CN113416777A - Biomarker related to occurrence and development of diabetic nephropathy and application thereof - Google Patents

Abstract

The invention discloses biomarkers related to the occurrence and development of diabetic nephropathy and application thereof, wherein the biomarkers comprise biomarker combinations of LPCAT1+ B4GALT5, FILIP1L + B4GALT5, LPCAT1+ FILIP1L + B4GALT5, and the invention further verifies that the biomarker combinations of LPCAT1+ B4GALT5, FILIP1L + B4GALT5, LPCAT1+ FILIP1L + B4GALT5 have better diagnosis efficiency and higher accuracy, sensitivity and specificity on the diabetic nephropathy.

Description

Biomarker related to occurrence and development of diabetic nephropathy and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a biomarker related to occurrence and development of diabetic nephropathy and application thereof.

Background

Diabetic Nephropathy (DN) is a common microvascular complication in diabetes, and is a vascular-damaging glomerulopathy mainly manifested by Diabetic glomerular sclerosis. Relevant statistics show that about 30-40% of type 2 diabetic patients eventually develop diabetic nephropathy (Fioretto P, Bruseghin M, Berto I, et al. Secondary protection in diabetes: role of diabetic control [ J ]. JAm Soc Nephrol,2006,17(4Suppl 2): S86-89.), diabetic nephropathy is mainly divided into 5 stages, the first stage mainly shows that Glomerular Filtration Rate (GFR) is increased, and is seen in the early stage of diabetes, and the stage has no special clinical expression; the second stage mainly shows that the Glomerular Filtration Rate (GFR) is increased or is close to a normal level, the urinary protein excretion rate (UAE) is normal and can be increased after strenuous exercise, renal biopsy shows that glomerular basement membrane is thickened, the blood pressure is normal, the stage can be reversed, and no special clinical manifestation exists; stage III is mainly characterized by normal or elevated Glomerular Filtration Rate (GFR), persistent microalbuminuria, mild elevated blood pressure, but still within the normal range, early diabetic nephropathy; continuous proteinuria appears in the fourth stage, urine protein can be detected conventionally, the quantity of the urine protein is more than 0.5 g/24 h, renal biopsy indicates that glomerular sclerosis, and the process can not be reversed even if strict treatment is carried out in the fourth stage; the fifth stage is mainly characterized by that the blood pressure is obviously raised, the urine protein is not like that of the kidney disease caused by other reasons, and can be reduced to the end stage of renal failure, the glomerular filtration rate can be reduced to below 10mL/min, urea nitrogen and creatinine are raised, edema and hypertension are further aggravated, and hypoproteinemia is produced.

To date, there is no specific treatment for diabetic nephropathy, focusing on prevention, early diagnosis and early intervention. The early clinical manifestation of diabetic nephropathy lacks specificity, has very large concealment, has no obvious clinical symptoms, the course of the patient is generally over 10 years until the occurrence of urine protein, once the diabetic nephropathy has persistent proteinuria in clinic, the state of renal function damage is usually irreversible, the renal failure finally develops to the end stage, and the early diagnosis can delay, block or even reverse the progress of the disease, so the early clinical diagnosis of the diabetic nephropathy is particularly important. The diabetic nephropathy is one of the common causes of end-stage nephropathy, but at present, there is no reliable biomarker for reflecting the occurrence and development of diabetic nephropathy in clinic, so that it is important for early diagnosis to find the biomarker related to early diagnosis of diabetic nephropathy and monitor in time.

Disclosure of Invention

The invention aims to provide a biomarker related to the occurrence and development of diabetic nephropathy, and the biomarker is applied to early diagnosis of the diabetic nephropathy, is proved to be closely related to the occurrence and development of the diabetic nephropathy by experiments, has better diagnosis efficiency when being applied to diagnosis of the diabetic nephropathy, has higher accuracy, sensitivity and specificity, and provides valuable biological information for early diagnosis and clinical treatment of the diabetic nephropathy.

