancreatic cancer.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of GCKR and its inhibitors in the preparation of drugs for treating pancreatic cancer, and to reveal for the first time that GCKR (Gene ID:2646, genomic version: GRCh38.p14) is a key metabolic switch driving liver metastasis of pancreatic cancer.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the application of GCKR as a target in screening or developing products that have the function of treating pancreatic cancer or inhibiting the proliferation and / or invasion and / or migration of pancreatic cancer cells.

[0008] Furthermore, treatment of pancreatic cancer or inhibition of the proliferation and / or invasion and / or migration of pancreatic cancer cells includes at least one of A1 to A3: A1. Inhibit the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the GCKR gene; A2. Inhibit or reduce the content, activity, and / or function of GCKR protein; A3. Inhibits the activity of GCKR gene remote enhancers; The GCKR gene remote enhancer is selected from any one of the nucleotide sequences shown in SEQ ID NO.7~11 and their reverse complementary sequences.

[0009] Secondly, the present invention provides the use of GCKR inhibitors in the preparation of products for treating pancreatic cancer or inhibiting the proliferation and / or invasion and / or migration of pancreatic cancer cells.

[0010] Furthermore, the GCKR inhibitor includes at least one of B1 to B3: B1. Substances that inhibit the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the GCKR gene; B2. Substances that inhibit or reduce the content, activity, and / or function of GCKR proteins; B3. Substances that inhibit the activity of GCKR gene remote enhancers; The GCKR gene remote enhancer is selected from any one of the nucleotide sequences shown in SEQ ID NO.7~11 and their reverse complementary sequences.

[0011] Furthermore, the GCKR inhibitor includes at least one of C1 to C3: C1. Nucleic acid molecules used to silence or knock out the GCKR gene; C2, Antibodies that bind to GCKR protein; C3. Nucleic acid molecules used to inhibit the activity of GCKR gene remote enhancers.

[0012] Furthermore, the GCKR inhibitor includes at least one of D1 to D7: D1. An RNA interference molecule targeting the GCKR gene, the RNA interference molecule being the sequence shown in SEQ ID NO.2; D2. sgRNA targeting exons of the GCKR gene, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO.3 and / or SEQ ID NO.4; D3, the DNA molecule encoding the sgRNA described in D2; D4. An expression cassette, recombinant vector, or recombinant host cell containing the sgRNA described in D2 and / or the DNA molecule described in D3; D5. sgRNA targeting the remote enhancer of the GCKR gene, wherein the nucleotide sequence of the sgRNA is at least one of those shown in SEQ ID NO.12~21; D6, the DNA molecule encoding the sgRNA described in D5; D7. An expression cassette, recombinant vector, or recombinant host cell containing the sgRNA described in D5 and / or the DNA molecule described in D6.

[0013] Among these, sgRNA targeting the GCKR gene exons ensures the singularity and purity of its mechanism of action through permanent inactivation at the gene level. This approach achieves potent tumor suppression while demonstrating good biosafety and promising prospects for clinical translation. sgRNA targeting the GCKR gene enhancer intervenes in the gene without affecting its structure, further enhancing targeting specificity and reducing toxicity to other tissues and organs.

[0014] Thirdly, the present invention provides a biomaterial, wherein the biomaterial is at least one of E1 to E10: E1, the aforementioned RNA interference molecules; E2, the above-mentioned sgRNAs targeting exons of the GCKR gene; E3, the aforementioned sgRNAs targeting the remote enhancers of the GCKR gene; E4, the DNA molecule encoding the sgRNA described in E2; E5, the DNA molecule encoding the sgRNA described in E3; E6. An expression cassette containing the DNA molecule described in E4 or E5; E7, a recombinant vector containing the DNA molecule described in E4 or E5, or a recombinant vector containing the expression cassette described in E6; E8, a recombinant host cell containing the DNA molecule described in E4 or E5, or a recombinant host cell containing the expression cassette described in E6, or a recombinant host cell containing the recombinant vector described in E7.

[0015] Specifically, a CRISPR technology system targeting pancreatic cancer metastasis-specific enhancers was developed, targeting the GCKR gene itself and its cis-regulatory elements. This system significantly reduced the expression level of GCKR in pancreatic cancer cells without damaging the GCKR protein structure, and significantly inhibited the invasion and migration of pancreatic cancer cells, thereby achieving the goal of more precisely treating pancreatic cancer metastasis.

[0016] Furthermore, vectors include, but are not limited to, phu6-gRNA-SV40-Neomycin (e.g., Wuhan Miaoling Biotechnology Co., Ltd. #P1715) and lenti-EF1a-dCas9-KRAB-Puro (e.g., Wuhan Miaoling Biotechnology Co., Ltd. #P2842).

[0017] Fourthly, the present invention provides a composition for editing remote enhancers of the GCKR gene, characterized in that it comprises the above-mentioned E3 or E5-containing biological material, and a fusion protein composed of dCas9 and a recruitable histone methyltransferase, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO.5; The biomaterials include expression cassettes, recombinant vectors, or recombinant host cells.

[0018] Fifthly, the present invention provides a pharmaceutical composition comprising the above-described biological material or the above-described composition.

[0019] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0020] Furthermore, pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-retarding agents, etc. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include isotonic agents, such as sugars, polyols, or sodium chloride, in the composition. Pharmaceutically acceptable carriers may also contain small amounts of excipients that can improve the shelf life or effectiveness of the antibody or antibody moiety, such as wetting agents or emulsifiers, preservatives, or buffers.

