PCSK7 enzyme iron metabolism related new substrate and application thereof

Abstract

The invention discloses a novel substrate of PCSK7 hydrolase and application of the novel substrate in detection of iron overload related diseases. The invention finds that IGF2R may be a new hydrolysis substrate of PCSK7 hydrolase, and IGF2R expression increase caused by IGF2R hydrolysis reduction caused by PCSK7 mutation of a hereditary hemochromic disease patient may be related to the attack of the hereditary hemochromic disease. In particular, the PCSK7 can be used for specifically hydrolyzing the extracellular part of the IGF2R, so that soluble IGF2R (sIGF2R) is formed and can be detected in serum. Therefore, the IGF2R can be used as a molecular marker for detecting and treating iron overload related diseases such as hemochromia, and can be used for detecting sIGF2R in serum of patients with the iron overload related diseases by preparing a clinical diagnostic kit. The kit comprises a solid-phase carrier, an IGF2R antibody coated on the solid-phase carrier, an enzyme-labeled secondary antibody, a standard substance, a chromogenic substrate, a washing solution and a reaction stop solution. The detection kit can be used for specifically and quantitatively detecting the level of sIGF2R in a serum specimen.

Description

[0001] summary

[0002] The present invention relates to a novel substrate of PCSK7 hydrolase and its application. Specifically, the present invention provides IGF2R as a novel substrate of PCSK7 hydrolase that competitively binds to HFE, and the use of IGF2R molecular markers in the preparation of detection reagents for iron overload-related diseases such as hereditary hemochromatosis and as therapeutic drug targets. The present invention also provides a corresponding soluble IGF2R (sIGF2R) detection kit preparation and detection method for the detection of iron overload-related diseases such as hereditary hemochromatosis. Technical Field

[0003] The present invention relates to the application field of diagnostic markers and drug targets. More specifically, the present invention relates to an IGF2R molecular marker that can be used for the detection and treatment of iron overload-related diseases and its application. Background Art

[0004] Hereditary hemochromatosis (HH) is an autosomal genetic disease caused by mutations in genes related to iron metabolism, which results in progressive deposition of excessive iron in the body, leading to dysfunction of organs such as liver cirrhosis. If not diagnosed and treated in time, it will lead to early death or lifelong disability of patients. HH is difficult to diagnose clinically, and genetic diagnosis helps early diagnosis and treatment. Data show that the incidence of HH worldwide is 1.5-3 / 1000 people, and it is a common disease among white people in Northern Europe. According to the latest classification standards, HH is divided into HFE and non-HFE types. Among them, HFE type HH caused by the homozygous mutation of HFE p.C282Y has a higher incidence in European and American countries, reaching 1 / 220-250, and the mutation rate can be as high as 85%-90%. In my country, HH is a rare disease, and there are few studies on mutations in genes related to HH iron metabolism. Most of the literature reports are individual cases or case series, and the gene mutation spectrum remains to be clarified. There are almost no reports on HFE p.C282Y mutations. In the early stage, our research group conducted exome sequencing on a group of primary iron overload patients with unknown pathogenic gene mutations and found that Chinese patients with primary iron overload had a high frequency of proprotein convertase family member PCSK7 gene mutations (5 / 32, 15.6%), including repeated PCSK7 p.V143F and PCSK7 p.T743N gene mutations in unrelated patients, suggesting that this new gene and new mutation found in primary iron overload patients may become potential pathogenic genes and hotspot mutations for HH in my country.