The biomarkers in the invention comprise genes LPCAT1, FILIP1L and B4GALT5, and the information of the genes LPCAT1, FILIP1L and B4GALT5 is respectively as follows:

gene ID in NCBI for LPCAT 1: 79888, located in the 5 th 3 rd sub-band of the 5 th chromosome short arm 1 region;

FILIP1L Gene ID in NCBI: 11259, located in the 1-band sub-band of 2 of long arm 1 region of chromosome 3;

b4GALT5 Gene ID in NCBI: 9334, located in 3 band 1 and 3 sub-bands of 3 bands of the long arm 1 region of chromosome 20.

The above object of the present invention is achieved by the following technical solutions:

the first aspect of the invention provides application of a reagent for detecting biomarkers in a sample in preparing a product for early diagnosis of diabetic nephropathy.

Further, the biomarkers include B4GALT5, in combination with LPCAT1, and/or FILIP 1L;

preferably, the biomarker is LPCAT1, B4GALT 5;

preferably, the biomarker is FILIP1L, B4GALT 5;

preferably, the biomarkers are LPCAT1, FILIP1L, B4GALT 5.

Further, the product comprises a reagent for detecting the expression level of the biomarker by a nucleic acid hybridization technology, a nucleic acid amplification technology, a protein immunization technology, a sequencing technology, a chromatography technology and a mass spectrometry technology.

Further, the nucleic acid hybridization technique refers to a process in which complementary nucleotide sequences (DNA to DNA, DNA to RNA, RNA to RNA, etc.) form noncovalent bonds through Watson-Crick base pairing, thereby forming stable homoduplex or heteroduplex molecules, which is also called nucleic acid hybridization.

Further, the nucleic acid amplification technology is a generic name of a large class of technical methods, and the existing nucleic acid amplification technology comprises conventional PCR, real-time fluorescence PCR, isothermal nucleic acid amplification technology and the like, can specifically amplify a trace amount of target DNA by millions of times, thereby greatly improving the analysis and detection capability of DNA molecules, detecting single-molecule DNA or samples containing only 1 target DNA molecule in every 10 ten thousand cells, and having the advantages of high sensitivity, strong specificity, rapidness, simplicity and the like.

Further, the protein immunization technique refers to a generic term for a class of methods including radioimmunoassay, direct, indirect or contrast enzyme-linked immunosorbent assay, enzyme immunoassay, fluorescence immunoassay, western blotting, immunoprecipitation, any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles or quantum dots) for the detection of a target, and the protein immunization can be performed in the form of a microtiter plate or strip.

Further, the sequencing technology includes (but is not limited to) first-generation sequencing, second-generation sequencing, and third-generation sequencing, wherein the first-generation sequencing is also called Sanger sequencing, and is a sequencing technology utilizing a DNA polymerase synthesis reaction, and the first-generation sequencing is a sequencing technology based on the Sanger method; the second generation sequencing is based on massively parallel sequencing technology (MPS), and can simultaneously complete the synthesis of a complementary strand of a sequencing template and the acquisition of sequence data; the third generation sequencing is based on single molecule sequencing and massively parallel sequencing technology.

Further, the chromatographic technique refers to a method for separating and analyzing each component in a complex mixture, and utilizes different substances having different distribution coefficients in a system composed of a stationary phase and a mobile phase, and when the two phases move relatively, the substances move together with the mobile phase and are repeatedly distributed between the two phases, so that each substance is separated.

Further, the mass spectrometry technique is a method of separating and detecting moving ions (charged atoms, molecules or molecular fragments, molecular ions, isotope ions, fragment ions, rearrangement ions, multiply-charged ions, metastable ions, negative ions, and ions generated by ion-molecule interaction) according to their mass-to-charge ratios using an electric field and a magnetic field. The accurate mass of the ions is measured, and the compound composition of the ions can be determined.

Further, the agent is selected from the group consisting of:

primers that specifically amplify the biomarkers; or

A probe that specifically recognizes the biomarker; or

Antibodies, antibody fragments, affinity proteins that specifically bind to the biomarkers.

Further, the product comprises a kit, a chip and a nucleic acid membrane strip.

Further, the sample comprises peripheral blood, serum, plasma, tissue.

Further, the kit comprises primers, probes or chips for detecting the expression level of the biomarker.

Further, the kit may further comprise instructions or labels for use, positive controls, negative controls, buffers, adjuvants or solvents.

Further, the instructions or labels indicate that the kit is for detecting diabetic nephropathy.

Further, the chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier.