[0021] This invention provides the application of GCKR and its inhibitors in the preparation of drugs for treating pancreatic cancer. It reveals for the first time that GCKR (Gene ID: 2646, genomic version: GRCh38.p14) is a key metabolic switch driving liver metastasis in pancreatic cancer, and that high expression of the GCKR gene promotes the growth and liver metastasis of pancreatic cancer in mice. The proliferation, invasion, and migration abilities of pancreatic cancer cells in the GCKR-low expression group were significantly lower than those in the normal control group. Using a pancreatic cancer orthotopic xenograft model constructed in highly immunodeficient mice, the invention demonstrated in vivo that GCKR knockout inhibits pancreatic cancer growth, indicating that GCKR inhibitors block pancreatic cancer progression by downregulating GCKR. Conversely, overexpression plasmids that promote GCKR expression can upregulate GCKR expression and promote pancreatic cancer progression. This provides a new application for GCKR and offers new insights for the treatment of pancreatic cancer. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This invention provides validation of GCKR gene editing efficiency based on Synthego ICE analysis in embodiments of the present invention. Specifically, A represents validation using a polyclonal cell line based on sg1, B represents validation using a polyclonal cell line based on sg2, and C represents validation using a monoclonal cell line based on sg1-13. Figure 2 The following are the results of GCKR expression levels in pancreatic cancer liver metastasis and primary lesion tissues provided in this embodiment of the invention. A represents the intersection of the sets of genes highly expressed in pancreatic cancer liver metastases (LMT) relative to primary lesions (PRM) and lactation-related genes in the three datasets GSE71729, GSE63124, and GSE42952 from the GEO public database. This intersection represents genes highly expressed in metastases and closely related to the lactation process, yielding GCKR. B is the immunohistochemical (IHC) staining map of GCKR in liver metastasis and primary lesion tissues from the PUMCH cohort. C is a statistical graph of GCKR protein expression levels in metastasis and primary lesion samples from the PUMCH cohort. Figure 3 The following are the invasion, migration, and proliferation results of the GCKR knockdown pancreatic cancer cell line provided in the embodiments of the present invention: A is a crystal violet staining image and quantitative statistical graph of CFPAC-1 cells; B is an EdU staining fluorescence image and quantitative statistical graph; and C is the RT-qPCR result of GCKR expression level detection of the GCKR knockdown pancreatic cancer cell line. Figure 4 The results of Western Blot verification of protein extraction from single-clone cells with knockout GCKR expression provided in this embodiment of the invention; Figure 5 The metabolomics analysis results of the GCKR-knockout CFPAC-1-luc cell line (GCKR-KO) provided in the embodiments of the present invention; Figure 6 This is the growth status of the GCKR knockout mouse pancreatic cancer orthotopic xenograft model provided in this embodiment of the invention. In this figure, A is the modeling process of the NPG mouse pancreatic cancer orthotopic xenograft model, B is the average bioluminescence signal intensity, C is the in vivo bioluminescence imaging of the NPG mouse pancreatic cancer orthotopic xenograft model, and D is the ex vivo pancreas (with spleen) photograph and orthotopic tumor weight statistics of the NPG mouse pancreatic cancer orthotopic xenograft model. Figure 7 The second part shows the growth of the mouse pancreatic cancer orthotopic xenograft model overexpressing GCKR provided in this embodiment of the invention. In this part, A is an ex vivo pancreas (with spleen) photograph of the NPG mouse pancreatic cancer orthotopic xenograft model and a statistical graph of the weight of the orthotopic transplanted tumor; B is the average bioluminescence signal intensity; C is the in vivo bioluminescence imaging of the NPG mouse pancreatic cancer orthotopic xenograft model; and D is the RT-qPCR result of the detection of GCKR expression level in the pancreatic cancer cell line overexpressing GCKR. Figure 8The growth of the liver metastasis model of mouse spleen injection with overexpressing GCKR provided in the embodiments of the present invention is shown in A, which is the modeling process of NPG mouse spleen injection model, B is a comparison and statistical diagram of the average bioluminescence signal intensity and mcherry fluorescence intensity of the liver, and C is a comparison of the liver of NPG mouse spleen injection model and a statistical diagram of the number of liver metastasis nodules. Figure 9 The results show the effects of key GCKR candidate enhancers on GCKR expression levels and the invasion and migration abilities of pancreatic cancer cell lines, as provided in the embodiments of the present invention. In this figure, A is the GCKR locus Hi-C interaction matrix, B is the relative expression level of GCKR, and C is the crystal violet staining images and corresponding cell count statistics of Transwell invasion and migration experiments. Detailed Implementation

[0024] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0025] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0027] Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0028] CFPAC-1 cells: human pancreatic ductal adenocarcinoma cells, manufactured by American Type Culture Collection (ATCC), product serial number: CRL-1918; Capan-1 cells: human pancreatic ductal adenocarcinoma cells, manufactured by American Type Culture Collection (ATCC), product serial number: HTB-79; PANC-1 cells: human pancreatic ductal adenocarcinoma cells, manufactured by American Type Culture Collection (ATCC), product serial number: CRL-1469; Serum-free medium (IMDM medium): Gibco#2581954.

[0029] Serum-free medium (DMEM medium): Gibco#8121728.

[0030] IMDM complete medium: IMDM medium / 20% fetal bovine serum (Gibco#1414426) / 1% penicillin-streptomycin solution (Gibco#15140122).

[0031] DMEM complete medium: DMEM medium / 10% fetal bovine serum (Gibco#1414426) / 1% penicillin-streptomycin solution (Gibco#15140122).

[0032] The EdU kit is Click-iT™ EdU Alexa Fluor™ 647, Invitrogen#C10424.

[0033] The RIPA cell lysis buffer was APPLYGEN#C1053.

[0034] The BCA protein quantification kit is Thermo Fisher Scientific #UE284362.

[0035] The protein loading buffer was GeneStar#20BB01.

[0036] The chemiluminescent developer is Thermo Fisher Scientific #VH311118.

[0037] The ChIP kit is CST#9005S.

[0038] VAHTS ® The Universal DNA Library Prep Kit for Illumina V3 is Vazyme#ND607.

[0039] RNA Rapid Extraction Kit: ES Science # RN001.

[0040] Reverse transcription kit: TaKaRa# RR047A.

[0041] This invention demonstrates, through Transwell experiments, animal experiments, and clinical specimen testing, that the expression level of the GCKR gene is positively correlated with the degree of pancreatic cancer metastasis, i.e., high expression of the GCKR gene can promote the invasion and migration of pancreatic cancer cells and liver metastasis of pancreatic cancer in mice.

