Biomarker for predicting or judging effect of KLK1 protein in treatment of type 2 diabetes mellitus by exenatide and application of biomarker

Abstract

The invention provides a biomarker for predicting or judging the effect of KLK1 protein on treatment of type 2 diabetes mellitus by exenatide, the biomarker is a GG genotype in a KLK1rs5516 site, and aims to predict or judge the clinical effect of treatment of the type 2 diabetes mellitus so as to help judge whether a diabetic patient can benefit from drug treatment or not. A new target spot is provided for the treatment of the type 2 diabetes mellitus by the exenatide, the application range of the exenatide in the treatment of the type 2 diabetes mellitus is widened, and the application conditions of the exenatide in the treatment of the type 2 diabetes mellitus are expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of clinical pharmacy and molecular biology, and specifically relates to a biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes by exenatide and its application. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by hyperglycemia, mainly due to target organ resistance to insulin or pancreatic beta cell dysfunction, resulting in relative insulin deficiency. In 2022, the American Diabetes Association (ADA) classified diabetes into four main types based on different pathogenesis: type 1 diabetes, type 2 diabetes, specific types of diabetes caused by other causes (such as monogenic diabetes syndrome, exocrine pancreatic disease, drug- or chemical-induced diabetes) and gestational diabetes, of which type 2 diabetes accounts for 90-95% of all cases. Type 2 diabetes (T2DM), also known as non-insulin-dependent diabetes, has a multi-gene and multi-factorial pathogenesis, and genetics, lifestyle habits and acquired health conditions affect the development of the disease. Studies have shown that diabetes has reached epidemic proportions, affecting more than 170 million people worldwide. Common symptoms of type 2 diabetes include frequent urination, thirst and excessive fluid intake, fatigue, blurred vision, abnormal weight loss, increased hunger and slow healing of ulcers. The medical and socioeconomic burden of the disease is caused by related complications, which puts tremendous pressure on the health care system. Environmental factors, lifestyle and genetic factors are the factors that induce the onset of type 2 diabetes.

[0003] Numerous studies have shown that islet inflammation and islet dysfunction are major factors leading to type 2 diabetes. When pancreatic β cells cannot adapt to the increased insulin demand due to insulin resistance, islet dysfunction occurs, leading to type 2 diabetes. Islet inflammation is characterized by the presence of cytokines and immune cells, β cell apoptosis, fibrosis, and amyloid deposition. This pathological change is associated with decreased β cell function and impaired insulin secretion. Patients with islet dysfunction have increased immune cells, cytokines, and chemokines, and islet immune cell infiltration has been observed in animal models of islet dysfunction. Cytokines play an important role in the damage and death of islet cells. The three major cytokines (IL-1β, TNF-a, and IFN-γ) most likely work together in the pancreatic immune infiltration process to induce β cell damage and apoptosis, and they play an important role in the pathogenesis of diabetes.

[0004] KLK1, full name Kallikrein-1, kallikrein 1, is 262 amino acids in size. Kallikrein is a subgroup of serine proteases with multiple physiological functions. Increasing evidence shows that many kallikreins are associated with carcinogenesis, and some have the potential to serve as new biomarkers for cancer and other diseases. This gene is one of the 15 kallikrein subfamily members located in the chromosome 19 cluster. This protein is functionally conserved because it can release the vasoactive peptide Lys-bradykinin from low molecular weight kinins. In 1909, Abelous et al. first reported that intravenous injection of human urine can cause a transient drop in blood pressure in dogs, and found antihypertensive substances in urine. In 1930, Kraut et al. discovered high concentrations of this substance in the pancreas and named it "Kallikrein", or kallikrein (KLK). In the past 30 years, with the development and application of molecular biology and cell biology techniques, it has been discovered that the kallikrein kinin system (KKS) is a complex endogenous multi-enzyme system that participates in regulating the physiological functions of the cardiovascular, kidney, and nervous systems, and is closely related to the occurrence of heart disease, kidney disease, inflammatory response, cancer and other diseases. Research on the cardiovascular system has progressed rapidly, and many clinical studies and basic experiments have confirmed that the occurrence of diseases such as diabetes, hypertension, heart failure, myocardial infarction and left ventricular hypertrophy is related to the reduction of KKS activity. Therefore, in-depth research on the role of KKS provides another new way to study the pathogenesis and treatment of cardiovascular-related diseases.

[0005] Literature reports show that KLK1 is expressed in 175 diseases including vascular disease, chronic kidney disease, prostatitis and aortic aneurysm, but there is no clear and detailed research on T2DM. According to NCBI query, KLK1 is highly expressed in the pancreas, but the mechanism of KLK1 in the treatment of type 2 diabetes with exenatide is not clear. Therefore, it is urgent to identify and develop biomarkers that can predict the efficacy of KLK1 protein in the treatment of type 2 diabetes with exenatide, so as to achieve the good purpose of personalized medication. Summary of the invention

[0006] The purpose of the present invention is to provide a biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes by exenatide on the basis of the prior art, aiming at the problem of the effect of exenatide in the treatment of type 2 diabetes. The biomarker is the GG genotype in the KLK1rs5516 locus, which is intended to predict or judge the clinical effect of the treatment of type 2 diabetes, so as to help judge whether the diabetic patient can benefit from the drug treatment, provide a new target for the treatment of type 2 diabetes by exenatide, and expand the scope of use of exenatide in the treatment of type 2 diabetes.

[0007] The second object of the present invention is to use the GG genotype in the KLK1rs5516 locus as a biomarker for predicting or judging the therapeutic effect of exenatide on patients with type 2 diabetes.

[0008] The third object of the present invention is a kit for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes with exenatide, the kit comprising a biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes with exenatide, wherein the biomarker is the GG genotype in the KLK1rs5516 site.