[0005] Proprotein Convertases (PCs) are a family of Ca 2+A general term for PCSK-dependent serine proteases that can restrictively hydrolyze precursor proteins, converting them from a single precursor into a variety of active or inactive products, thereby exerting different physiological functions. Currently known PCs include 9 members: PCSK1 / 3, PCSK2, Furin, PCSK4, PCSK5 / 6, PACE4, PCSK7, SK-1 / S1P and PCSK9, which exert proteolytic effects in different ways. The first 7 members can cleave substrates at single or paired basic residues. Proprotein Convertase Subtilisin / Kexin Type 7 (PCSK7) is a widely expressed transmembrane protease that is highly enriched in the liver. Little is known about PCSK7-specific substrates. TFR1, E-cadherin, IGF1R precursor and EGF precursor are the only reported potential substrates. The body's iron homeostasis is mainly regulated by the liver. HFE and the iron-carrying ferroportin (TF) competitively bind to TFR1. When iron is overloaded, free HFE affects the expression of hepcidin by binding to other ligands, thereby regulating the uptake of iron in the circulatory system. TFR1 is a homodimeric type II membrane protein that can mediate cellular iron uptake, thereby regulating cellular iron homeostasis. When iron is overloaded, TFR1 is released from the HFE / β2 microglobulin / TFR1 complex and binds to transferrin. The released free HFE upregulates the expression of hepcidin to maintain the body's iron homeostasis. However, none of these substrates can explain the relationship between PCSK7 gene mutations and hemochromatosis. There may be other PCSK7 iron metabolism-related hydrolysis substrates.

[0006] The insulin-like growth factor receptor family (IGFRs) includes insulin-like growth factor 1 receptor (IGF1R) and 2 receptor (IGF2R). Tumor-related studies have reported that IGF1R precursor may be one of the hydrolysis substrates of PCSK7 enzyme. It is worth noting that IGF2R is another iron metabolism-related ligand of HFE. When the plasma iron-loaded TF concentration increases, iron-loaded TF competes with HFE for binding to TFR1. Once separated from TFR1, HFE can bind to IGF2R in the mannose-6-phosphate / IGF2R complex (M6P / IGFI2R) and release M6P at the same time. Our previous study further screened potential iron metabolism-related substrates of PCSK7 through transcriptomics combined with mass spectrometry analysis. The results showed that IGF2R, as another iron metabolism-related ligand of HFE, may be a new hydrolysis substrate of PCSK7 and may be related to the pathogenesis of HH.

[0007] In particular, the increased expression of IGF2R due to the reduced hydrolysis of IGF2R caused by PCSK7 mutation in patients with hereditary hemochromatosis may be related to the onset of hereditary hemochromatosis. Since PCSK7 can specifically hydrolyze the extracellular part of IGF2R, soluble IGF2R is formed and can be detected in serum. Based on this, IGF2R can be used as a molecular marker for the detection and treatment of iron overload-related diseases such as hemochromatosis, and can be used to prepare a clinical diagnostic kit for the detection of sIGF2R in the serum of patients with iron overload-related diseases.

[0008] At present, there is still a lack of screening and diagnostic methods for iron overload-related diseases with high sensitivity, good specificity, and clinical operability in clinical practice. Among various clinical detection methods, serum iron alone lacks specificity. The combined detection of serum transferrin saturation and serum ferritin is a method with high sensitivity and specificity, but inflammation, infection, cancer, metabolic syndrome, etc. can lead to increased serum ferritin, so its specificity is limited. Genetic testing is mainly used to determine whether it is primary hemochromatosis, but its cost is high and the results are uncertain. Liver biopsy is the gold standard for determining some iron overload-related diseases such as secondary hemochromatosis, but it is complicated and expensive to operate, and there are problems such as invasiveness and poor patient compliance, which makes it inconvenient for screening of iron overload-related diseases.

[0009] In summary, there is still a lack of satisfactory diagnostic markers for iron overload-related diseases in the art, and even more of a lack of methods for detecting iron overload by serological methods for iron overload-related diseases. Therefore, there is an urgent need to develop markers and methods for detecting iron overload (state) in iron overload-related diseases (especially serum detection). Summary of the invention

[0010] 1. The purpose of the present invention is to provide a marker for iron overload (state) and its application that can be used to detect iron overload-related diseases (especially serum detection).

[0011] 2. In the first aspect of the present invention, there is provided a use of insulin-like growth factor 2 receptor (IGF2R), which is used for preparing an iron overload detection reagent.

[0012] (1) As a preferred embodiment of the present invention, the iron overload detection is serum detection of soluble IGF2R;

[0013] (2) As a preferred embodiment of the present invention, the serum detection is performed by ELISA method.

[0014] 3. In the second aspect of the present invention, a diagnostic kit for iron overload detection is provided, which can specifically and quantitatively detect the level of soluble IGF2R in serum samples. The kit contains:

[0015] (1) a solid phase carrier, an IGF2R antibody coated on the solid phase carrier, an enzyme-labeled secondary antibody, a standard substance, a colorimetric substrate, a washing solution, and a reaction termination solution;

[0016] (2) a label or instructions, wherein the label or instructions indicate that the kit is used for iron overload detection.