[0042] Example 1 I. RT-qPCR assay to detect the knockdown efficiency of siRNA on GCKR expression 1. CFPAC-1 cells were cultured in IMDM complete medium in six-well plates and divided into a control group and a GCKR low expression group. si-NC and si-GCKR small interfering RNA were transfected using Lipo3000 transfection reagent (Invitrogen#L3000015).

[0043] Among them, si-NC uses a non-targeted control siRNA (Guangzhou Ruibo Biotechnology Co., Ltd., product number: siN0000001-1-5), denoted as GCKR-NC, and si-GCKR is shown in Table 1.

[0044] Table 1

[0045] 2. After 24 hours of transfection, the culture medium was replaced with complete medium, and the cells were cultured for another 24 hours. Then, total RNA was extracted from different groups of cells using an RNA rapid extraction kit. 3. The concentration and purity of RNA were determined by an ultra-micro spectrophotometer (A260 / A280 ratio between 1.8 and 2.0), and 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit. 4. Using cDNA as a template, real-time quantitative PCR was performed using SYBR Green qPCR premix. The reaction system contained: 2 μL cDNA, 0.4 μL each of forward and reverse primers (GCKR and internal control GAPDH), 10 μL SYBR premix, and nuclease-free water to a final volume of 20 μL. Amplification was performed on a real-time quantitative PCR instrument. The reaction program was: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds; 60℃ annealing / extension for 30 seconds, for a total of 40 cycles. Finally, melting curve analysis was performed (95℃ for 15 seconds, 60℃ for 1 minute, 95℃ for 15 seconds). The primers used are shown in Table 2. 5. The relative expression level of the GCKR gene was calculated using the 2^(-ΔΔCt) method, with the GCKR-NC group as the calibration control group, and the changes in GCKR mRNA expression were detected.

[0046] The results are as follows Figure 3 As shown in Figure C, the siRNAs shown in Table 1 can significantly reduce the RNA expression level of GCKR.

[0047] Table 2

[0048] II. The Transwell experimental verification was conducted in the following steps: 1. After transfecting as described above for 24 hours, change to complete culture medium, culture for another 24 hours, digest with trypsin to collect cells, resuspend in serum-free medium (IMDM medium), and count.

[0049] 2. Two groups of CFPAC-1 cells were seeded into the upper chamber (Costar #3422) of a Transwell chamber without Matrigel coating (Corning #356237) to assess migration ability. Matrigel was diluted 1:5 with serum-free medium (IMDM), and 50 μL was evenly spread onto the membrane of the upper chamber of the Transwell chamber. The chamber was incubated at 37°C for 2 hours to solidify into a gel, and then seeded with the two groups of CFPAC-1 cells to assess invasion ability. The seeding density of CFPAC-1 cells was 80,000 cells / chamber, with a seeding volume of 200 μL. 500 μL of complete culture medium containing 20% ​​fetal bovine serum was added to the lower chamber.

[0050] 3. Place the cells in a 37℃ constant temperature CO2 incubator. After 48 hours, fix and stain the cells in the chambers with 0.1% crystal violet methanol solution. After 30 minutes, wash and dry the chambers. Observe the cancer cells on the bottom of the chambers under a regular optical microscope, count the cancer cells and perform statistical analysis.

[0051] III. EdU staining assay for CFPAC-1 cell proliferation was performed according to the following steps: 1. Cultured CFPAC-1 cells in six-well plates, the cells adhered overnight, and were allowed to recover to normal growth state.

[0052] 2. Prepare 2×EdU working solution: Dilute EdU 1:500 with preheated cell culture medium (10 mM EdU diluted to 20 μM, i.e., 2× working solution). Add 2×EdU working solution to a six-well plate at a 1:1 volume ratio to make the final EdU concentration 10 μM, and continue to incubate the cells for 2 hours.

[0053] 3. Discard the supernatant, fix the cells with 4% paraformaldehyde at room temperature for 20 minutes, then wash 3 times with PBS; permeabilize with PBS containing 0.5% Triton X-100 for 20 minutes, then wash 3 times with PBS.

[0054] 4. Prepare the click reaction mixture according to the EdU kit instructions. Add an appropriate amount of Click reaction solution to each well and incubate at room temperature in the dark for 30 minutes to allow EdU to undergo a click chemical reaction with the fluorescent azide dye.

[0055] 5. Discard the Click reaction solution, wash three times with PBS, add DAPI nuclear dye and incubate at room temperature in the dark for 10–15 minutes for nuclear counterstaining.

[0056] 6. Observe and photograph using a fluorescence microscope. Detect the proportion of EdU-positive cells (proliferating cells) at a wavelength of 647nm. Calculate the EdU positivity rate using the total number of cell nuclei stained with DAPI as an internal reference.

[0057] The Edu staining kit mentioned above is Invitrogen #C10424.

[0058] The results are as follows Figure 3 As shown in Figures A and B, the proliferation, invasion, and migration abilities of pancreatic cancer cells in the GCKR low expression group were significantly lower than those in the normal control group.

[0059] Example 2 Construction of GCKR knockout cell line Two different sgRNA targets (sg1 and sg2) were designed targeting the exon region of the GCKR gene (Gene ID: 2646, genome version: GRCh38.p14). The target recognition sequences of this sgRNA include sg1: 5'-GACCCCGGAGCCTGGCAAGT-3' (SEQ ID NO.3) and sg2: 5'-GGGTTTGACTTCTCCGTGAT-3' (SEQ ID NO.4).

[0060] 1. Preparation of polyclonal cells with GCKR knocked out by CRISPR-Cas9 Add 1 mL of complete culture medium to a 24-well plate of CFPAC-1 cells (CFPAC-luc cells) that have been pre-inoculated with stable transfection of Luciferase lentivirus (Hanheng Biotechnology (Shanghai) Co., Ltd., # HH20230518GX-LP01) and incubate at 37°C, 5% CO2 for 30 min in a cell culture incubator.

