CN117230187A - Application of POFUT1 in preparation of glioma diagnostic reagent and prognosis evaluation - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of fucosyltransferase POFUT1 in preparation of glioma early diagnosis reagents and screening of prognosis evaluation targets. The application greatly complements and perfects the technical problem that the prior art has fewer molecular targets for early diagnosis methods and prognosis information of glioma patients. According to the invention, the early diagnosis and prognosis evaluation of glioma patients are assisted by researching POFUT1 protein expression level through an immunological method, and a novel target and a novel method are provided for solving the current situation of lack of early diagnosis and prognosis evaluation of glioma patients.

Description

Application of POFUT1 in preparation of glioma diagnostic reagent and prognosis evaluation

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of fucosyltransferase POFUT1 in preparation of glioma early diagnosis reagent and screening prognosis evaluation.

The application greatly complements and perfects the technical problem that the prior art has fewer molecular targets for early diagnosis methods and prognosis information of glioma patients. According to the invention, the early diagnosis and prognosis evaluation of glioma patients are assisted by researching POFUT1 protein expression level through an immunological method, and a novel target and a novel method are provided for solving the current situation of lack of early diagnosis and prognosis evaluation of glioma patients.

Background

Gliomas are one of the most common malignant tumors of the central nervous system, accounting for about 26.5% of all primary brain and other central nervous system (Central Nervous System, CNS) tumors and 80.7% of malignant tumors, with GBM being the most common highly malignant tumor, accounting for 47.1% of primary malignant brain tumors. Gliomas have higher morbidity, mortality and recurrence rates, and men have higher morbidity than women, and the etiology of gliomas is related to the interaction of congenital genetic high risk factors and environmental carcinogen factors. The prognosis of glioma patients is poor, the treatment of high-grade glioma mainly follows the traditional Stupp scheme at present, namely surgery is mainly used for cutting tumor, temozolomide is treated and combined with synchronous radiotherapy after surgery, even if a plurality of new treatment schemes such as combined chemotherapy, electric field treatment, molecular targeted therapy, delivery oncolytic adenovirus and the like are researched on the basis of the scheme along with the development of medicine, the glioma patients have no typical clinical manifestation at early stage, and the early diagnosis method is not short, the conventional physical examination project does not involve intracranial tumor detection, so that the glioma patients have already developed malignant progress to a great extent when finding out, and the survival time of the glioma patients is not obviously improved, so that a long path is needed for finding out early diagnosis methods and finding effective clinical targets.

In protein biosynthesis, the modification of amino acids by covalent attachment to various sugar chain groups is known as glycosylation modification. Protein glycosylation is largely classified into O-linked glycosylation and N-linked glycosylation, and is classified into fucosylation, sialylation, mannosylation, and the like depending on the kind of linked glycosyl. Glycosyltransferases are key enzymes for sugar chain addition during glycosylation and can modify a particular glycoprotein. Glycans covalently attached to biomolecules regulate protein function through indirect mechanisms such as direct interactions such as recognition of glycan structures by the conjugate or control of protein conformation and stability. Alterations in aberrant glycosylation are achieved by genetic mutations in glycoproteins, nucleotide sugar donor changes, abnormalities in expression or localization of glycosyltransferases, and the like, which can lead to increased N-glycan branching, increased O-glycan density, and the generation of aberrant forms of sialylated and fucosylated terminal structures, ultimately leading to dysfunction of the associated protein. Abnormal glycosylation is involved in various pathophysiological processes such as tumor invasion and infiltration, angiogenesis, chemotherapy resistance and the like, is considered as a characteristic change mark of various cancers, and research shows that the high expression of the abnormal glycosylation in various body fluids is closely related to prognosis of patients, and the abnormal glycosylation is widely applied to the field of clinical diagnosis markers.