[0017] 4. In the third aspect of the present invention, the use of IGF2R is provided: (a) as a new substrate of PCSK7 hydrolase that competitively binds to HFE, soluble IGF2R is used as a detection marker for iron overload-related diseases such as hereditary hemochromatosis; or (b) the use of sIGF2R in the preparation of a kit for the detection of serum iron overload for iron overload-related diseases such as hereditary hemochromatosis; (c) the use of IGF2R as a drug target in the treatment of iron overload-related diseases such as hereditary hemochromatosis.

[0018] 5. Within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions.

[0019] The main advantages of the present invention include:

[0020] (1) It was first proposed that IGF2R is an iron metabolism-related binding protein of HFE. It may be a new iron metabolism-related hydrolysis substrate of PCSK7 and may be related to the pathogenesis of iron overload-related diseases such as hereditary hemochromatosis. It can be used as a diagnostic and therapeutic marker for iron overload-related diseases such as hereditary hemochromatosis.

[0021] (2) For the first time, a new serum marker was provided for the detection and diagnosis of iron overload-related diseases. It can specifically and quantitatively detect the level of soluble IGF2R in serum samples, which is helpful for the detection or auxiliary detection of the iron overload status of iron overload-related diseases such as hereditary hemochromatosis, thereby helping to take corresponding treatment measures as early as possible.

[0022] (3) The serum testing method is more convenient, faster and easier for patients to accept.

[0023] (4) It is convenient for dynamically monitoring the disease progression of patients with iron overload-related diseases such as hereditary hemochromatosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .IGF2R may be a new iron metabolism-related hydrolysis substrate of PCSK7 enzyme. A. MSMS spectrum of three peptides matching IGF2R; B. Western blot detection of cell IGF2R and soluble IGF2R in cell culture medium; C. Molecular structure of the full-length IGF2R protein and its potential PCSK7 hydrolysis site.

[0025] Figure 2 .Knockdown of PCSK7 expression increases cellular IGF2R expression and promotes the binding of HFE-β2 microglobulin-IGF2R complex. A. Huh-7 cells were treated with siPCSK7, and the mRNA expression level of IGF2R in cells was detected by q-RT-PCR;

[0026] B. Western blot was used to detect the levels of cellular IGF2R and soluble sIGF2R in cell culture medium, as well as the levels of HFE and β2-microglobulin; C. Statistical analysis of the expression levels of cellular IGF2R protein and soluble sIGF2R protein in culture medium after siPCSK7 treatment; D. IP experiment was used to detect the binding of HFE to IGF2R and TFR2 after PCSK7 knockdown; E. Histograms showed the ratio of IGF2R and TFR2 expression levels after immunoprecipitation to those in the control group.

[0027] Figure 3 PCSK7 p.V143F and p.T743N mutations affect the expression of HAMP and iron metabolism signaling pathways such as ERK, and increase cellular IGF2R by inhibiting hydrolysis, thereby promoting the binding of the HFE-β2 microglobulin-IGF2R complex and correspondingly reducing the level of extracellular soluble sIGF2R.

[0028] A, F. Huh-7 cells were transfected with PCSK7 wild type (WT) and PCSK7 p.V143F plasmid or PCSK7 p.T743N plasmid. The results showed that compared with cells transfected with PCSK7WT, PCSK7 p.V143F and p.T743N mutations reduced the expression of HAMP; B, G. PCSK7 p.V143F and p.T743N mutations inhibited iron metabolism-related molecular pathways such as ERK and BMP / SMAD; C, H. PCSK7 p.V143F and p.T743N mutations did not change the mRNA expression level of IGF2R; D, I. PCSK7 p.V143F and p.T743N mutations increased the expression of cellular IGF2R and reduced the content of soluble sIGF2R in the culture medium, while increasing the expression of cellular HFE and β2 microglobulin; E, J. Histograms show that PCSK7 Statistical comparison of grayscale values ​​of cellular IGF2R expression and soluble sIGF2R expression after p.V143F and p.T743N mutations.