[0009] The technical solution of the present invention is as follows:

[0010] A biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes with exenatide, wherein the biomarker is the GG genotype in the KLK1rs5516 site, and the gene sequence of the KLK1rs5516 site is shown in SEQ ID No. 1, specifically as follows: GTGAGATGCA GTTGTCCCTG GATGGGCGGG GGAAGGAGGG GGAGCTGGGGCACTGATGGG GCGAGTGGCT ACAGCTAGCT GGGAAGCAGT GAAGCAGATG CCTGGTTAGC TCTCAGAAGCCAGTTCAGAG GCTGAGTCCC CTCCCTCAGC CCTTCCAGAC CCTGGGGGCA GGGCTGCCTC ACCACACAGGTGTCTTTGCC ACCTTCCAGG TGTCCGACAC ACAGCATGAAGTCTGTCACC TTCTGGACGT GGGCTTTTTTGCACTCATCA TTAGGCAGGA TTTTGAGGTC CACACACTGG AGATCATCTG GAAATGAGAC TACGAACCCGGGAAAAAGGGCTGCAGCC CGACCTCACC TGCTTCCCTG GCCCTTTCTC CCTTGGACGCAGGAGTCCCCATCCCGCCT TGGGCTACAC AGTCTGCCCC CACATACAATTCTCTGGTTC GATGCTGCCC CAGCCGGAAGCCAAACAGGT GCTCCCCACTTCGGGTTCCT SGGTGGGCAA CTCCACGACC TTCACAGCAT CTGTGATGGTATCAGCAGGC TCTGTCAGGC GGAGCAGCAT GAGGTCGTGG CTGTAGTCCT CGTCTGCTTG GCGGGTGTGGTTCTCCAGGA GGCTCATGTT GAAGCCAGGG TGTGGGAAGC TCTCACTGAC ATGAACAAAC TGGGCTGTGTTTTCGTCGTC AAACAAGTTG TGGCGACCCAGCCAGAGCTG GTAATTGCTG GGGAAAGATG GGAATGGAGGGATGAGAAGACGGGAAGGGG AAAAGGGGAT GGGGCAGGGG AGCATGGGGGG AGGGTCCCCAAAGAGAGAAAGGGAAAGAGAAAAATGTGGG TGGGCGGAGA CAGAGGAAGA AAGATATAGT TATGGAGAAA TACAGACAGCGAATGGGAAG ATGAAAGATT TTGAGAGTGC AAAAGGCAAATGAAGAGACA GAGCAAAAAA GAGCTCAAAAGGACAGAGGG GTGAAAAAGG CGGAGGGGAG GGCAGTGAGAA; in the above sequence, the S at the 501st base position is an allele mutation of C or G, which causes enzyme cleavage site polymorphism; wherein, the CC genotype is the wild gene of the nucleic acid chain; the CG genotype and the GG genotype are the mutant genes of the nucleic acid chain.

[0011] The present invention also provides the use of the GG genotype in the KLK1rs5516 site as a biomarker for predicting or judging the therapeutic effect of exenatide on patients with type 2 diabetes. The gene sequence of the KLK1rs5516 site is shown in SEQ ID No. 1. In the above sequence, S at the 501st base position is an allele mutation of C or G, and the mutation causes polymorphism of the enzyme cleavage site; wherein the CC genotype is the wild gene of the nucleic acid chain; the CG genotype and the GG genotype are mutant genes of the nucleic acid chain.

[0012] The present invention also provides a kit for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes by exenatide, the kit comprising a biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes by exenatide, the biomarker being a GG genotype in the KLK1rs5516 site, the gene sequence of the KLK1rs5516 site being as shown in SEQ ID No. 1, in the above sequence, S at the 501st base position is an allele mutation of C or G, and the mutation causes a polymorphism of an enzyme cleavage site; wherein the CC genotype is a wild gene of the nucleic acid chain; the CG genotype and the GG genotype are mutant genes of the nucleic acid chain.

[0013] In one embodiment of the present invention, we collected 84 blood samples from T2DM patients from the Affiliated Hospital of Xuzhou Medical University, performed SNP genotyping, and found that the KLK1rs5516 locus was GG, GC, and CC. After analyzing these three genotypes, we found that compared with the GC+CC genotype, patients carrying the GG genotype were able to improve T2DM patients' blood sugar, blood lipids, islet function and other functions after using exenatide for 6 months. Therefore, it is concluded that patients with the GG genotype in the KLK1rs5516 locus have significant effects after treatment with exenatide.

[0014] In a specific embodiment, we first performed Western Blot experiments on the pancreatic tissue of mice with successful modeling to verify that KLK1 was significantly expressed in the pancreatic tissue of diabetic mice. The diabetic mice were purchased from Nanjing Jicui Yaokang Biological Co., Ltd. and were divided into three groups: db / m, db / db, and db / db+Ex. The verification method specifically includes the following steps:

[0015] To verify the effects of high glucose environment and exenatide treatment on KLK1 protein expression in pancreatic tissue of mice, immunofluorescence double staining and Western-blot experiments were used to detect KLK1 protein expression in pancreatic tissue of db / m mice, db / db mice and exenatide-treated db / db mice (db / db+Ex). Western-blot results showed that compared with db / m mice, the expression level of KLK1 protein in pancreatic tissue of db / db mice was significantly increased (P<0.05), while the expression level of KLK1 protein in db / db mice was significantly decreased after exenatide treatment (P<0.05).

[0016] To verify the effects of high-glucose environment and exenatide on KLK1 protein expression in INS-1 cells, we planned to divide them into a control group (11.1mmol / L glucose), a MA group (33.3mmol / L mannitol), a HG group (33.3mmol / L glucose) and an exenatide treatment group (33.3mmol / L glucose + 100nmol / L exenatide). INS-1 cells were treated with complete medium containing high glucose for 48h to simulate the high-glucose environment in vivo. At the same time, exenatide was treated for 48h in the same high-glucose environment. To exclude the influence of osmotic pressure, a mannitol group was set up simultaneously. The changes in intracellular KLK1 protein expression were detected by Western-blot experiments. The results showed that compared with the control group, there was no significant difference in KLK1 protein expression in the MA group, and the KLK1 protein expression in the HG group was upregulated. After exenatide treatment, the upregulation of KLK1 protein expression caused by high glucose could be reversed, and the difference was statistically significant. The above results indicate that under high glucose conditions, KLK1 protein expression is upregulated, and exenatide can reverse the downregulation of KLK1 protein expression caused by high glucose.