Further, the oligonucleotide probes comprise oligonucleotide probes for the biomarkers for detecting the level of expression of the biomarkers.

"primer" as used herein refers to a nucleic acid fragment comprising 5 to 100 nucleotides, preferably 15 to 30 nucleotides, capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction).

The "probe" as used herein refers to a nucleic acid sequence comprising at least 5 nucleotides, for example, 5 to 100 nucleotides, which can hybridize to an expression product of a target gene or an amplification product of the expression product under a specified condition to form a complex, and a label for detection, including but not limited to a label for fluorescent quantitative PCR or fluorescent in situ hybridization, may be included on the hybridization probe.

The term "antibody" as used herein refers to a specific immunoglobulin directed against an antigen site, and the antibody of the present invention refers to an antibody that specifically binds to a biomarker polypeptide and/or protein as described herein, and can be produced according to conventional methods in the art, and includes polyclonal or monoclonal antibodies, antibody fragments (e.g., Fab ', F (ab')2, and Fv fragments), single chain Fv (scfv) antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site, as long as the antibody exhibits the desired biological binding activity.

In a second aspect of the invention, a product is provided for early diagnosis of diabetic nephropathy.

Further, the product comprises reagents for detecting biomarkers in a sample, the biomarkers comprising B4GALT5, in combination with LPCAT1, and/or FILIP 1L;

preferably, the biomarker is LPCAT1, B4GALT 5;

preferably, the biomarker is FILIP1L, B4GALT 5;

preferably, the biomarkers are LPCAT1, FILIP1L, B4GALT 5;

preferably, the product comprises a kit, a chip and a nucleic acid membrane strip.

Further, the reagents include reagents that detect mRNA expression levels of the biomarkers;

preferably, the agent is an agent that detects the level of cDNA complementary to mRNA transcribed from the biomarker;

more preferably, the reagents comprise a probe that specifically recognizes the biomarker, a primer that specifically amplifies the biomarker;

preferably, the product also comprises a reverse transcription reagent, a second generation sequencing reagent and a total RNA extraction reagent.

Further, the reagents include reagents for detecting the expression level of the polypeptide and/or protein encoded by the biomarker;

preferably, the agent is an antibody, antibody fragment, affinity protein that specifically binds to the polypeptide and/or protein encoded by the biomarker.

As used herein, the term "polypeptide" refers to a compound consisting of amino acids linked together by a peptide bond, including full-length or amino acid fragments of a polypeptide, and the expression level of the polypeptide encoded by the gene can be normalized based on the amount of total protein in a sample or the amount of the polypeptide encoded by a housekeeping gene.

The "expression level" and "level" as used herein refer to the absolute or relative amount of a biomarker of the present invention, and the expression level of any of the biomarkers of the present invention can be determined by a variety of techniques, and in particular, the absolute or relative amount of a biomarker of the present invention can be detected by methods well known to those skilled in the art.

The third aspect of the present invention provides an early diagnosis device for diabetic nephropathy.

Further, the apparatus comprises:

(1) a data acquisition module: obtaining expression profile data for a biomarker according to the first aspect of the invention in a sample from a subject;

(2) an evaluation judgment module: the system is used for providing the expression profile data of the biomarkers as input data to a trained evaluation model, and the evaluation model carries out evaluation judgment on whether the subject has diabetic nephropathy and/or the risk of having diabetic nephropathy based on the expression profile data of the biomarkers in a subject sample;

(3) a result output module: and the evaluation judging module is used for outputting the result of the evaluation judging module to obtain the result of whether the subject suffers from the diabetic nephropathy and/or the risk of suffering from the diabetic nephropathy.

The fourth aspect of the invention provides the use of a reagent for detecting a biomarker in a sample for the preparation of a device for the early diagnosis of diabetic nephropathy;

preferably, the biomarker comprises B4GALT5, in combination with LPCAT1, and/or FILIP 1L;

preferably, the biomarker is LPCAT1, B4GALT 5;

preferably, the biomarker is FILIP1L, B4GALT 5;

preferably, the biomarkers are LPCAT1, FILIP1L, B4GALT 5.