[0029] Figure 4.The effect of PCSK7 intervention on soluble IGF2R. A. The ELISA method was used to detect the expression of soluble IGF2R protein in the culture supernatant of cells after PCSK7 intervention; B. The ELISA method was used to detect the expression of sIGF2R protein in mouse and rat serum, in which IGF2R recombinant protein was used as a positive control and blank wells as a negative control. Rat and mouse serum were diluted 1000 times respectively. The results showed that soluble sIGF2R protein could be detected in both rat and mouse serum. DETAILED DESCRIPTION

[0030] After extensive and in-depth research, the inventor unexpectedly discovered for the first time that PCSK7 is related to the regulation of body cell iron balance through the hydrolysis of iron metabolism-related substrates, and IGF2R may be a new PCSK7 iron metabolism-related substrate and may be related to the occurrence of HH. Further analysis shows that as another iron metabolism-related binding protein of HFE, IGF2R may be a new substrate that competitively binds to PCSK7 hydrolase with HFE. IGF2R is a member of the insulin-like growth factor receptor family. Similar to plasma soluble sTFR1, high levels of serum soluble sIGF2R can be detected in mice, rats and humans using ELISA, and the method is simple. Therefore, serum soluble sIGF2R can be used as a marker for iron overload detection. The present invention was completed on this basis.

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0032] Experimental methods

[0033] 1. Construction of vectors for PCSK7 overexpression / knockout / knockdown, mutants, and IGF2R potential hydrolysis site mutants

[0034] Using the CRISPR Design online tool, three pairs of sequence-specific gDNA oligos were designed and synthesized for the first and second exons of the PCSK7 gene, cloned into the pLenti-CRISPR v2 vector, and the successfully connected plasmids were sequenced and verified. Using cell lines such as QSG hepatocytes and Huh-7 hepatoma cells, the constructed CRISPR plasmids were transfected into cells, and puromycin (Puro) was added for screening. Cells that survived 48-72 hours were collected and diluted in multiples, the cell density was adjusted to 2 / 100ul, and inoculated in 96-well plates, 50ul per well (i.e., each well contained a single cell). After the cell clones were formed, they were amplified and cultured step by step in 24-well plates to 6-well plates, and a small amount of cells were taken to extract total protein. The knockout of PCSK7 protein was detected by Western Blot to obtain a PCSK7 knockout stable cell line.

[0035] PCSK7 small interfering RNA was purchased commercially. PCSK7 interfering lentiviral particles pLV-shPCSK7-Flag were constructed using conventional gene recombination technology. After the vector construction was completed, the correctness of the target fragment carried was verified by gene sequencing. Using human leukocyte cDNA as a template, PCSK7 or IGF2R gene was amplified and cloned into CMV-MCS-3FLAG-SV40-Neomycin vector, and the constructed PCSK7 or IGF2R expression plasmid was used as a template to construct Flag-PCSK7 p.V143F, p.T743N mutant expression plasmids and Flag-IGF2R p.K2234A, p.K2206A, p.R2159A, p.R2107A, p.K2071A, p.R2048A mutant expression plasmids using Gene Tailor Site-Directed Mutagenesis System (Invitrogen).

[0036] 2. Cell Culture, Transfection, and Construction of Iron Overload Model

[0037] It is planned to use Huh-7, HepG2 liver cancer cell lines and other cell lines for cell model research. The cells are cultured in DMEM medium containing double antibodies and 10% fetal bovine serum and cultured in a carbon dioxide incubator at 37°C. When the adherent cells are 50-70% full, the PCSK7 small interfering RNA, PCSK7 overexpression, PCSK7 p.V143F mutant and other plasmids are transfected by liposome transfection method; pLV-shPCSK7-Flag and empty vector pLV-Flag are transfected by conventional lentiviral vector transfection methods to infect cultured Huh-7, HepG2 and other liver cancer cell lines. The culture medium is replaced 5 hours after transfection. After 24h-48h of cell culture, the cells are collected and PCSK7 expression is detected by real-time quantitative PCR and Western blot to clarify the transfection efficiency. The above cells are treated with 2mg / ml Holo-transferrin (combined with iron cell model), and the cells are collected after 24h of culture for downstream detection and analysis.