[0061] Preparation of the RNP complex (CRIPSR-Cas9 / gRNA): Maintain the volume of the Cas9 / gRNA complex less than 10% of the total reaction volume, with each electroporation system containing 10 μL of Neon. TM tip. Add 1.0 μL TrueCut TM Cas9 Protein v2 (30 pmol / μL) and 0.2 μL of sgRNA (150 pmol / μL) were added to 5 μL of resuspension buffer R and gently mixed (molar ratio of Cas9 protein: sgRNA = 1:1); incubated at room temperature for 20 min.

[0062] Collect cells in the logarithmic growth phase; gently pipette the cell suspension and collect the suspension; centrifuge at 350 g for 3 min and discard the supernatant; resuspend the cells in 1 mL PBS; count the cells using a cell counter and collect 2 × 10⁶ cells. 5 Cells / samples were divided into two groups: a KO group and a control group. After centrifugation at 300 g for 3 min, the supernatant was discarded. Each sample was then resuspended in 5 µL of Buffer R. For the KO group, the Cas9 / gRNA complex was gently added to the cell suspension and mixed well, while for the control group, the cells were added to the corresponding volume of Buffer R.

[0063] 2. Use Neon TM Electroporation of the transfection system Add 3 mL of Electrolytic Buffer E to Neon TM Tubes; using Neon TM Pipe 10 μL of the cell and Cas9 / gRNA mixture into a 10 μL tip; gently place into Neon. TM In Tubes, electroporate the above system using the preset electroporation program (select 1400V / 20ms / 2pulses); immediately place the electroporated cells gently into a pre-incubated 24-well plate; place the cell plate in an incubator at 37°C and 5% CO2 for 72 h.

[0064] 3. CRISPR-Cas9 knockout GCKR efficiency verification Genomic DNA was extracted from transfected cell pellets and sequenced using Sanger sequencing to assess GCKR knockout efficiency at the genomic level. Sequencing analysis of polyclonal cells transfected with sg1 and sg2 using the Synthego ICE platform showed that both sg1 and sg2 transfected polyclonal cells achieved highly efficient gene editing, with insertion / deletion rates exceeding 90% and high knockout efficiency scores, indicating a strong ability to induce DNA breaks and functional mutations at target sites. Figure 1 As shown in Figures A and B, this laid the foundation for the preparation of monoclonal cells.

[0065] 4. Preparation of monoclonal cells by CRISPR-Cas9 knockout of GCKR Polyclonal cells transfected with sg1 and sg2 were seeded into two 384-well cell culture plates using a multipipe pipette at a concentration of 80 µL, 1 cell / well. The wells were numbered sequentially as GCKR-sg1-1~384 and GCKR-sg2-1~384, and placed in a cell culture incubator for 4~6 weeks, starting from day 1. On days 7 and 30, the cells were observed using IncuCyte scanning. Single-clonal cells were further transferred to new 96-well cell culture plates for further culture; single-clonal cells were then further expanded to 24-well cell culture plates. A portion of the cells from the 24-well plates was collected, resuspended in 500 μL in 1.5 mL EP tubes, centrifuged at 350g for 3 min, and the supernatant was discarded. Genomic DNA was extracted from the cell pellet and amplified using primers for Sanger sequencing to identify the target fragment at the genomic level. Another portion of the cell pellet was subjected to Western blotting for protein identification.

[0066] 5. Western blot identification 1) Total protein was extracted using RIPA cell lysis buffer.

[0067] 2) Protein concentration was determined using the BCA protein quantification kit. 5× protein loading buffer was added, and total protein was separated by electrophoresis using a 10% SDS-PAGE gel. The protein was then electrotransferred onto an NC membrane.

[0068] 3) Block the NC membrane with 5% skim milk for 1 hour, and incubate overnight at 4°C with GCKR primary antibody.

[0069] 4) Wash three times with TBST solution, then incubate with HRP-labeled rabbit secondary antibody at room temperature for 1 hour.

[0070] 5) Use chemiluminescent developing solution to develop protein bands and detect GCKR expression.

[0071] The primary antibodies against GCKR are a rabbit polyclonal antibody (Affinity # AF0569) and a rabbit monoclonal antibody (CST#14328S).

[0072] The results are as follows Figure 4As shown, lane 1 is GCKR-blank, lanes 2-4 are GCKR-sg1-1, GCKR-sg1-13, and GCKR-sg1-19 respectively, and lanes 5-8 are GCKR-sg2-1, GCKR-sg2-7, GCKR-sg2-14, and GCKR-sg2-20 respectively. The cell line with better knockout effect and growth status (GCKR-sg1-13) was selected and named GCKR-KO, while GCKR-blank served as the control group and was named GCKR-NC for subsequent functional verification.

[0073] Analysis of Sanger sequencing results of GCKR-sg1-13 monoclonal cells using Synthego ICE showed that the insertion / deletion rate of this monoclonal cell was as high as 99%, with a model fit R² of 0.99 and a knockout efficiency score of 99, indicating that the genome was almost completely edited at the target site with no wild-type sequence residue. The sequencing peak diagram clearly showed a single mutation type (+1 bp insertion), confirming that this clone is a homozygous frameshift mutant, capable of achieving complete knockout of GCKR gene function, suitable for subsequent functional studies and phenotypic analysis, such as... Figure 1 As shown in C.

[0074] Example 3 Metabolomics Analysis This embodiment performs metabolomics analysis on the CFPAC-1-luc cell line (GCKR-KO) with GCKR knocked out in Example 2.

[0075] 1. Metabolite extraction: A mixed solvent (acetonitrile:methanol:water = 2:2:1, volume ratio) pre-cooled in an ice bath was used, and an isotope-labeled internal standard mixture (ISTD-MIX, BIOTREE) was added.

[0076] 2. Sample processing: Collect 1*10 7 The cells of GCKR-KO and its control (GCKR-NC) were precipitated, vortexed, sonicated at 4°C for 30 minutes, and then placed at -20°C for 10 minutes to settle.

[0077] 3. Centrifugation and concentration: Centrifuge at 4°C and 14,000×g for 20 minutes, take 800 μL of supernatant, concentrate using a vacuum concentrator (CentriVap, Labconco), and finally redissolve with 100 μL of 50% acetonitrile / 0.1% formic acid.