Further, the samples include (but are not limited to): tissue (e.g., kidney tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, peripheral blood, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tissue culture fluids, tissue extracts, and combinations thereof;

preferably, the sample is selected from peripheral blood, serum, plasma, tissue.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a boxplot showing differential expression of the LPCAT1 gene, in which panel A: training set, B picture: a verification set;

FIG. 2 is a boxplot showing differential expression of FILIP1L gene, in which panel A: training set, B picture: a verification set; fig. 3 shows a boxplot of the B4GALT5 gene differential expression, in which panel a: training set, B picture: a verification set;

FIG. 4 shows the ROC plot for the gene combination LPCAT1+ FILIP1L, in which panel A: training set, B picture: a verification set;

FIG. 5 shows the ROC plot for the gene combination LPCAT1+ B4GALT5, in which panel A: training set, B picture: a verification set;

FIG. 6 shows ROC plots for the gene combination FILIP1L + B4GALT5, where panel A: training set, B picture: a verification set;

FIG. 7 shows ROC plots for the gene combination LPCAT1+ FILIP1L + B4GALT5, in which panel A: training set, B picture: and (5) verifying the set.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 screening of genes differentially expressed in diabetic nephropathy

1. Data source

The data come from GEO database, download diabetic nephropathy data set GSE142153 from GEO database as training set, sample size is Control: case 10: 23; and downloading a diabetic nephropathy data set GSE30122 from a GEO database to serve as a verification set, wherein the sample size is Control: case 50: 19.

2. data pre-processing

And standardizing the original data of the training set and the verification set downloaded from the GEO database, annotating the standardized gene expression matrix through a Platform file, and taking an average value of a plurality of probes corresponding to the same gene as the expression quantity of the gene.

3. Differential expression analysis

And respectively carrying out differential expression analysis on the data in the data sets GSE142153 and GSE30122 by using a 'limma' packet in R language software, wherein the screening standard of the differential expression genes is as follows: value <0.05, | logFC | > 0.5.

4. Results of the experiment

The result shows that 1391 differentially expressed genes in the training set obtained by screening, 740 differentially expressed genes in the verification set and 84 shared differentially expressed genes are obtained by intersecting the differentially expressed genes in the two data sets, wherein the differentially expressed genes LPCAT1, FILIP1L and B4GALT5 in the training set and the verification set are respectively shown in figures 1-3, the differentially expressed genes LPCAT1, FILIP1L and B4GALT5 in the diabetic nephropathy are all up-regulated, and the difference has statistical significance (P < 0.05).

Example 2 diagnostic Performance validation

1. Experimental methods

For the differentially expressed genes LPCAT1, FILIP1L, B4GALT5 selected in example 1, a receiver operating curve (ROC curve) was plotted using the R package "pROC" (version 1.15.0) and analyzed for sensitivity, specificity, AUC values to determine their diagnostic potency in the training set and validation set, alone and in combination;

when the diagnostic efficacy of the single index in the training set and the verification set is judged, the expression quantity of the gene is directly used for analysis, the level corresponding to the one point with the maximum Youden index is selected as the cutoff value of the gene, and the gene with the AUC of 0.5< AUC <0.8 is used for joint analysis;

and when the judgment indexes are combined with the diagnosis efficiency in the training set and the verification set, performing Logitics regression on the expression level of each gene, calculating the probability of whether each individual is ill or not through a fitted regression curve, determining different probability division thresholds, and calculating the sensitivity, specificity, accuracy and the like of the combined diagnosis scheme according to the determined probability division thresholds.

2. Results of the experiment

The results are shown in tables 1-3 and FIGS. 4-7, and the results show that the gene combination LPCAT1+ B4GALT5, FILIP1L + B4GALT5, LPCAT1+ FILIP1L + B4GALT5 has better diagnostic effect on diabetic nephropathy than a single marker, and has higher sensitivity and specificity, which indicates that the gene combination can be applied to the diagnosis of diabetic nephropathy.