The significant change of cell glycosylation modification is a common characteristic of malignant tumors, and fucosylation is an important mode of glycosylation modification, is the most common modification mode in glycoprotein and glycolipid oligosaccharide modification, and GDP-L-fucose is the only donor of fucosylation and has close relation with tumors. Fucosylation is mainly a process of adding fucose units to a substrate to form oligosaccharides by means of the catalysis of fucosyltransferases, which can be divided into two major classes, N-fucosyltransferases and O-fucosyltransferases. Of these, 11N-fucosyltransferases and 2O-fucosyltransferases have been shown to be effective tools for determining cancer diagnosis and prognosis in a variety of tumors. It plays an important role in many biological functions, such as ABO blood group determination, host-microorganism interaction, selectin-dependent leukocyte endothelial adhesion, ontogenesis, early diagnosis of tumors, and development and progression of tumors. Such as: the cell surface fucosylated Le Y oligosaccharide is a tumor-associated antigen, which is highly expressed in many tumors of epithelial origin such as breast cancer, ovarian cancer, liver cancer and colorectal cancer, and monoclonal antibodies (IGN-311 and hu3s 193) directed against this antigen have been used as potential drugs for the immunotherapy of tumors of epithelial origin; the fucosylation modification of the Notch receptor can influence the combination of a Notch Dll1 ligand and the Notch receptor, so that the abnormal activation of a Notch signal pathway promotes the invasion and metastasis capacity of liver cancer, and the core fucosylated alpha fetoprotein (AFP-L3) has more specific diagnostic value on the liver cancer than AFP; fucosylated Haptoglobin (HPT) is elevated in levels in malignant tumors such as ovarian, lung, and breast cancers, and becomes a molecular marker for a variety of tumors.

POFUT1 is one of O-fucosyltransferases, and this gene is located on chromosome 20 and encodes a protein comprising 393 amino acids, GDP-protein O-fucosyltransferase 1, belonging to the family of fucosyltransferases. The GDP-Fuc is used as glycosyl donor to transfer the L-Fuc to the side chain hydroxyl of the peptide chain Ser/Thr, and the O-fucoside bond is synthesized by catalysis, so that the fucosylation of the EGF-like domain can be modified. Which catalyzes the reaction of fucose via an O-glycosidic bond to a conserved serine or threonine residue found in the consensus sequence C2-X (4, 5) - [ S/T ] -C3 of the EGF domain, wherein C2 and C3 are the second and third conserved cysteines. The gene finds related researches in other tumors, POFUT1 can activate MAPK and PI3K/Akt signal paths so as to promote proliferation, migration and invasion of trophoblast cells, and the gene finds that the increased expression of POFUT1 can cause the increased expression of related proteins such as cyclin D1, cyclin E and the like, and simultaneously the level of cyclin-dependent kinase inhibitors P21 and P27 is reduced, which indicates that the proliferation promotion effect is related to the increase of cell cycle progress by promoting the cells to enter S phase; in liver cancer cells, POFUT1 is down-regulated, cell proliferation and adhesion are obviously inhibited, and cell cycle retardation is obvious; in colorectal cancer, downregulation of POFUT1 inhibits cell proliferation, reduces cell invasion and migration, inhibits cell cycle progression, and stimulates colorectal cancer apoptosis in vitro. Meanwhile, in lung cancer, plasma POFUT1 is a biomarker with diagnostic properties; bioinformatics analysis in gastric cancer finds that POFUT1 is related to cell cycle, cancer progress and invasive tumor phenotype, and can be used as a potential biomarker for early diagnosis of gastric cancer; in pancreatic ductal adenocarcinoma, POFUT1 may affect prognosis by affecting the density of microvascular generation; also in colorectal, breast, oral cancers, it has been shown to be associated with the progression of cancer, affecting prognosis.

POFUT1 has close relation with tumors, is proved to influence the progress of the cancers in various cancers and is related to prognosis, POFUT1 has fresh research in the field of glioma, the research proves that the POFUT1 is highly expressed in glioma and is obviously higher than that of peripheral glioma tissues, the expression of the POFUT1 is also increased along with the increase of glioma grade, and meanwhile, compared with the survival time of a glioma patient with the high expression of the POFUT1, the survival time of the glioma patient with the high expression of the POFUT1 is obviously reduced, so that theoretical basis is provided for applying the POFUT1 to early diagnosis reagents and screening prognosis evaluation targets. Therefore, in order to determine the relation between the POFUT1 expression level and the clinical prognosis of glioma patients, the application value of the POFUT1 expression level as a molecular marker for predicting glioma cancer prognosis is discussed.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides the application of the fucosyltransferase POFUT1 in preparation of glioma diagnostic reagents and prognosis evaluation for the first time, and the application solves the technical problems of single early diagnosis method, less hysteresis and less molecular targets for prognosis evaluation and single judgment standard of glioma patients in the prior art.