[0078] 4. LC-MS / MS analysis: An Agilent 1290 Infinity UHPLC system coupled with an AB Sciex TripleTOF 6600 mass spectrometer was used for analysis, which was performed by Wuhan Maiwei Technology Co., Ltd.

[0079] 5. Global metabolite analysis: Mass spectrometry detection was performed using an Orbitrap Exactive series mass spectrometer (Thermo) with an electrospray ionization (ESI) source, and signals were acquired in both positive and negative ion modes.

[0080] 6. Metabolite Annotation: The detected metabolite signal information (mass-to-charge ratio, fragmentation pattern, retention time) is compared with the METLIN, HMDB, and MassBank databases to complete metabolite identification.

[0081] 7. Screening of differential metabolites: Partial least squares discriminant analysis (PLS-DA) model was used to evaluate the differences in metabolites between GCKR-KO and GCKR-NC groups.

[0082] Example 4 Construction of GCKR overexpression cell line 1. Vector digestion: Based on GV661 vector (element sequence: CMV-MCS-3FLAG-EF1a-mCherry-T2A-puromycin; cloning site: EcoRI / BamHI; control number: CON474, Shanghai Jikai Biotechnology Co., Ltd.).

[0083] 2. Obtaining the target gene fragment: Use the primers shown in Table 3 to retrieve the target gene fragment.

[0084] Table 3

[0085] Primer description: Contains exchangeable base pairs, restriction enzyme sites, and the 5' end sequence of the target gene for PCR extraction of the target gene.

[0086] 3. The product was exchanged into a linearized expression vector. Following the steps above, the PANC-1 pancreatic cancer cell line, namely PANC-1-luc cells, which had been pre-inoculated with stable Luciferase lentivirus (Hanheng Biotechnology (Shanghai) Co., Ltd., # HH20230518GX-LP01), was transfected with the vector. After that, positive clones were sequenced and compared. The inserted sequence is shown in SEQ ID NO.46.

[0087] The alignment results show that the sequence alignment indicates that the fragment has 100% homology with the expected overexpression vector insertion sequence, and the sequencing results confirm the sequence alignment.

[0088] 4. Drug screening was performed on transfected cells using 2 μg / mL puromycin (MCE#HY-K1057), and GCKR expression levels were verified using RT-qPCR. Figure 7As shown in Figure D, this method demonstrates that it can achieve significant overexpression of the GCKR gene, named GCKR-OE (PANC-1-luc cells with the control vector introduced are named GCKR-CON), which is suitable for subsequent functional studies and phenotypic analysis.

[0089] Example 5 I. Animal Experiments 1. The GCKR-KO CFPAC-1-luc pancreatic cancer cell line and the corresponding negative control group (GCKR-NC) were seeded in IMDM complete medium containing 20% ​​serum, and the GCKR-OE PANC-1-luc pancreatic cancer cell line and the corresponding negative control group (GCKR-CON) were seeded in DMEM complete medium containing 10% serum. The cells were cultured and expanded in a 37°C CO2 incubator.

[0090] 2. Animals: Mice were selected, specifically 4-6 week old male NOD-PrkdcscidIl2rgtm1 / Vst (NPG) mice (from Vitonda Biotechnology Co., Ltd.).

[0091] 3. Two groups of cells were collected after digestion and injected into the pancreas and spleen of NPG mice. The injection volume for the pancreas was 5 × 10⁻⁶ cells / mL. 6 Cells / animal, spleen injection volume is 1×10⁻⁶ 6 Cell / each

[0092] The specific surgical procedure is as follows: Mice were anesthetized with 2.5% Avertin. A transverse incision of about 0.5 cm was made under the left rib to expose the pancreas or spleen. Cancer cells were injected into the pancreas and spleen respectively using an insulin injector. Then, the injection site was compressed with a 75% alcohol swab for 2 minutes to kill any leaked cancer cells. The pancreas or spleen was returned to its original position, and the peritoneum, muscles and skin were sutured layer by layer using 5-0 absorbable sutures. After disinfection, the mice were placed in a warm environment to recover.

[0093] 4. Mice were housed in an SPF-grade animal facility. In the fourth week after modeling, the model mice were injected intraperitoneally with 10 μL / g of D-fluorescein potassium salt (MERCK#50227). IVIS in vivo imaging of the animals was performed 10 minutes later.

[0094] For the spleen-injected liver metastasis model, the liver was dissected after in vivo imaging to observe liver metastases and perform ex vivo imaging of the liver. The liver tissue was then fixed, sectioned, and stained with hematoxylin and eosin (HE staining) to count the number of cancerous nodules. For the pancreatic cancer orthotopic xenograft model, the orthotopic xenograft was dissected after in vivo imaging, photographed, and weighed.

[0095] Example 6 Clinical Specimen Detection 1. Pancreatic cancer tissue specimens were collected from the Pathology Specimen Bank of Peking Union Medical College Hospital (PUMCH) to form a pancreatic cancer sample cohort, and 4μm thick tissue sections were prepared.

[0096] 2. The tissue sections were dewaxed with xylene and dehydrated with ethanol of different concentrations (100%, 85% and 75% ethanol solutions by mass concentration), and then boiled in citric acid antigen retrieval solution for 20 minutes for antigen retrieval.

[0097] 3. Each tissue section was blocked with 10% sheep serum for 30 minutes, and then incubated with anti-GCKR primary antibody (Affinity # AF0869) at room temperature for 1 hour to obtain incubated tissue sections.

[0098] 4. Each incubated tissue section was incubated with HRP-labeled secondary antibody dilution buffer for 30 minutes, then incubated with DAB imaging buffer for 20 seconds, and then stained with hematoxylin to obtain stained tissue sections.

[0099] 5. Finally, the stained tissue sections were placed in ethanol solutions of different concentrations (75%, 85% and 100% ethanol solutions by mass) for dehydration and then mounted in neutral resin.

[0100] 6. The expression level of GCKR in the tissue sections was assessed by two independent pathologists.