[0017] We subsequently found that the genotypes of the KLK1rs5516 locus are divided into three types: GG, CC and CG. Among them, different genotypes of the KLK1rs5516 locus have significantly different effects on the efficacy of exenatide. Compared with the KLK1rs5516 CC and CG genotypes, patients with the GG genotype had significantly improved islet function, and significantly reduced fasting blood glucose and postprandial blood glucose after exenatide treatment, which has significant guiding significance for the use of exenatide. For example, after detecting different genotypes of the KLK1rs5516 locus, patients with the GG genotype can be guided in medication, and patients with the CC or CG genotype can be guided in the use of other types of drugs.

[0018] The technical solution of the present invention has the following advantages:

[0019] The present invention provides a biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes by exenatide. The biomarker is the GG genotype in the KLK1rs5516 locus, and is intended to predict or judge the clinical effect of using exenatide to treat type 2 diabetes, so as to help judge whether the diabetic patient can benefit from drug treatment, provide a new target for exenatide to treat type 2 diabetes, broaden the scope of use of exenatide to treat type 2 diabetes, and expand the conditions for use of exenatide to treat type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the genotyping spectrum of the KLK1rs5516 locus;

[0021] Figure 2 The effect of different genotypes of KLK1rs5516 on the efficacy of exenatide;

[0022] Figure 3 The effect of exenatide on body weight, blood glucose and pancreatic islet function of db / db mice; the data are expressed as mean ± standard error; Figure 3 A is the dynamic changes of the body weight of mice after eight weeks of administration; Figure 3 B is AUC, the area under the curve of the change in mouse body weight after 8 weeks of administration; Figure 3 C is the weight of pancreatic tissue of mice after eight weeks of administration; Figure 3 D is AUC, the area under the curve of pancreatic tissue weight of mice after eight weeks of administration; Figure 3 E is the dynamic changes of blood glucose in mice after eight weeks of administration; Figure 3 F is the area under the curve of the weight change of mice after eight weeks of administration; Figure 3 G is the fasting insulin level of mice after eight weeks of administration; Figure 3 H: OGTT experiment, blood glucose time curve; Figure 3 I: AUC, area under the blood glucose level curve; Figure 3J: ITT experiment, blood glucose time curve; Figure 3 K is AUC, area under the blood glucose level curve; compared with the db / m group, *P<0.05, **P<0.01, compared with the db / db group, # P<0.05, ## P < 0.01;

[0023] Figure 4 is the expression level of KLK1 in the pancreatic tissue of diabetic mice: Figure 4 A is Western Blot (WB) analysis of KLK1 in pancreatic tissue of diabetic mice; Figure 4 B is the immunofluorescence double staining (IF) experiment of KLK1 in the pancreatic tissue of diabetic mice; compared with the db / m group mice, *** P<0.001; compared with db / db group mice, # P < 0.05;

[0024] Figure 5 is the expression level of KLK1 in INS-1 cells of the diabetic model: Figure 5 A is to verify the effects of high glucose environment and exenatide on KLK1 protein expression in INS-1 cells. Figure 5 B shows the expression changes of KLK1 in INS-1 cells in immunofluorescence experiments;

[0025] Figure 6 Based on the overexpression of KLK1, WB analysis of KLK1 in INS-1 cells was performed to simulate the disease state and treat with exenatide;

[0026] Figure 7 The effect of overexpression of KLK1 in INS-1 cells on exenatide regulation of pancreatic islet function; Figure 7 AA is the KSIS value (the ratio of the insulin content produced by high sugar stimulation to the insulin content produced by low sugar); Figure 7 B is (orange bar graph, low sugar 3.3mmol / L; green bar graph, high potassium 50mmol / L), ELISA method to detect insulin secretion, and corrected by insulin content; Figure 7 C is the insulin content of INS-1 cells, corrected by the total amount of cellular DNA; mean ± standard error, n = 4. * P<0.05, ** P<0.01; compared with KLK1-OE, # P<0.05, ## P<0.01; compared with Ex, $ P<0.05, $$ P < 0.01;

[0027] Figure 8 Based on the knockdown of KLK1, WB analysis of KLK1 in INS-1 cells was performed by simulating the disease state and then treating with exenatide;

[0028] Fig. 9 The effect of knocking down KLK1 on exenatide regulation of islet function in INS-1 cells; Fig. 9 A is the KSIS value (the ratio of the insulin content produced by high sugar stimulation to the insulin content produced by low sugar); Fig. 9 B is (orange bar graph, low sugar 3.3mmol / L; green bar graph, high potassium 50mmol / L), ELISA method to detect insulin secretion, and corrected by insulin content; Fig. 9 C is the insulin content of INS-1 cells, corrected by the total amount of cell DNA. Mean ± standard error, n = 4; compared with veh, * P<0.05, ** P<0.01; compared with KLK1-KD, # P<0.05, ## P<0.01; compared with Ex, $ P<0.05, $$ P<0.01. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of them. 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.

[0030] The present invention provides KLK1rs5516 as a protein target for predicting the efficacy of exenatide in treating T2DM. The present invention studies the role of KLK1 in the efficacy of exenatide in treating type 2 diabetes from the perspective of clinical, diabetic model tissue and in vitro cell levels. First, SNP site typing was performed on clinical blood samples of patients with type 2 diabetes treated with exenatide, and the results showed that the KLK1rs5516 site had significant differences in the process of exenatide treating T2DM. Secondly, at the level of pancreatic tissue in mice of the diabetic model, WB experiments verified that KLK1 was significantly upregulated in the diabetic model. Finally, at the rat INS-1 cell level, a diabetic disease cell model was constructed using HG and exenatide co-stimulation, and it was found that KLK1 expression was upregulated under HG, and after exenatide treatment, the upregulation of KLK1 protein expression caused by high sugar could be reversed, that is, KLK1 was used as a new protein target for detecting exenatide in treating T2DM, which provided a new idea for the efficacy of exenatide in treating T2DM.