[0038] 3.RT-PCR, Western blot, ELISA and immunohistochemistry

[0039] Real time PCR, Western blot, ELISA and immunohistochemistry detection of IGF2R, sIGF2R, HFE, β2-microglobulin, hepcidin, ERK pathway molecule p-ERK and BMP / SMAD pathway molecule p-Smad1 / 5 were performed as follows:

[0040] Real time PCR: Total RNA was extracted using Trizol reagent and then reverse transcribed. Primers for the above molecules were designed according to conventional methods and PCR amplification was performed on an ABI real-time quantitative PCR instrument.

[0041] Western blot: The cells were treated with lysate, total protein was extracted, and then electrophoresed to PVDF membrane after SDS-PAGE electrophoresis, blocked with 5% BSA at room temperature for 1 hour, and the above target molecule antibodies were purchased commercially, and then the membrane was incubated with the above target molecule antibodies at 4°C overnight, GAPDH was used as a control, and then incubated with HRP-conjugated secondary antibodies at 37°C for 1 hour. Detection was performed by chemiluminescence method.

[0042] Immunohistochemistry: Paraffin-embedded tissue specimens collected from patients with primary hemochromatosis were sliced ​​into 4 μm thick sections, and the expression levels of IGF2R, hepcidin and other molecules were detected using conventional immunohistochemistry techniques. Specimens without hemochromatosis were used as controls to observe the expression levels of IGF2R, hepcidin and other molecules in hemochromatosis specimens carrying PCSK7 mutations.

[0043] ELISA test: The concentration determination kit for soluble IGF2R in cell culture medium supernatant and animal model serum can be prepared by yourself or carried out according to the commercial kit.

[0044] 4. Co-immunoprecipitation

[0045] According to the above method, IGF2R, HFE, and β2 microglobulin were overexpressed by constructing overexpression plasmids, and PCSK7 knockdown / knockout and various PCSK7 mutant cells were added to pre-cooled cell lysis buffer containing protease inhibitors and phosphatase inhibitors (containing 50mM Tris (pH7.5), 150mM KCl, 1% P-40 detergent, 8% glycerol, 1mM EDTA, 1mM DTT, 1mM protease and phosphatase inhibitors). After the cells are fully lysed, centrifuge at 12000rpm for 20min in a 4℃ refrigerated centrifuge. The supernatant was added to 3 volumes of 50mM Tris (pH7.5), 150mM KCl, 8% glycerol, 1mM EDTA, 1mM DTT, and 1mM protease inhibitor mixed solution, and placed on a 4℃ silent mixer with protein A beads and corresponding antibodies (HFE, β2 microglobulin, IGF2R, TFR2, etc.) to combine overnight. After binding, centrifuge at 2000 rpm for 1 min in a 4°C centrifuge, add immunoprecipitation rinse solution (50mM Tris (pH7.5), 150mM KCl, 0.1% Triton X-100, 1mM EDTA, 1mM DTT, 1mM protease inhibitor) and wash 3-4 times. Remove the supernatant, add an appropriate amount of 20ml SDS loading buffer, heat at 98°C for 5min, and then perform SDS-PAGE and Western hybridization to analyze the expression of the target protein.

[0046] 5. Immunofluorescence

[0047] After cell culture and intervention, cells grown on coverslips were washed three times with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100 for 10 minutes. They were blocked with 5% calf serum to remove nonspecific binding. After washing, the cells were incubated with primary antibodies of target antibodies (HFE, β2 microglobulin, and IGF2R, etc.) at 4°C overnight. After washing three times with PBS for 5 minutes, the cells were incubated with a mixture of anti-mouse Alex 488 and anti-Rabbit Alex 594 secondary antibodies at room temperature for 1 hour. After washing with PBS, the cells were incubated with rhodamine-labeled phalloidin for 30 minutes. After washing with PBS, the cells were placed on a slide in a mounting medium and observed and photographed with a confocal microscope.

[0048] 6. Data processing

[0049] The experimental data were collated and statistically analyzed according to conventional methods. The quantitative data were expressed as mean ± standard deviation, and the statistical significance was analyzed by Student's t-test; the categorical data were analyzed by Chi-square test. The probability obtained by the statistical test was P < 0.05, which was considered statistically significant.