[0101] By analyzing the GCKR expression level in tumor tissues of a pancreatic cancer sample cohort, the results are as follows: Figure 2 As shown, the GCKR protein level in pancreatic cancer liver metastases was significantly higher than that in pancreatic cancer in situ lesions. Simultaneously, the effects of intervening in GCKR gene expression levels on pancreatic cancer cell invasion, migration, and liver metastasis in mice were explored at both in vitro cellular and in vivo animal levels. The Transwell results are as follows: Figure 3 As shown, the invasive and migratory abilities of pancreatic cancer cells in the GCKR low-expression group were significantly lower than those in the normal control group; and as... Figure 5 As shown, knocking out GCKR can inhibit the production of lactate, a core metabolite of glycolysis in pancreatic cancer cells, thus disrupting a key link in the regulation of glucose metabolism in tumor cells. This prevents cancer cells from adapting to the local metabolic environment of the tumor, thereby inhibiting their growth. This explains the inhibitory effect of this invention on pancreatic cancer growth from a metabolic perspective. Pancreatic cancer orthotopic injection models and spleen injection models were constructed using highly immunodeficient mice. After four weeks of rearing, bioluminescence imaging of isolated liver tissue and in vivo animals was performed. Figure 6 As shown, in vivo studies have demonstrated that GCKR knockout expression can inhibit pancreatic cancer growth. Figure 7 and Figure 8As shown, overexpression plasmids can promote GCKR expression levels, thereby promoting pancreatic cancer growth and liver metastasis. Therefore, GCKR (glucokinase regulatory protein), as a core molecule of metabolic regulation, acts as a metabolic switch in the progression of pancreatic cancer cells.

[0102] Example 7 This embodiment utilizes Hi-C sequencing and ChIP-seq sequencing to discover the remote enhancer region of the GCKR gene and demonstrates that intervening in the enhancer region can affect the expression level, invasion, and migration ability of the GCKR gene.

[0103] I. Hi-C Library Construction and Sequencing Steps 1. Cell cross-linking: Digest and collect cells, 1×10 7 Cells per sample were fixed in a 1% formaldehyde solution for 10 min, cross-linking was terminated with glycine solution for 5 min, and then the cells were washed three times with pre-cooled PBS.

[0104] 2. Cell lysis: The cell pellet was resuspended in Hi-C lysis buffer containing 10 mM Tris-HCl, 10 mM NaCl, 0.2% NP-40, 1 / 10 volume of PIC, and pH=8.0. The pellet was then placed on ice for 15 min and centrifuged to obtain the pellet.

[0105] 3. Restriction enzyme digestion: Use 50 μL of 0.5% SDS and incubate at 62℃ for 5-10 min. Add 145 μL of water and 25 μL of 10% Triton X-10 to quench the SDS. Mix gently to avoid generating bubbles and incubate at 37℃ for 15 min. Then add 25 μL of 10×NEBuffer2 and 100 U of MboI restriction endonuclease and incubate at 37℃ for at least 2 hours in a thermal mixer.

[0106] 4. End labeling: Incubate at 62℃ for 20 min to inactivate MboI, then cool to room temperature; add 50 μL MasterMix, mix well by pipetting, and incubate at 37℃ for 1 hour by rotation; the formula of the Master Mix is: 37.5 μL of 0.4 mM biotin-14-dATP, 1.5 μL of 10 mM dCTP, 1.5 μL of 10 mM dGTP, 1.5 μL of 10 mM dTTP, and 8 μL of 5 U / μL DNA polymerase I.

[0107] 5. Proximal ligation: Add 900 μl of ligation master mix, mix well, and incubate at room temperature for 4 hours by rotation. The formulation of the ligation master mix is ​​as follows: 663 μL of water, 120 μL of 10×NEB T4 DNA ligase buffer, 100 μL of 10% Triton X-100, 12 μL of 10 mg / ml BSA, and 5 μL of 400 U / μL T4 DNA ligase.

[0108] 6. Decross-linking: Add 50 μl Proteinase K and 120 μl 10% SDS, incubate at 55 °C for 30 min, add 130 μL of 5 M NaCl solution and incubate at 68 °C for at least 2 hours, then extract DNA using the phenol-chloroform method.

[0109] 7. Extracted DNA was fragmented, DNA fragments were enriched with Biotin, library constructed, sequencing was performed, and Hi-C upstream analysis was conducted. Paired-end 150bp sequencing was performed using the Illumina HiSex X Ten sequencing platform (PE150), with a data volume of 250Gb / sample. FastQC was used for quality control, and Hi-C-Pro was used to remove low-quality reads. The data were aligned to the hg19 human genome, and a 1M raw interaction matrix was constructed. Finally, the Hi-C interaction matrix was visualized using Juicer software or WashU Epigenome Browser.

[0110] II. Detailed Procedure of ChIP-seq Experiment 1. Cell cross-linking: Digest and collect cells, 4 × 10⁻⁶ 6 Cells per sample were fixed in a 1% formaldehyde solution for 10 min, cross-linking was terminated with glycine solution for 5 min, and then the cells were washed three times with pre-cooled PBS.

[0111] 2. Preparation of cell nuclei and chromatin fragments: Using an enzymatic ChIP kit, each IP sample was lysed with 1 ml of Buffer A for 10 min, centrifuged at 2000g for 5 min at 4°C to precipitate the cell nuclei, resuspended in 1 ml of Buffer B, and then 0.5 μL of Micrococcal Nuclease was added to each IP sample and incubated at 37°C for 20 min. 10 μL of 0.5 M EDTA was added to each IP sample, and digestion was stopped by placing it on ice for 2 min. The sample was centrifuged at 16000g for 1 min at 4°C to obtain the cell nucleus pellet. 100 μL of ChIP Buffer was added to each IP unit and incubated on ice for 10 min. The nuclear membrane was disrupted using a Bioruptor® Plus sonicator at 4°C on high speed, with 15 s sonication followed by 45 s intervals, for 40 cycles. The sample was centrifuged at 9400g for 10 min at 4°C, and the supernatant was collected.