[0031] Example 1: SNP site for KLK1 genotyping

[0032] Experimental steps:

[0033] Blood samples from 84 patients with T2DM were collected from the Affiliated Hospital of Xuzhou Medical University. 1 μl of each DNA sample was subjected to 1% agarose electrophoresis for quality inspection and concentration estimation, and then the sample was diluted to a working concentration of 10-30 ng / μl based on the estimated concentration.

[0034] 1. Multiplex PCR reaction:

[0035] rs5516

[0036] Upstream primer: seq(5′-3′):AGGTGTCTTTGCCACCTTCCA;

[0037] Downstream primer: seq(5′-3′):GCCTGACAGAGCCTGCTGATA;

[0038] Concentration of each primer pair in multiplex PCR primers (Probe Mix) (μM):

[0039] Preparation of PCR reaction premix: Prepare the premix according to the table below. Considering the loss caused by adsorption of the pipette tip during aliquoting, it is recommended to prepare 100 samples for each 96 samples.

[0040]

[0041] 2. PCR reaction: Prepare the PCR reaction premix, shake and centrifuge thoroughly to mix, then dispense into 96-well plates, 19 μl per reaction well, then add 1 μl of DNA sample to each reaction well, cover with film, shake and mix slightly, then centrifuge at 3000 rpm for 0.5 minutes, then immediately (the sooner the better) place the 96-well plate on the PCR instrument after centrifugation, remove the film, then cover with the 96-well rubber cover mat, cover the PCR instrument hot cover, and run according to the following program:

[0042]

[0043]

[0044] 3. Multiplex PCR product purification

[0045] 5U SAP enzyme and 2U exonuclease I enzyme were added to 20μl PCR product, incubated at 37℃ for 1 hour, and then inactivated at 75℃ for 15 minutes.

[0046] SNaPshot multiplex single base extension reaction

[0047] a) Extension primer

[0048] SNP site ID number: rs5516;

[0049] Primer name: rs5516SF;

[0050] seq(5'-3'):TTTTTTTTTTTTTTTTTTTTTTGCTCCCCACTTCGGGTTCCT;

[0051] Function: Encoding the formation of rs5516 gene.

[0052] b) Concentration of each pair of primers in the extension reaction multiple extension primers (Extended Primer Mix) (μM): Prepare the extension reaction premix according to the table below. Considering factors such as loss caused by adsorption of the pipette tip during aliquoting, it is recommended to prepare 100 samples for each 96 samples.

[0053] Reagents Dosage Dosage SNaPshot Multiplex Kit (ABI) 5.00μl 500μl Extension substrate mixture 1.00μl 100μl Nuclease-free water 2.00μl 200 μl Total volume 8μl 800μl

[0054] 4. Extension reaction: After fully shaking and centrifuging the prepared extension reaction premix, dispense it into a 96-well plate, dispense 8 μl into each reaction well, then add 2 μl of the multiplex PCR purified product into each reaction well, cover with a film, shake lightly and mix, centrifuge at 3000 rpm for 0.5 minutes, then immediately (the sooner the better) place the 96-well plate on the PCR instrument after centrifugation, remove the film, cover with a 96-well rubber cover mat, cover the PCR instrument hot cover, and run according to the following program:

[0055]

[0056]

[0057] 5. Purification of extension products

[0058] 1U SAP enzyme was added to 10μl of the extension product, incubated at 37℃ for 1 hour, and then inactivated at 75℃ for 15 minutes.

[0059] 6. Load the extension product onto ABI3730XL sequencer

[0060] Take 0.5 μl of each purified extension product, mix it with 0.5 μl Liz120 SIZE STANDARD and 9 μl Hi-Di, denature it at 95°C for 5 minutes, and then load it on ABI3730XL sequencer.

[0061] 7. Data Analysis

[0062] The raw data collected on the ABI3730XL sequencer were analyzed using the software GeneMapper 4.1 (Applied Biosystems, USA).

[0063] like Figure 1 As shown, the genotypes of the KLK1rs5516 locus are divided into three types: GG, CC and CG, and the genotype maps of the KLK1rs5516 locus GG, GC, and CC are shown in the figure.

[0064] Example 2: Effects of high glucose environment and exenatide treatment on KLK1 protein expression in mice

[0065] Experimental methods

[0066] 1. Reagents

[0067] RIPA lysis buffer, PMSF, and SDS-PAGE protein loading buffer were purchased from Shanghai Biotech Research Institute; Western primary antibody diluent and Western secondary antibody diluent were purchased from Wuhan Savill Biotechnology Co., Ltd.; colored pre-stained protein markers were purchased from Thermo Fisher, USA; NC membranes were purchased from Millipore, USA; bovine serum albumin was purchased from Beijing Solebold Technology Co., Ltd.; Tween-20 was purchased from Biosharp; SDS-PAGE gel preparation kit was purchased from Nanjing Novozymes Biotech Co., Ltd.; KLK1 antibody was purchased from Affinity Biosciences; β-actin antibody was purchased from Proteintech Group; glucose was purchased from Beijing Sinopharm Group; methanol was purchased from Shanghai Test; RPMI 1640 medium, fetal bovine serum, PBS, trypsin-EDTA digestion solution, and penicillin-streptomycin solution (100×) were purchased from Nanjing Senbega Biotechnology Co., Ltd.