The invention provides an application of fucosyltransferase POFUT1 in preparation of glioma diagnostic reagents and prognosis evaluation.

In the early stage, the invention carries out bioinformatics analysis on glioma related data in a public database to obtain that the expression level of POFUT1 in glioma tissues is obviously higher than that of other normal tissues, and the prognosis of a high-expression POFUT1 glioma patient is obviously worse than that of a low-expression patient; meanwhile, through immunohistochemical analysis of clinical glioma tissues and unpaired normal tissues, the expression level of the glioma tissues is obviously higher than that of other normal tissues, the higher the tumor level is, the higher the expression of POFUT1 is, the difference has statistical significance, and further, the clinical information of the tumor tissues is subjected to statistical analysis to obtain that the prognosis of a high-expression POFUT1 glioma patient is obviously worse than that of a low-expression patient, the expression condition of the POFUT1 has correlation with factors such as age, sex, disease state, clinical stage, IDH mutation type and the like of the patient, and the prognosis model of the POFUT1 factors is considered to evaluate the prognosis of the patient better.

The invention is realized by the following technical scheme: and performing immune judgment through POFUT1 protein expression, and determining the relation between POFUT1 protein expression and diagnosis and prognosis of the patient through POFUT1 protein expression, so as to assist in diagnosis and prognosis evaluation of glioma patients. Compared with the prior art, the invention assists diagnosis and prognosis evaluation of glioma patients through the expression level of the immunological index POFUT1 protein, and provides an accurate and rapid prediction method for solving the current situation of lack of diagnosis and prognosis evaluation means of glioma patients.

Drawings

FIG. 1 is a graph showing the threshold value of POFUT1 expression in glioma tissue screened by ROC curve.

FIG. 2 is a graph showing protein expression levels of POFUT1 in glioma tissues and paracancerous normal tissues (115 gliomas and 8 unpaired paraneoplastic tissues), wherein A is a tumor peripheral tissue immunohistochemical image (x 200); WHO grade II glioma tissue immunohistochemical pictures (x 200); WHO III grade glioma tissue immunohistochemical pictures (. Times.200); WHO grade IV glioma tissue immunohistochemical pictures (. Times.200).

FIG. 3 is a box plot showing the difference in protein expression levels of POFUT1 in glioma tissue and paraneoplastic normal tissue (115 gliomas and 8 unpaired paraneoplastic tissues).

FIGS. 4 and 5 show survival analysis of POFUT1 high and low expression status in the overall patient group, different age groups, different gender groups, different IDH status group, different grade group, different MGMT methylation status group, different 1p19q status group, and different Ki67 expression group.

Figure 6 is a schematic representation of a multi-factor COX regression analysis.

Fig. 7 is a combination of fig. 2 and 3.

Detailed Description

The invention will now be further illustrated by means of specific examples in conjunction with the accompanying drawings without limiting the invention.

The pathological tissue specimens used in the examples of the present invention were obtained from the pathological tissue of 115 glioma patients resected by neurosurgery in the Shanxi national hospitals during the period of 2015 1 month-2018 month, and from the tissue specimens around the tumor of 8 GBM patients, and were paraffin-embedded and prepared into tissue sections as study subjects, and were confirmed by pathological histology examination using WHO classification criteria, wherein 28 patients of class II, 37 patients of class III, and 50 patients of class IV. All patients did not receive any form of related treatment prior to surgery, including radiation, chemotherapy, and immunotherapy; collecting complete medical record information and pathological data of a patient; the patient survival information is followed up by telephone, the follow-up is stopped by 2021 at 11 months and 24 days, the observation starting point time is the operation date, the ending is the death date or the follow-up stopping date, and the total survival time (OS) is the main observation end point. The study was approved by the ethical committee of the fifth clinical medical college of the university of mountain western medicine (2021, medical science and lun review No. 320).