[0112] 3. Chromatin Immunoprecipitation: Add 400 μl ChIP Buffer, 10 μL of target antibody and 2 μL of IgG to each IP sample, mix thoroughly, and incubate at 4°C for 4 hours to overnight. Then add 30 μL of Protein A / G magnetic beads to each IP sample, incubate at 4°C for 2 hours, and then place on a magnetic rack to wash 3 times with 1 mL of low-salt buffer (1×ChIP Buffer) and wash once with 1 mL of high-salt buffer (1000 μL 1×ChIP Buffer, 70 μL 5M NaCl).

[0113] 4. Chromatin elution and decrosslinking: Place on a magnetic rack, remove the supernatant, add 150 μl of 1×ChIP elution buffer to each IP sample, elute at 65°C with shaking for 30 min, take the supernatant and add 6 μL of 5M NaCl and 2 μL of Proteinnase K to decrosslink, incubate at 65°C for at least 2 hours.

[0114] 5. DNA purification: Add 750 μL of DNA Binding Buffer to each DNA sample, centrifuge through a centrifuge column, discard the liquid in the collection tube, add 750 μL of DNA Wash Buffer to the centrifuge column and centrifuge; replace with a new collection tube, add 50 μL of DNA Elution Buffer to the centrifuge column and centrifuge for 30 seconds to obtain purified ChIP-DNA. 6. ChIP-seq: using VAHTS ®The Universal DNA Library Prep Kit for Illumina V3 was used to construct ChIP-DNA libraries. Paired-end 50bp sequencing (PE50) was performed using the Illumina HiSex X Ten sequencing platform, with a data volume of 6Gb / sample. FastQC was used for data quality control. The sequencing reads were aligned to the human genome, and the genomic location of all peaks was annotated using Bedtools. Finally, the bigwig file was obtained using deepTools and visualized using WashU Epigenome Browser.

[0115] III. Design Steps for Small Guide RNAs (sgRNAs) Targeting GCKR Gene Remote Enhancers 1. The pancreatic cancer liver metastasis cell line (Capan-1) and the pancreatic cancer primary lesion cell line (PANC-1) were derived from liver metastases of a 40-year-old male pancreatic head cancer patient and primary lesions of a 56-year-old female pancreatic head cancer patient, respectively. Based on joint analysis of Hi-C and H3K27ac CHIP-seq data of the Capan-1 and PANC-1 cell lines, eight candidate enhancers of GCKR, Enh#1 to Enh#8, were identified (e.g., ...). Figure 9 (E1~E8 shown in A).

[0116] 2. Select five candidate enhancers (Enh#3~Enh#7) from those whose H3K27ac peak value of Capan-1 is significantly higher than that of PANC-1 (e.g., Capan-1). Figure 9 As shown in Figure A (E3-E7), small guide RNAs (sgRNAs) targeting the remote enhancer of the GCKR gene were designed using the CRISPOR tool website (CRISPOR (tefor.net)) and epigenetic editing technology (CRISPR / dCas9-KRAB), and the sgRNA with the highest silencing efficiency (sgEnh#4-1) was identified. The epigenetic editing technology used is based on the CRISPR / dCas9-KRAB principle. This system achieves transcriptional repression of specific genes by fusing dCas9 (deactivated Cas9, dCas9) with a repressor factor (KRAB, which can recruit histone methyltransferases, etc.) under the mediation of sgRNA. The amino acid sequence and nucleotide sequence of the fusion protein of dCas9 and recruitable histone methyltransferase are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0117] 3. The nucleotide sequences of the five selected GCKR gene enhancer sites Enh#3 to Enh#7 are shown in SEQ ID NO.7 to 11. The target sequences for sgRNA binding to all GCKR gene enhancer sites Enh#3 (chr2:27666790-27669083), Enh#4 (chr2:27711085-27712580), Enh#5 (>chr2:27749200-27751500), Enh#6 (>chr2:27805433-27807286), and Enh#7 (>chr2:27850000-27852700) and the template sequences for sgRNA transcription are shown in Table 4.

[0118] Table 4

[0119] 4. Construction of CRISPR / dCas9-KRAB knockout system using co-transfection + two-drug screening method 1) Vector preparation: The phU6-gRNA-SV40-Neomycin plasmid (Wuhan Miaoling Biotechnology Co., Ltd. #P1715) was digested with Bbsl restriction endonuclease (NEB#R3539S). After DNA electrophoresis, the DNA fragment was recovered from the agarose gel using a DNA recovery and purification kit (ABclonal# RK30100).

[0120] 2) Ligation and transformation: The annealed double-stranded sgRNA and the enzyme-digested vector backbone were ligated and then transformed using DH5α competent cells (Shanghai Beyotime Biotechnology Co., Ltd. #D1031S).

[0121] 3) Co-transfection and selection: After extracting plasmids using a plasmid miniprep kit (Tiangen Biotech (Beijing) Co., Ltd. #DP103), the constructed sgRNA-Neo plasmid and lenti-EF1a-dCas9-KRAB-Puro plasmid (Wuhan Miaoling Biotechnology Co., Ltd. #P2842) were mixed and introduced into target CFPAC-1 cells using liposomes (Lipo3000, Invitrogen #L3000015). Forty-eight hours after transfection, 2 μg / mL Puromycin (MCE #HY-K1057) and 500 μg / mL G418 (MCE #HY-17561) were added simultaneously for selection. After the control group cells completely died, the surviving cells were considered to have successfully integrated the dual systems and were used for subsequent verification of CRISPR / dCas9-KRAB knockout efficiency targeting different enhancer sites.

[0122] IV. RT-qPCR assay to detect the silencing efficiency of GCKR expression at targeted enhancer sites Using the sgCtrl group as a calibration control, and referring to the RT-qPCR experimental method in Example 1, the changes in GCKR RNA expression levels in CRISPR / dCas9-KRAB knockout systems with different enhancer target sites were detected.

[0123] V. Transwell assay to verify the effect of targeted enhancer sites on pancreatic cancer invasion and migration. CFPAC-1dCas9-KRAB cells were seeded in six-well plates and divided into a control group (sgCtrl) and a stable sgRNA expression group (sgEnh#4-1). Other steps were the same as the Transwell experiment in Example 1.