[0068] 2. Animals

[0069] Before conducting animal experiments, all experimental personnel obtained the Jiangsu Province Laboratory Animal Training Certificate, and the animal experiment plan was approved by the Animal Ethics Committee of Xuzhou Medical University and obtained the ethics number. db / m micedb / db mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and male mice C57BL / 6J were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0070] 3. Cell Culture

[0071] INS-1 cells were cultured in RPMI 1640 complete medium (complete medium contains 10% fetal bovine serum, 11.1 mmol / L glucose, 50 μmol / L β-mercaptoethanol, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 mmol / L HEPES, 1 mmol / L sodium pyruvate) and placed in a 5% CO2, 37°C constant temperature incubator. The medium was changed every other day and the cells were passaged depending on their growth status.

[0072] Cell model construction

[0073] 3.1 Cell treatment

[0074] (1) High-glucose environment: When the cell density was between 80% and 90%, glucose or mannitol was added to the INS-1 cells in the high-glucose group and the mannitol group to a concentration of 33.3 mmol / L. The cells in the control group were blank treated to keep the same operation steps. After treatment, the cells were cultured in an incubator for 48 h and the cells were collected after treatment.

[0075] (2) Exenatide treatment: When the cell density was between 80% and 90%, exenatide (100 nmol / L) was administered. The control group was given a blank treatment to keep the same operation steps. After treatment, the cells were placed in an incubator and cultured for 48 h. After the treatment, the cells were collected.

[0076] 3.2 Cell lentiviral transfection

[0077] (1) Before preparing for the infection experiment, the cells were passaged and transferred to a 6-well plate to ensure that the cell density would not be too high during the infection process.

[0078] (2) Before the infection experiment begins, take the lentivirus out of the -80°C freezer and place it in an incubator filled with ice to slowly thaw.

[0079] (3) Calculate the required lentiviral dose according to the MOI value recommended in the instructions, dilute with culture medium, and transfer to a 6-well plate for transfection.

[0080] (4) After the infection operation is completed, the time is set and the culture medium is replaced after 12 hours. During the transfection period, the cell status is carefully observed.

[0081] (5) Observe the transfection efficiency under a fluorescence microscope 48-72 hours after infection and add puromycin for screening if necessary.

[0082] (6) After successful transfection, cells should be frozen in time.

[0083] 4. Western-blot experiment

[0084] 4.1 Protein determination

[0085] (1) Prepare lysis buffer according to the ratio of RIPA:PMSF:phosphatase inhibitor = 100:1:2.

[0086] (2) Add lysis buffer and mix well by pipetting. Vortex on a vortexer for 15 seconds. Place on ice or in a 4°C refrigerator for lysis. Vortex every 10 minutes for a total of 20 minutes.

[0087] (3) Centrifuge at 12000g, 4°C for 15 min.

[0088] (4) After centrifugation, use a pipette to aspirate the supernatant, trying to avoid contact with the precipitate during the aspiration process, and record the volume of the aspirated supernatant.

[0089] (5) Use an instrument to measure its concentration.

[0090] (6) Add 1 / 4 volume of SDS-PAGE protein loading buffer (5X) and vortex to mix.

[0091] (7) 100℃ metal bath for 15 min, then store in a -20℃ refrigerator.

[0092] 4.2 Electrophoresis

[0093] (1) Leak detection: After the glue rack is assembled, add distilled water. After 5 minutes, check whether the liquid level in the glass plate has dropped significantly. If not, pour out the distilled water and invert the glue rack to drain the water.

[0094] (2) Glue preparation: prepare the lower layer of glue according to the formula in the instruction manual. After mixing on a vortex machine, slowly add it to the center of the glass plate. Add about 5 mL to each glass plate. Add distilled water or anhydrous ethanol to the upper layer. After standing for 30 min, when a clear boundary appears between the lower and upper layers of liquid, pour out the upper layer of liquid and invert the glue preparation rack to drain the water. Prepare the upper layer of glue according to the instruction manual. After adding the upper layer of glue, insert the sample comb. When a gap appears between the sample comb and the upper layer of glue, remove the glass plate.

[0095] (3) Electrophoresis: Assemble the configured glass plate in the electrophoresis tank (with the short plate facing inward), pour the electrophoresis solution so that it covers the short plate, and pull out the sample comb vertically; complete the sample loading according to the experimental design, and move it into the outer electrophoresis tank after the sample loading is completed. Add an appropriate amount of electrophoresis solution to the outer tank, preferably covering the inner end of the electrophoresis tank. Set the program, S1: 60V, 30min, S2: 100V, 70min.

[0096] (4) Transfer solution: Add 20 ml of methanol to 1× transfer solution. Soak the filter paper and NC membrane in the prepared transfer solution.

[0097] (5) After the electrophoresis process is completed, remove the glass plate and cut the gel according to the target molecular weight.

[0098] (6) Transfer membrane, 100 V, 50 min.

[0099] (7) After the transfer is completed, the NC membrane is placed in a blocking solution (30 ml PBS plus 0.9 g bovine serum albumin) and blocked for 1 h.

[0100] (8) Dilute the primary antibody at a ratio of 1:1000. Place the blocked strips in a primary antibody incubation box and place in a 4°C refrigerator overnight.

[0101] (9) The next day, remove the strips and place them on a shaker for washing. Wash with PBST for 5 min each time, for a total of 3 times.

[0102] (10) Prepare the secondary antibody in the right proportion under light-proof conditions. After washing, place the strips in an incubation box containing the secondary antibody and incubate for 1 h.

[0103] (11) After incubation, place the membrane on a shaker to wash twice with PBST for 5 min and once with PBS for 5 min.

[0104] (12) After washing, the NC membrane was immersed in PBS liquid and then transferred to the instrument for scanning.

[0105] 5. Potassium chloride stimulated insulin secretion (KSIS) experiment

[0106] 5.1 Solution preparation

[0107] (1) Prepare KRBH solution.

[0108] KRBH solution formulation

[0109]

[0110] After each component was dissolved, it was sterilized by filtration and stored at 4°C.

[0111] (2) Preparation of acid ethanol solution: 100 ml of the solution contains 74 ml of anhydrous ethanol, 1.4 ml of hydrochloric acid and 24.6 ml of double distilled water.