[0050] Example 1

[0051] IGF2R may be a new iron metabolism-related hydrolysis substrate of PCSK7

[0052] The control group and the PCSK7 knockdown group were simultaneously subjected to transcriptome sequencing and proteomics testing and identified by mass spectrometry analysis. The molecules with no difference in mRNA level but increased protein expression after PCSK7 knockdown were screened out. It was found that IGF2R protein level, another iron metabolism-related ligand of HFE, increased by 4.34 times, which is likely to be a new hydrolysis substrate of PCSK7 enzyme ( Figure 1 A). The Uniprot protein database was used to search for the IGF2R amino acid sequence and sIGF2R protein structure. The results showed that compared with IGF2R, soluble sIGF2R lacked the cytoplasmic and transmembrane domains at the carboxyl terminal, but retained the extracellular domain and ligand binding site, suggesting that hydrolases can cut IGF2R near the transmembrane region to form soluble sIGF2R. The TMHMM Server online tool predicted the IGF2R transmembrane region and found that the IGF2R transmembrane region was located at amino acids 2305-2327. Because PCSK7 can be in the motif (R / K)X n (R / K)↓cleaves the substrate at specific single or paired basic amino acids within (arrows indicate cleavage positions, X n Corresponding to 0-, 2-, 4- or 6-amino acid spacer), by comparing the amino acid sequences near the transmembrane region of IGF2R, it was found that KCGK 2234 The sites met the above characteristics, and combined with Western blot analysis of cell IGF2R (calculated based on the average molecular weight of amino acids 128, the theoretical molecular weight is about 319KDa) and the molecular weight of soluble sIGF2R in cell culture medium ( Figure 2 B), RR 2048 ,KLGVIGDK 2071 , KR 2107 , KLFR 2159 , RK 2206 , KCGK 2234 It may be a potential hydrolysis site, corresponding to a theoretical molecular weight of about 262-286KDa.

[0053] Example 2

[0054] Knockdown of PCSK7 increases cellular IGF2R expression and reduces the content of soluble sIGF2R in culture medium

[0055] Given that IGF2R may be a potential enzymatic substrate of PCSK7, its enzymatic products are shed from cells to form soluble sIGF2R and enter the circulatory system. In this example, Huh-7 cells were treated with PCSK7 small interfering RNA to analyze whether knocking down PCSK7 expression affects cellular IGF2R expression. The results showed that, consistent with the transcriptome sequencing results, knocking down PCSK7 expression did not affect the mRNA level of IGF2R ( Figure 2 A), but knocking down PCSK7 expression can increase the expression of cellular IGF2R protein and reduce the content of soluble sIGF2R in the culture medium ( Figure 2 BC), indicating that knocking down PCSK7 expression can inhibit the hydrolysis of IGF2R protein. At the same time, it was also observed that the expression of HFE and β2 microglobulin in the cells increased simultaneously ( Figure 2 B). In addition, Huh7 cells were transfected with HFE-GFP plasmid after PCSK7 knockdown, and immunoprecipitation (IP) was performed using magnetic beads with tag-GFP. The results showed that the binding of HFE to IGF2R was enhanced after PCSK7 knockdown, while the binding of HFE to TFR2 was weakened ( Figure 2 D, E), suggesting that knockdown of PCSK7 can reduce the hydrolysis of IGF2R and promote the binding of HFE-β2 microglobulin-IGF2R complex, which may affect the dissociation of HFE and its binding to TFR2.

[0056] Example 3

[0057] PCSK7 p.V143F and p.T743N mutations affect the iron metabolism signaling pathway and inhibit cellular IGF2R hydrolysis