[0124] H3K27ac modification is a marker of active enhancers. High levels of H3K27ac modification indicate high enhancer activity, and the corresponding target gene is in a transcriptionally activated state. This invention analyzed H3K27ac modification information and DNA interaction information at the whole genome level of pancreatic cancer cells. The modification map and interaction map of H3K27ac in the GCKR gene were obtained using the WashU tool website (http: / / epigenomegateway.wustl.edu / ). The results showed that compared with pancreatic cancer primary tumor cells, the GCKR gene remote enhancer region had a higher level of H3K27ac modification in liver metastasis cells.

[0125] The silencing efficiency of sgRNAs targeting enhancer sites Enh#3-1, Enh#3-2, Enh#4-1, Enh#4-2, Enh#5-1, Enh#5-2, Enh#6-1, Enh#6-2, Enh#7-1, and Enh#7-2 on GCKR expression was detected using RT-qPCR experiments. The results showed that all sgRNAs had a significant effect on silencing GCKR expression (e.g., ...). Figure 9 As shown in Figures A and B), sg#Enh#4-1 showed the most significant inhibitory effect. Transwell assays verified that the aforementioned CRISPR / dCas9-KRAB system and the identified sgRNAs significantly inhibited the invasion and migration abilities of pancreatic cancer cells (e.g., ...). Figure 9 As shown in Figure C, this suggests the great potential of targeting the GCKR gene activity enhancer for the treatment of pancreatic cancer metastases.

[0126] In summary, specific knockout of the GCKR gene in pancreatic cancer cells using CRISPR / Cas9 technology significantly inhibits the growth of in situ pancreatic cancer tumors. Tumor burden is significantly reduced: in a mouse in situ tumorigenesis model, GCKR knockout resulted in a statistically significant decrease in both in vivo bioluminescence intensity and ex vivo tumor weight; in a mouse liver metastasis model, GCKR overexpression promotes liver metastasis formation. Sequences targeting the GCKR enhancer significantly inhibit GCKR expression levels and the invasive and migratory abilities of pancreatic cancer cell lines. Therefore, sgRNAs designed targeting the GCKR gene and their intervention methods can successfully inhibit pancreatic cancer progression, providing important technical solutions and application value for clinical research on targeted metabolic therapy for pancreatic cancer.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of GCKR as a target in screening or developing products that have the function of treating pancreatic cancer or inhibiting the proliferation and / or invasion and / or migration of pancreatic cancer cells.

2. The application according to claim 1, characterized in that, Treatment of pancreatic cancer or inhibition of the proliferation and / or invasion and / or migration of pancreatic cancer cells includes at least one of A1 to A3: A1. Inhibit the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the GCKR gene; A2. Inhibit or reduce the content, activity, and / or function of GCKR protein; A3. Inhibits the activity of GCKR gene remote enhancers; The GCKR gene remote enhancer is selected from any one of the nucleotide sequences shown in SEQ ID NO.7~11 and their reverse complementary sequences.

3. Use of GCKR inhibitors in the preparation of products for the treatment of pancreatic cancer or for inhibiting the proliferation and / or invasion and / or migration of pancreatic cancer cells.

4. The application according to claim 3, characterized in that, The GCKR inhibitor includes at least one of B1 to B3: B1. Substances that inhibit the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the GCKR gene; B2. Substances that inhibit or reduce the content, activity, and / or function of GCKR proteins; B3. Substances that inhibit the activity of GCKR gene remote enhancers; The GCKR gene remote enhancer is selected from any one of the nucleotide sequences shown in SEQ ID NO.7~11 and their reverse complementary sequences.

5. The application according to claim 4, characterized in that, The GCKR inhibitor includes at least one of C1 to C3: C1. Nucleic acid molecules used to silence or knock out the GCKR gene; C2, Antibodies that bind to GCKR protein; C3. Nucleic acid molecules used to inhibit the activity of GCKR gene remote enhancers.

6. The application according to claim 5, characterized in that, The GCKR inhibitor includes at least one of D1 to D7: D1. An RNA interference molecule targeting the GCKR gene, the RNA interference molecule being the sequence shown in SEQ ID NO.2; D2. sgRNA targeting the exon of the GCKR gene, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO.3 and / or SEQ ID NO.4; D3, the DNA molecule encoding the sgRNA described in D2; D4. An expression cassette, recombinant vector, or recombinant host cell containing the sgRNA described in D2 and / or the DNA molecule described in D3; D5. sgRNA targeting the remote enhancer of the GCKR gene, wherein the nucleotide sequence of the sgRNA is at least one of those shown in SEQ ID NO.12~21; D6. The DNA molecule encoding the sgRNA described in D5; D7. An expression cassette, recombinant vector, or recombinant host cell containing the sgRNA described in D5 and / or the DNA molecule described in D6.

7. A biomaterial, characterized in that, The biomaterial is at least one of E1 to E10: E1, the RNA interference molecule as described in claim 6; E2, the sgRNA targeting the GCKR gene exons as described in claim 6; E3, the sgRNA targeting the GCKR gene remote enhancer as described in claim 6; E4, the DNA molecule encoding the sgRNA described in E2; E5, the DNA molecule encoding the sgRNA described in E3; E6. An expression cassette containing the DNA molecule described in E4 or E5; E7, a recombinant vector containing the DNA molecule described in E4 or E5, or a recombinant vector containing the expression cassette described in E6; E8, a recombinant host cell containing the DNA molecule described in E4 or E5, or a recombinant host cell containing the expression cassette described in E6, or a recombinant host cell containing the recombinant vector described in E7.

8. A composition for editing the remote enhancer of the GCKR gene, characterized in that, Includes the E3 or E5-containing biomaterials as described in claim 7, and a fusion protein composed of dCas9 and a recruitable histone methyltransferase, the amino acid sequence of which is shown in SEQ ID NO. 5; The biomaterials include expression cassettes, recombinant vectors, or recombinant host cells.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the biomaterial of claim 7 or the composition of claim 8.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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