The statistical analysis method adopted is as follows: the difference between glioma and paraneoplastic normal tissue groups adopts t test, one-way Anova test and analysis of variance test, and the best demarcation value of POFUT1 protein high expression and low expression is found by using the working characteristic curve (receiver operating characteristic curve, ROC curve) of the subject. Correlation analysis of the expression of POFUT1 and the clinically relevant factors was examined by four-grid-table chi-square. And comparing the influence of the high and low expression of the POFUT1 gene on the survival prognosis of the patient by adopting a Kaplan-Meier analysis method, log-rank, breslow and Tarine-Ware test, and analyzing the effect of the POFUT1 expression in the prognosis prediction of the glioma patient by adopting a single-factor and multi-factor Cox proportional risk regression model. The statistics and corresponding drawings were performed using SPSS 25.0 software and Graphpad Prism software. P < 0.05 was considered statistically significant.

The reagents, main laboratory instruments and consumables used in the examples of the present invention are shown in table 1.

Table 1: instrument, reagent and consumable.

The specific experimental procedure is as follows.

(1) Fresh brain glioma tissue is taken in an operating room, and 10% paraformaldehyde is fixed.

(2) Is sent to a pathology department for dehydration and embedding to prepare wax blocks and 2 mu m slices.

(3) The sections were placed in a 65 ℃ oven for 3 h to deparaffinize the tissue surface.

(4) Tissue sections were immersed in 3 identical xylene solutions in sequence, each for 10 min to completely dewax.

(5) The tissue slices are sequentially put into absolute ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol and 70% ethyl alcohol, and soaked for 2min respectively to hydrate the tissues.

(6) Tissue sections were placed in 3% H ₂ O ₂ Three distilled water washes the pieces after 15 min of soaking, and removes false positive results caused by endogenous peroxidase.

(7) The pieces were soaked in immunohistochemistry and washed with 1×pbs for 2min and repeated 3 times.

(8) Placing the tissue slice into antigen retrieval liquid, maintaining the pressure for 3min by an autoclave, and preserving the temperature for 5min to retrieve the antigen.

(9) Soaking the pieces in PBST for 5min, cleaning the pieces, repeating for 3 times, and drying the residual PBST on the pieces.

(10) Primary antibodies were diluted with PBST according to the instructions, added dropwise to the tissues, and incubated overnight in a wet box at 4 ℃.

(11) Soaking the pieces in PBST for 5min, cleaning the pieces, repeating for 3 times, and drying the residual PBST on the pieces.

(12) The ready-to-use secondary antibody is dripped on the tissue and put into a wet box for incubation for 20min at 37 ℃.

(13) Soaking the pieces in PBST for 5min, cleaning the pieces, repeating for 3 times, and drying the residual PBST on the pieces.

(14) DAB is dripped for dyeing, the film is observed under a microscope for 3-5min, and after the tissue turns to be yellow brown, tap water is used for stopping dyeing.

(15) The pieces were placed in hematoxylin for 3min for nuclear staining. If the dyeing is too deep, placing the flakes in 1% hydrochloric acid alcohol for 2-3s, and washing the flakes with tap water for decoloring; if the color is too light after decolorization, the flakes are placed in ammonia water for 2-3s, and then the flakes are washed by tap water for blue returning.

(16) Sequentially placing the slices into 70% ethanol, 80% ethanol, 90% ethanol, anhydrous ethanol 1 and anhydrous ethanol 2 for dehydration.

(17) The pieces were soaked in xylene for 10 min, then replaced with fresh xylene and soaked, and repeated 3 times.

(18) And (3) sealing the sheet with neutral resin.

And (3) value analysis: the slide was scanned using a K-Viewer digital pathology scanning system. Five fields are randomly selected under the high power mirror (200×) of each piece, and as POFUT1 is expressed in cytoplasm in glioma cells, image J software is selected to analyze the expression quantity of cytoplasmic protein, the percentage of positive area and staining intensity scores will be derived separately, the score was assigned 3 (+++), 2 (++), 31 min (+) and 0min (-). The percent positive area was multiplied by the staining intensity score to calculate a histochemical score (histochemistry score, H-score), which is representative of the POFUT1 protein expression level.

Is prepared by common reagents.

(1) Immunohistochemistry with 1×pbs: three distilled water dissolution (NaH) ₂ PO ₄ 5.29 g，Na ₂ HPO ₄ 57.8 g, naCl 16 g), constant volume to 2L, and diluting the three distilled water according to the proportion of 1:9 for later use.

(2) Immunohistochemistry with PBST: tween-20:1 x pbs=1:1000.