[0058] Without knocking out the endogenous expression of PCSK7, we conducted a preliminary analysis of whether PCSK7 p.V143F and p.T743N mutations might affect the expression of hepcidin. By constructing PCSK7 p.V143F and p.T743N mutants and transfecting Huh-7 cells (using PCSK7 and GAPDH as controls, respectively), the results showed that both PCSK7 p.V143F and p.T743N mutations could downregulate the expression of hepcidin, and the difference was statistically significant ( Figure 3 A, 3F), PCSK7 p.V143F and p.T743N mutations can affect ERK and BMP / SMAD and other iron metabolism-related molecular pathways ( Figure 3 B, 3G). In addition, PCSK7 p.V143F and p.T743N mutations did not affect IGF2R mRNA expression ( Figure 3C, 3H), further proving that the reduction of IGF2R is due to proteolysis; PCSK7 overexpression also reduced the expression of cellular IGF2R, HFE, and β2-microglobulin, suggesting its effect on the HFE-β2-microglobulin-IGF2R complex ( Figure 3 D, 3I). Western blot also showed that PCSK7 p.V143F and p.T743N mutations could increase the expression of IGF2R in cells and reduce the content of soluble sIGF2R in the culture medium, while increasing the expression of HFE and β2 microglobulin in cells ( Figure 3 DE, 3I-J), suggesting that both PCSK7 p.V143F and p.T743N mutations can reduce the degradation of IGF2R and promote the binding of HFE-β2 microglobulin-IGF2R complex, which may affect the dissociation of HFE.

[0059] Example 4

[0060] Intracellular IGF2R is hydrolyzed to form extracellular soluble IGF2R and effectively released into the culture supernatant or serum, and can be detected by ELISA

[0061] The ELISA method was used to detect the effect of PCSK7 intervention on the soluble IGF2R in the supernatant. The results showed that the concentration of soluble IGF2R in the supernatant of liver cancer cells cultured with various PCSK7 interventions could be detected by ELISA, and its level was consistent with the expression trend in the supernatant of cell culture medium detected by Western blot method ( Figure 4 A). In addition, the expression of sIGF2R protein in mouse and rat serum was detected by ELISA method, in which IGF2R recombinant protein was used as positive control and blank wells were used as negative control. Rat and mouse serum were diluted 1000 times respectively. The results showed that soluble sIGF2R protein could be detected in both rat and mouse serum.

[0062] Example 5

[0063] Preparation and detection of soluble IGF2R (sIGF2R) detection kit

[0064] A kit for detecting soluble IGF2R is prepared, the kit comprising: (1) a solid phase carrier, an IGF2R antibody coated on the solid phase carrier, an enzyme-labeled secondary antibody, a standard substance, a colorimetric substrate, a washing solution, and a reaction stop solution; and (2) a label or instructions, the label or instructions indicating that the kit is used for iron overload detection. The above detection kit can be used to quantitatively detect the content of soluble IGF2R in an unknown serum sample by ELISA method, and compared with the standard value (the content of IGF2R in a normal population), when the content of sIGF2R is significantly higher than the standard value, it can be assessed that there is a high probability of iron overload.

[0065] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. IGF2R as a novel iron metabolism-related substrate of PCSK7 hydrolase and its application.

2. Application of soluble IGF2R (sIGF2R) in the detection of iron overload-related diseases such as hereditary hemochromatosis.

3. Application of IGF2R as a drug target in the treatment of iron overload-related diseases such as hereditary hemochromatosis.

4. Application of PCSK7 molecular marker in the detection and treatment of iron overload-related diseases such as hereditary hemochromatosis.

5. The use according to claim 2, characterized in that: The level of sIGF2R in the serum of patients with iron overload-related diseases such as hereditary hemochromatosis is lower than that in the serum of a healthy control group.

6. The use according to claim 2, characterized in that: The sIGF2R is of high value in the screening and early diagnosis of patients with iron overload-related diseases such as hereditary hemochromatosis.

7. The use according to claim 2, characterized in that: The reagent for detecting sIGF2R is an enzyme-linked immunosorbent assay (ELISA) detection kit; the kit comprises a solid phase carrier, an IGF2R antibody coated on the solid phase carrier, an enzyme-labeled secondary antibody, a standard substance, a color developing substrate, a washing solution and a reaction stopping solution; the detection kit can specifically and quantitatively detect the level of sIGF2R in a serum sample.

8. Use of the serum sIGF2R described in claims 5-6 in the preparation of a product for screening and diagnosing patients with iron overload-related diseases such as hereditary hemochromatosis.

9. Use of the IGF2R described in claim 3 in developing a product for treating patients with iron overload-related diseases such as hereditary hemochromatosis.

10. Use of the PCSK7 described in claim 4 in the development of diagnostic and therapeutic products for patients with iron overload-related diseases such as hereditary hemochromatosis.

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