TABLE 1 statistics of diagnostic potency of individual genes

Gene	Training set AUC	Validation set AUC
			LPCAT1	0.796	0.762
FILIP1L	0.761	0.697
			B4GALT5	0.778	0.728

TABLE 2 diagnostic Performance statistics of Gene combinations in training sets

Gene	AUC	Sensitivity of the composition	Specificity of
				LPCAT1+FILIP1L	0.848	0.652	1.000
LPCAT1+B4GALT5	0.861	0.696	1.000
				FILIP1L+B4GALT5	0.817	0.913	0.700
LPCAT1+FILIP1L+B4GALT5	0.883	0.783	0.900

TABLE 3 diagnostic Performance statistics of Gene combinations in validation set

Gene	AUC	Sensitivity of the composition	Specificity of
				LPCAT1+FILIP1L	0.777	0.737	0.800
LPCAT1+B4GALT5	0.823	0.632	0.940
				FILIP1L+B4GALT5	0.804	0.737	0.800
LPCAT1+FILIP1L+B4GALT5	0.842	0.684	0.940

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (10)

1. Use of a reagent for detecting a biomarker in a sample for the manufacture of a product for the early diagnosis of diabetic nephropathy, wherein the biomarker comprises a combination of B4GALT5, with LPCAT1, and/or FILIP 1L;

preferably, the biomarker is LPCAT1, B4GALT 5;

preferably, the biomarker is FILIP1L, B4GALT 5;

preferably, the biomarkers are LPCAT1, FILIP1L, B4GALT 5.

2. The use according to claim 1, wherein the product comprises reagents for detecting the expression level of the biomarkers of claim 1 by nucleic acid hybridization techniques, nucleic acid amplification techniques, protein immunization techniques, sequencing techniques, chromatography techniques, mass spectrometry techniques.

3. Use according to claim 2, wherein the agent is selected from the group consisting of:

a primer that specifically amplifies the biomarker of claim 1; or

A probe that specifically recognizes the biomarker of claim 1; or

An antibody, antibody fragment, affinity protein that specifically binds to the biomarker of claim 1.

4. The use of claim 1, wherein the product comprises a kit, a chip, a nucleic acid membrane strip.

5. The use of claim 1, wherein the sample comprises peripheral blood, serum, plasma, tissue.

6. A product for use in the early diagnosis of diabetic nephropathy, the product comprising reagents for detecting biomarkers in a sample, the biomarkers comprising B4GALT5, in combination with LPCAT1, and/or FILIP 1L;

preferably, the biomarker is LPCAT1, B4GALT 5;

preferably, the biomarker is FILIP1L, B4GALT 5;

preferably, the biomarkers are LPCAT1, FILIP1L, B4GALT 5;

preferably, the product comprises a kit, a chip and a nucleic acid membrane strip.

7. The product of claim 6, wherein the reagents comprise reagents to detect mRNA expression levels of the biomarkers;

preferably, the agent is an agent that detects the level of cDNA complementary to mRNA transcribed from the biomarker;

more preferably, the reagents comprise a probe that specifically recognizes the biomarker, a primer that specifically amplifies the biomarker;

preferably, the product also comprises a reverse transcription reagent, a second generation sequencing reagent and a total RNA extraction reagent.

8. The product of claim 6, wherein the reagents comprise reagents for detecting the level of expression of polypeptides and/or proteins encoded by the biomarkers;

preferably, the agent is an antibody, antibody fragment, affinity protein that specifically binds to the polypeptide and/or protein encoded by the biomarker.

9. An early diagnosis device for diabetic nephropathy, comprising:

(1) a data acquisition module: obtaining expression profile data for the biomarker of claim 1 in a sample from a subject;

(2) an evaluation judgment module: the system is used for providing the expression profile data of the biomarkers as input data to a trained evaluation model, and the evaluation model carries out evaluation judgment on whether the subject has diabetic nephropathy and/or the risk of having diabetic nephropathy based on the expression profile data of the biomarkers in a subject sample;

(3) a result output module: and the evaluation judging module is used for outputting the result of the evaluation judging module to obtain the result of whether the subject suffers from the diabetic nephropathy and/or the risk of suffering from the diabetic nephropathy.

10. The application of the reagent for detecting the biomarkers in the sample in the preparation of the diabetic nephropathy early diagnosis device;

preferably, the biomarker comprises B4GALT5, in combination with LPCAT1, and/or FILIP 1L;

preferably, the biomarker is LPCAT1, B4GALT 5;

preferably, the biomarker is FILIP1L, B4GALT 5;

preferably, the biomarkers are LPCAT1, FILIP1L, B4GALT 5.

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