[0112] 5.2KSIS Functional Experiment

[0113] (1) After the islets or cells are treated, wash the culture medium in the plate with a pipette (taking a 24-well plate as an example), add 400 μl of KRBH solution to each well, move to a constant temperature incubator, and time for 1 hour.

[0114] (2) After the timing is over, the supernatant in the plate is aspirated with a pipette, and 400 μl of KRBH solution containing 3.3 mmol / L glucose is added. After incubation in a constant temperature incubator for 1 h, the liquid in the plate is collected with a pipette and stored in a -80°C refrigerator.

[0115] (3) After washing the supernatant, add blank KRBH solution for cleaning. Add 400 μl of KRBH solution containing 50 mmol / L potassium chloride to each well and incubate in a constant temperature incubator for 1 h. Collect the clean supernatant with a pipette and store in a -80°C refrigerator.

[0116] (4) Digest the cells with enzymes, transfer to EP tubes and centrifuge, discard the supernatant, add 400 μl of acid ethanol, and place in a 4°C refrigerator overnight; centrifuge the next day, collect the supernatant and cells, and store in a -80°C refrigerator.

[0117] (5) Insulin content determination: Strictly follow the instructions of the ELISA kit and DNA extraction kit to detect insulin content, extract intracellular DNA, and use an instrument to detect the DNA content for correction of insulin content.

[0118] 6. Tissue sample collection of diabetic model mice

[0119] Nanjing Jicui Pharmaceutical Technology Co., Ltd. purchased diabetic mice, three groups: db / m, db / db and db / db+Ex, and subcutaneously injected exenatide at a dose of 50μg / 10g for animal modeling. The blood glucose and body weight of the mice were measured every week, and the OGTT experiment was performed in the seventh week, and the ITT experiment was performed in the eighth week. After two days of stabilization, the pancreatic tissue of the mice was obtained and stored in a -80℃ refrigerator for later use.

[0120] 7. Extraction and concentration determination of total protein in mouse pancreatic tissue samples

[0121] Weigh a certain weight of mouse pancreatic tissue (generally about 0.02g) in a 1.5ml Eppendorf tube using an analytical balance, add 9 times the volume of tissue weight containing PMSF tissue lysis buffer (radioimmunoprecipitation buffer: phenylmethylsulfonyl fluoride = 50:1), chop the tissue with scissors in an ice water bath, homogenize with a homogenizer, incubate on ice for 30min (vibrate once every 10min), then centrifuge at 4°C, 12000g for 15min, carefully aspirate the supernatant, and record its volume. Use Nano Drop one micro-volume UV-Vis spectrophotometer to determine the protein concentration.

[0122] 8. Protein expression in pancreatic tissue samples detected by Western blotting

[0123] Add SDS-PAGE protein loading buffer (5X) to the protein sample with a determined concentration, boil it at 100℃ for 12min to denature it, and store it in a -20℃ refrigerator waiting for loading. Prepare gel according to the instructions of the SDS-PAGE gel rapid preparation kit. After gelation, add different volumes of protein samples according to the total amount of 100μg of protein, add electrophoresis solution, and perform constant voltage electrophoresis (100V, 80min). After running, cut the gel according to the protein molecular weight.

[0124] Transfer: Use a wet transfer instrument to transfer the membrane and set the transfer conditions (100V, 60min)

[0125] Blocking: Place the NC membrane in 3% BSA blocking solution and incubate on a shaker for 1 h;

[0126] Incubate with primary antibody: Place the blocked NC membrane in the primary antibody diluted in advance (1:1000) and incubate overnight at 4°C. The next day, take out the NC membrane from the refrigerator and place it on a shaker. Wash it quickly on a shaker with PBST, 5 minutes each time, for a total of 3 times.

[0127] Place the secondary antibody incubation solution (dilution ratio 1:10000) in the dark box and soak the NC membrane, incubate at room temperature for 1 hour. After the end, quickly wash the NC membrane with PBST on a shaker, wash for 5 minutes each time, for a total of 2 times, and then wash once with PBS for 5 minutes, and protect from light throughout the process. After washing, place the strips in a dark box filled with PBS. Scan with the Odyssey CLX dual-color infrared laser imaging system. Result analysis: Use Image J to calculate the gray value of the protein bands and statistically analyze the relative expression of the target protein.

[0128] 9. Immunofluorescence detection of KLK1 protein expression in pancreatic tissue samples

[0129] The pancreatic tissue was removed from the 4% paraformaldehyde fixative and cleaned with PBS. It was placed in a pre-frozen cryostat and sliced ​​when the OCT embedding medium changed from transparent to milky white. The slices were cut into 10 micron thickness, then removed and placed on a glass slide, rewarmed for 30 minutes, washed with PBS 3 times, 5 minutes each time, dried, circled with a histochemical pen, blocked with 5% BSA at room temperature for 1h 30mim, dried with 5% BSA and applied with primary antibody, and kept in a refrigerator at 4°C overnight. The next day, the tissue sections were equilibrated at room temperature for 30 minutes, washed with PBS for 5 minutes, a total of 3 times, excess water was absorbed with qualitative filter paper, secondary antibody was applied, kept at room temperature away from light for 2 hours, washed with PBS 4 times, 5 minutes each time, a fluorescent quencher containing DAPI was added, and the sections were sealed.

[0130] 10. Data processing and statistical analysis

[0131] SPSS 25.0 software was used to perform statistical analysis on the experimental data, and the data were expressed as mean ± standard error. The independent sample t test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. Both tests were performed with two-sided tests, and P < 0.05 indicated that the difference was statistically significant.

[0132] like Figure 3 As shown: The diabetic db / db model mice were given exenatide for 8 weeks, and OGTT and ITT experiments were performed in the seventh and eighth weeks respectively. Subsequent insulin level tests and other blood sugar and body weight data were analyzed. The results showed that exenatide increased the pancreatic tissue weight of db / db mice and improved the islet function of db / db mice.

