Use of ABCG1 in the diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to the use of ABCG1 in the diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia. After testing, the expression of ABCG1 was significantly downregulated in the blood of HIV combined with hyperlipidemia patients, and it was verified that ABCG1 has the characteristics of high sensitivity and high specificity. By detecting the expression of ABCG1, it can be used to diagnose or auxiliary diagnosis whether hyperlipidemia exists in HIV-infected groups, provide guidance for the early intervention of hyperlipidemia in HIV-infected groups, and have broad clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of ABCG1 in diagnosing or assisting in diagnosing HIV combined with hyperlipidemia. Background Art

[0002] After HIV infects the human body, it mainly attacks CD4+T lymphocytes in the immune system, causing damage to the immune system. As the disease progresses, patients not only face the risk of serious immunodeficiency-related diseases, but also suffer from a series of metabolic disorders, among which hyperlipidemia is more common. According to statistics, the incidence of hyperlipidemia in HIV-infected people is significantly higher than that in the general population. This concurrent condition not only aggravates the patient's condition, but also greatly increases the risk of complications such as cardiovascular disease, seriously affecting the patient's quality of life and prognosis.

[0003] HIV-infected people themselves may experience a variety of symptoms due to viral infection, such as fever, night sweats, weight loss, fatigue, diarrhea, etc., which can easily mask the early symptoms of hyperlipidemia. Therefore, exploring accurate diagnostic markers will help provide guidance for early intervention of hyperlipidemia in HIV-infected populations. ABCG1, as a member of the G superfamily of ATP-binding cassette transporters, plays a key role in lipid metabolism. It is mainly responsible for transporting intracellular cholesterol and phospholipids to extracellular high-density lipoprotein (HDL), and plays an indispensable role in maintaining intracellular lipid homeostasis. A large number of studies have shown that the expression level of ABCG1 is closely related to lipid metabolism in various cell types, such as macrophages, hepatocytes, and vascular endothelial cells. However, in the current research on the use of ABCG1 for the diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia, there is a lack of research on whether it can meet the two important diagnostic indicators of sensitivity and specificity, and no systematic conclusion has been formed. Summary of the invention

[0004] The purpose of the present invention is to provide a use of ABCG1 in diagnosing or assisting in diagnosing HIV combined with hyperlipidemia.

[0005] The inventors found in their own sequencing data that the expression of ABCG1 was significantly reduced in the HIV combined with hyperlipidemia group, suggesting that it plays a key role in lipid metabolism imbalance, and verified through experiments that ABCG1 has the characteristics of high sensitivity and high specificity. Based on this, the inventors completed the following invention.

[0006] The first aspect of the present invention is to provide the use of ABCG1 as a biomarker in the preparation of a product for diagnosing or assisting in the diagnosis of HIV combined with hyperlipidemia.

[0007] In this use, the product includes a product for detecting the expression level of ABCG1 in a sample; further, detecting the expression level of ABCG1 in a sample may be detecting the expression level of the ABCG1 gene in the sample and / or detecting the expression level of the ABCG1 protein in the sample.

[0008] In this use, the product can rely on methods such as qRT-PCR, RT-PCR or gene chips to perform quantitative or semi-quantitative analysis on the mRNA transcripts of ABCG1 and detect the expression level of the ABCG1 gene in the sample; or rely on enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunoturbidimetry and other methods to measure the concentration of ABCG1 protein and detect the expression level of ABCG1 protein in the sample.

[0009] In this application, the product used to detect the expression level of ABCG1 in a sample can be an oligonucleotide probe targeting the ABCG1 encoding DNA sequence, which can detect the mRNA of ABCG1 through hybridization techniques such as Northern hybridization or in situ hybridization; or it can be a PCR primer used for PCR-related amplification techniques, such as ordinary PCR, real-time quantitative PCR or nested PCR, to analyze the mRNA level of ABCG1; or it can be an antibody targeting ABCG1, which detects the expression of ABCG1 protein through immunological techniques such as Western Blot, immunohistochemistry, immunofluorescence, etc.; or it can be an ABCG1-specific nucleic acid aptamer, which uses its specific binding ability to recognize the nucleic acid molecule of ABCG1 molecules; or it can be an ABCG1-targeted molecular imprinting polymer, which specifically recognizes and detects ABCG1 molecules in applications such as affinity chromatography or sensor construction.

[0010] In this application, under the premise that ABCG1 can be used as a biomarker for diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia, the above-mentioned probes, PCR primers, antibodies or nucleic acid aptamers, etc. can be obtained by methods known to those skilled in the art. For example, the probe can be directly synthesized by chemical synthesis based on the known ABCG1 encoding DNA sequence, or prepared from biological materials.

[0011] In this application, the sample is derived from blood; although the inventors discovered the results in the examples through blood, it is clear to those skilled in the art that other samples derived from subjects, such as plasma, cerebrospinal fluid, saliva, tissue fluid, urine or lymph fluid, may also be suitable for the determination of the present invention if the detection conditions are met. The present invention does not place any particular limitation on the source of the sample.

[0012] In this use, the product includes a reagent for detecting the expression level of ABCG1, and the reagent may include the various detection components mentioned above, such as probes, primers, antibodies, etc., and the corresponding buffers, reaction substrates and other auxiliary reagents are also within the scope of protection of the present invention.