(3) Sodium citrate antigen retrieval solution: 2000 2 g sodium citrate is added into the three distilled water ml to be dissolved, and the three distilled water is stored at room temperature.

（4）3% H ₂ O ₂ ：30% H ₂ O ₂ : methanol=1:9, stored protected from light.

(5) 1% hydrochloric acid alcohol: absolute ethyl alcohol: concentrated hydrochloric acid=125:1.

(6) Immunohistochemistry with ammonia: three steamed water: concentrated ammonia = 500:3.

Experimental results.

Immunohistochemical staining was performed on 28 cases of grade II gliomas, 37 cases of grade III gliomas, 50 cases of grade IV gliomas, and 8 cases of paraneoplastic tissue sections as controls, and the cut-off values were determined by using the expression level (H-score) of POFUT1 in gliomas and the survival state of the patient as ROC curves, thereby distinguishing high-low expression groups, see ROC curves (FIG. 1). Analysis of the results shows that: POFUT1 is mainly localized in cytoplasm and expressed at each level and around tumor, tumor tissue is obviously higher than that of paraneoplastic tissue, the expression increases with the increase of level, and the difference has statistical significancep <0.05 (fig. 2 and 3).

And (3) analyzing the correlation between POFUT1 expression and clinical pathological factors by using chi-square test. Analysis results show that POFUT1 expression level has correlation with age, WHO classification, IDH mutation type, 1p19q deletion type, ki-67 value and survival statep <0.05 (table 2). Wherein, the age of the patient is greater than or equal to 50 years old, GBM, IDH wild type, 1p19q is not deleted, ki-67 is greater than 20%, and the proportion of POFUT1 high-expression patients in dead patients is higher. It is further suggested that POFUT1 may be involved in the development of glioma.

Table 2 correlation analysis of POFUT1 and clinical pathology information in clinical samples.

The Kaplan-Meier analysis method is adopted to analyze the correlation between the high and low expression of POFUT1 and the survival time of patients in different groups, and the analysis results show that the survival time of the POFUT1 high-expression patients is obviously shortened in the general patient group, the IDH wild group and the mutant group, the high-grade group, the different age groups, the different gender groups, the MGMT methylation and unmethylation groups, the 1p19q deletion and no deletion group and the Ki67 high-expression groupp <0.05 (fig. 4 and 5). Above mentionedThe results further suggest that the expression level of POFUT1 may be a predictive marker of post-operative survival in glioma patients.

And carrying out multi-factor COX regression analysis on the survival time of the patient by combining the clinical pathology information of the patient and the expression quantity of POFUT 1. Analysis results show that comprehensively analyzing factors such as age, sex, disease state, clinical stage, IDH mutation type and the like of patients, the high expression POFUT1 is a risk factor compared with the low expression (HR= 2.296, 95% confidence interval=1.210-4.358,p =0.011), the expression level of POFUT1 in the patient is an independent prognostic evaluation factor (fig. 6).

According to the invention, through an immunohistochemical test, the relationship between the POFUT1 expression condition and the clinical pathological parameters of glioma patients is researched, and the prediction value of POFUT1 on glioma prognosis is evaluated. The expression of POFUT1 was observed to be significantly higher in tumor tissue than in normal control tissue. The high expression level of POFUT1 is related to factors such as age, sex, disease state, clinical stage, IDH mutation type, etc. of patients. Patients with high POFUT1 expression have shorter survival time and poorer prognosis. More importantly, the POFUT1 high expression can be used as an independent predictor of poor prognosis of glioma patients.

In summary, the invention is an application of the immunological index POFUT1 protein expression level to assist diagnosis and prognosis evaluation of glioma patients, namely, diagnosis and prognosis information of the patients are directly determined according to the expression level of POFUT1 protein in tumor tissues of glioma patients.

The foregoing is merely illustrative of embodiments of this invention and it will be appreciated by those skilled in the art that variations may be made without departing from the principles of the invention, which is also intended to be considered as a scope of the invention.

Claims (2)

1. Use of the fucosyltransferase POFUT1 for the preparation of a glioma diagnostic reagent, characterized in that: the use of the fucosyltransferase POFUT1 in the preparation of a reagent for diagnosing glioma patients.

2. Use of fucosyltransferase POFUT1 in the preparation of a reagent for prognostic evaluation of glioma patients.