[0133] like Figure 4As shown: In db / db mice, KLK1 protein expression was upregulated, and after treatment with exenatide, KLK1 protein expression was downregulated; the fluorescence experimental results were consistent with the WB results.

[0134] like Figure 5 As shown: KLK1 protein expression was upregulated in INS-1 cells under high glucose environment. After administration of exenatide, KLK1 protein expression was downregulated. The fluorescence experimental results were consistent with the WB results.

[0135] like Figure 6 Shown: A KLK1 overexpression model was established in INS-1 cells, based on the overexpression of KLK1, to simulate the disease state, and WB analysis of KLK1 in INS-1 cells was performed by administering exenatide for treatment.

[0136] like Figure 7 As shown: KSIS experiment was performed on INS-1 cells overexpressing KLK1. The results showed that overexpression of KLK1 could reduce the function of exenatide in promoting insulin secretion, and the effect was more obvious under high potassium conditions;

[0137] like Figure 8 Shown: A KLK1 knockdown model was established in INS-1 cells, based on the knockdown of KLK1, to simulate the disease state, and WB analysis of KLK1 in INS-1 cells was performed by administering exenatide for treatment.

[0138] like Fig. 9 As shown: KSIS experiment verification was carried out on INS-1 cells with KLK1 knockdown. The results showed that knocking down KLK1 can enhance the function of exenatide in promoting insulin secretion, and the effect is more significant under high potassium conditions.

[0139] Example 3: Treatment effect of patients with different genotypes in the KLK1rs5516 locus after using exenatide

[0140] Statistical analysis and observation were performed on 84 samples of T2DM patients collected from the Affiliated Hospital of Xuzhou Medical University, and the blood sugar, blood lipids, pancreatic islet function and other functions of each patient were observed after using exenatide for 6 months. It was found that the 84 T2DM patients met the Hardy-Weinberg equilibrium test, as shown below.

[0141]

[0142]

[0143] Note: Observed frequency is: the actual observed genotype frequency

[0144] Expected frequency is: the frequency of a genotype expected under ideal conditions

[0145] At the same time, the data of various indicators of 84 patients were statistically analyzed by SPS25.0S, and the data were expressed as mean ± standard error. The independent sample t test was used for comparison between the two groups, and the one-way analysis of variance was used for comparison between multiple groups. Both two-sided tests were used, and P < 0.05 indicated that the difference was statistically significant. The results showed that exenatide can improve blood sugar, blood lipids, and pancreatic islet function in patients with T2DM, thereby improving the occurrence and development of type 2 diabetes. The results are as follows:

[0146]

[0147] Note: BMI, body mass index; WHR, waist-to-hip ratio; FPG, fasting blood glucose; PPG, postprandial plasma glucose; HbA1c, glycosylated hemoglobin; HOMA-IR, insulin resistance index; HOMA-B, islet function; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

[0148] like Figure 2 The figure shows a statistical analysis of different genotypes of the KLK1rs5516 locus, and it was found that different genotypes of the KLK1rs5516 locus had different effects on the efficacy of exenatide. Specifically, compared with the KLK1rs5516 CC+CG genotype, patients with the GG genotype had significantly improved islet function after exenatide treatment, and their fasting blood glucose and postprandial blood glucose were significantly reduced.

[0149] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements 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. A biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes with exenatide, characterized in that: The biomarker is the GG genotype at the KLK1 rs5516 locus, and the gene sequence of the KLK1 rs5516 locus is as shown in SEQ ID No.1, specifically as follows: GTGAGATGCAGTTGTCCCTGGATGGGCGGG GGAAGGAGGGGGAGCTGGGG CACTGATGGG GCGAGTGGCTACAGCTAGCT GGGAAGCAGT GAAGCAGATG CCTGGTTAGCTCTCAGAAGCCAGTTCAGAG GCTGAGTCCC CTCCCTCAGC CCTTCCAGAC CCTGGGGGCAGGGCTGCCTCACCACACAGG TGTCTTTGCC ACCTTCCAGG TGTCCGACACACAGCATGAA GTCTGTCACC TTCTGGACGTGGGCTTTTTT GCACTCATCATTAGGCAGGATTTTGAGGTC CACACACTGG AGATCATCTGGAAATGAGACTACGAACCCG GGAGAAAAAG GGCTGCAGCC CGACCTCACC TGCTTCCCTGGCCCTTTCTCCCTTGGACGC AGGAGTCCCC ATCCCCGCCT TGGGCTACACAGTCTGCCCC CACATACAAT TCTCTGGTTCGATGCTGCCC CAGCCGGAAGCCAAACAGGT GCTCCCCACT TCGGGTTCCT SGGTGGGCAACTCCACGACCTTCACAGCAT CTGTGATGGT ATCAGCAGGC TCTGTCAGGC GGAGCAGCATGAGGTCGTGGCTGTAGTCCT CGTCTGCTTG GCGGGTGTGG TTCTCCAGGAGGCTCATGTT GAAGCCAGGG TGTGGGAAGCTCTCACTGAC ATGAACAAACTGGGCTGTGT TTTCGTCGTC AAACAAGTTG TGGCGACCCAGCCAGAGCTGGTAATTGCTG GGGAAAGATG GGAATGGAGG GATGAGAAGA CGGGAAGGGGAAAAGGGGATGGGGCAGGGG AGCATGGGGG AGGGTCCCCA AAGAGAGAAAGGGAAAGAGAAAAATGTGGG TGGGCGGAGACAGAGGAAGA AAGATATAGTTATGGAGAAATACAGACAGC GAATGGGAAG ATGAAAGATTTTGAGAGTGCAAAAGGCAAA TGAAGAGACA GAGCAAAAAA GAGCTCAAAA GGACAGAGGGGTGAAAAAGGCGGAGGGGAG GGCAGTGAGAA; in the above sequence, the S at the 501st base position is an allele mutation of C or G, which causes enzyme cleavage site polymorphism; wherein, the CC genotype is the wild gene of the nucleic acid chain; the CG genotype and the GG genotype are the mutant genes of the nucleic acid chain.

2. Use of the GG genotype in the KLK1rs5516 locus as a biomarker for predicting or judging the therapeutic effect of exenatide on patients with type 2 diabetes, wherein the gene sequence of the KLK1rs5516 locus is shown in SEQ ID No. 1, and is specifically as follows: GTGAGATGCAGTTGTCCCTG GATGGGCGGG GGAAGGAGGGGGAGCTGGGG CACTGATGGG GCGAGTGGCTACAGCTAGCT GGGAAGCAGTGAAGCAGATG CCTGGTTAGC TCTCAGAAGC CAGTTCAGAGGCTGAGTCCCCTCCCTCAGC CCTTCCAGAC CCTGGGGGCAGGGCTGCCTC ACCACACAGGTGTCTTTGCCACCTTCCAGG TGTCCGACAC ACAGCATGAAGTCTGTCACCTTCTGGACGT GGGCTTTTTTGCACTCATCATTAGGCAGGA TTTTGAGGTCCACACACTGG AGATCATCTG GAAATGAGAC TACGAACCCGGGAAAAAGGGCTGCAGCC CGACCTCACC TGCTTCCCTG GCCCTTTCTC CCTTGGACGCAGGAGTCCCCATCCCCGCCT TGGGCTACAC AGTCTGCCCC CACATACAATTCTCTGGTTC GATGCTGCCC CAGCCGGAAGCCAAACAGGT GCTCCCCACTTCGGGTTCCT SGGTGGGCAA CTCCACGACC TTCACAGCATCTGTGATGGTATCAGCAGGC TCTGTCAGGC GGAGCAGCAT GAGGTCGTGG CTGTAGTCCTCGTCTGCTTGGCGGGTGTGG TTCTCCAGGA GGCTCATGTT GAAGCCAGGGTGTGGGAAGC TCTCACTGAC ATGAACAAACTGGGCTGTGT TTTCGTCGTCAAACAAGTTG TGGCGACCCAGCCAGAGCTG GTAATTGCTGGGGAAAGATGGGAATGGAGG GATGAGAAGACGGGAAGGGGG AAAAGGGGAT GGGGCAGGGGAGCATGGGGGAGGGTCCCCAAAGAGAGAAAGGGAAAGAGAAAAATGTGGGTGGGCGGAGACAGAGGAAGAAAGATATAGTTATGGAGAAATACAGACAGCGAATGGGAAG ATGAAAGATT TTGAGAGTGC AAAAGGCAAATGAAGAGACAGAGCAAAAAAGAGCTCAAAAGGACAGAGGG GTGAAAAAGGCGGAGGGGAG GGCAGTGAGAA; in the above sequence, S at the 501st base position is an allele mutation, S at the 501st base position is C or G, and the mutation causes a polymorphism in the restriction site; wherein, The CC genotype is the wild-type gene of the nucleic acid chain; the CG genotype and the GG genotype are the mutant genes of the nucleic acid chain.

3. A kit for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes with exenatide, characterized in that: The kit includes a biomarker for predicting or judging the effect of KLK1 protein in the treatment of type 2 diabetes with exenatide, wherein the biomarker is a GG genotype in the KLK1rs5516 site, and the gene sequence of the KLK1rs5516 site is shown in SEQ ID No. 1, specifically as follows: GTGAGATGCAGTTGTCCCTGGATGGGCGGG GGAAGGAGGG GGAGCTGGGGCACTGATGGG GCGAGTGGCTACAGCTAGCT GGGAAGCAGT GAAGCAGATG CCTGGTTAGCTCTCAGAAGCCAGTTCAGAG GCTGAGTCCC CTCCCTCAGC CCTTCCAGAC CCTGGGGGCAGGGCTGCCTCACCACACAGG TGTCTTTGCC ACCTTCCAGG TGTCCGACACACAGCATGAAGTCTGTCACC TTCTGGACGTGGGCTTTTTT GCACTCATCA TTAGGCAGGATTTTGAGGTC CACACACTGG AGATCATCTG GAAATGAGACTACGAACCCG GGAGAAAAAG GGCTGCAGCC CGACCTCACC TGCTTCCCTGGCCCTTTCTC CCTTGGACGCAGGAGTCCCC ATCCCCGCCT TGGGCTACACAGTCTGCCCC CACATACAAT TCTCTGGTTC GATGCTGCCCCAGCCGGAAGCCAAACAGGT GCTCCCCACT TCGGGTTCCT SGGTGGGCAA CTCCACGACC TTCACAGCATCTGTGATGGT ATCAGCAGGC TCTGTCAGGC GGAGCAGCATGAGGTCGTGG CTGTAGTCCT CGTCTGCTTGGCGGGTGTGG TTCTCCAGGAGGCTCATGTT GAAGCCAGGG TGTGGGAAGC TCTCACTGACATGAACAAACTGGGCTGTGT TTTCGTCGTC AAACAAGTTG TGGCGACCCA GCCAGAGCTG GTAATTGCTGGGGAAAGATG GGAATGGAGG GATGAGAAGA CGGGAAGGGG AAAAGGGGAT GGGGCAGGGG AGCATGGGGGAGGGTCCCCA AAGAGAGAAAGGGAAAGAGAAAAATGTGGG TGGGCGGAGA CAGAGGAAGA AAGATATAGTTATGGAGAAA TACAGACAGC GAATGGGAAG ATGAAAGATT TTGAGAGTGC AAAAGGCAAA TGAAGAGACAGAGCAAAAAA GAGCTCAAAA GGACAGAGGG GTGAAAAAGG CGGAGGGGAG GGCAGTGAGAA; in the above sequence, the S at the 501st base position is an allele mutation of C or G, which causes enzyme cleavage site polymorphism; wherein, the CC genotype is the wild gene of the nucleic acid chain; the CG genotype and the GG genotype are the mutant genes of the nucleic acid chain.

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