[0013] In this use, the product also includes a tool capable of detecting the expression of ABCG1 in a sample, and the tool may be a chip, a test strip, a test kit, etc.; further, in an embodiment of the present invention, RNA sequencing (RNA-seq) is implemented based on a high-throughput sequencing platform, and thus the high-throughput sequencing platform, or others such as an ELISA reader, a fluorescence quantitative PCR instrument, etc., are also within the scope of protection of the present invention.

[0014] The second aspect of the present invention is to provide a kit for diagnosing hyperlipidemia in HIV-infected people by measuring the expression level of ABCG1 in a sample. The kit may include the various reagents mentioned above for detecting the expression level of ABCG1, such as primers and probes for the ABCG1 gene, or antibodies for the ABCG1 protein and corresponding markers; it can be understood by those skilled in the art that the kit may also include positive controls, negative controls and calibrators, and these products are also within the scope of protection of the present invention.

[0015] The third aspect of the present invention is to provide a method for diagnosing or assisting hyperlipidemia in HIV-infected people, comprising the following steps: (1) obtaining a sample from a subject; (2) detecting the expression level of ABCG1 gene or protein in the sample; (4) if the expression level of ABCG1 gene or protein is lower than that of a normal HIV-infected patient, then the HIV-infected patient suffers from hyperlipidemia or has a tendency to suffer from hyperlipidemia.

[0016] The fourth aspect of the present invention is to provide a method for evaluating the efficacy of treatment for hyperlipidemia in HIV-infected people, comprising the following steps: (1) obtaining a sample from a subject; (2) detecting the expression level of ABCG1 gene or protein in the sample; and (3) comparing the changes in ABCG1 expression before and after treatment, and comprehensively evaluating the treatment in combination with changes in clinical symptoms (such as blood lipid levels, viral load, immune indicators, etc.).

[0017] The significant advantages of the present invention are:

[0018] The present invention provides a biomarker with high sensitivity and good specificity for the diagnosis of HIV-related hyperlipidemia. According to the expression level of ABCG1 in the test sample, it can be used to diagnose or assist in diagnosing whether hyperlipidemia exists in the HIV-infected population, and provide guidance for the early intervention of hyperlipidemia in the HIV-infected population. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a volcano plot of gene expression levels in blood samples of patients with HIV combined with hyperlipidemia and patients with simple HIV infection according to Example 1 of the present invention; red dots in the figure represent up-regulated genes, and blue dots represent down-regulated genes;

[0021] Figure 2 This is a scatter plot of ABCG1 expression levels in the HIV-only group and the HIV combined with hyperlipidemia group in Example 2 of the present invention;

[0022] Figure 3 The ROC curve diagram drawn according to the expression level of ABCG1 in the simple HIV group and the HIV combined with hyperlipidemia group in Example 2 of the present invention;

[0023] Figure 4 This is a scatter plot of ABCG1 expression levels in the HIV-only group and the HIV combined with hyperlipidemia group in Example 3 of the present invention;

[0024] Figure 5 This is a ROC curve diagram drawn based on the ABCG1 expression levels in the HIV-only group and the HIV combined with hyperlipidemia group in Example 3 of the present invention. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. 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.

[0026] The terms "first", "second" and "third" in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined as "first", "second" and "third" can explicitly or implicitly include multiple features. In the description of this application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. All directional indications (such as up, down, left, right, front, back...) in the embodiments of the present application are only used to explain the relative position relationship, movement, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication also changes accordingly. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or modules is not limited to the listed steps or modules, but optionally also includes steps or modules that are not listed, or optionally also includes other steps or modules inherent to these processes, methods, products or devices.

[0027] Reference to "embodiments" herein means that the specific features, structures, or characteristics described in conjunction with the embodiments may be included in multiple embodiments of the present application. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0028] Design process of the present invention: The inventors found that the gene expression profiles of patients with HIV infection and hyperlipidemia were significantly different from those of patients with simple HIV infection. In the applicant's own sequencing data, a total of 156 genes were found to be significantly upregulated and 102 genes were significantly downregulated, indicating that the gene expression regulation mechanism of patients with HIV infection and hyperlipidemia was changed compared with simple HIV patients. Among these differentially expressed genes, the expression level of the ABCG1 gene was significantly reduced. The ABCG1 protein, also known as ATP-binding cassette transporter G1, belongs to the ATP-binding cassette (ABC) transporter superfamily. Its main function is to participate in the transport of intracellular cholesterol and phospholipids, promote the transport of intracellular cholesterol and phospholipids to high-density lipoprotein (HDL) particles, play a key role in the reverse cholesterol transport (RCT) process, help maintain the dynamic balance of cholesterol in the body, and is crucial to the regulation of lipid metabolism.

[0029] Based on the above findings, the inventors further designed experiments to compare patients with HIV combined with hyperlipidemia with patients with simple HIV infection. The results showed that ABCG1 showed good specificity and sensitivity when used to diagnose HIV combined with hyperlipidemia. Based on these research results, the inventors completed the use of ABCG1 in the diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia. The following will describe the process in detail through specific examples.

[0030] It should be noted that the experimental methods used in the embodiments of the present invention, unless otherwise specified, are all conventional methods, and the reagents and materials used, unless otherwise specified, can be obtained from commercial channels.

[0031] Example 1: Screening of molecular markers for diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia:

[0032] RNA sequencing (RNA-seq) analysis was used to detect the expression differences of different genes in blood samples of patients with HIV combined with hyperlipidemia and patients with simple HIV infection, and to preliminarily screen molecular markers for diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia, which specifically included the following steps:

[0033] 1) Sample collection: Blood samples were collected from 16 patients in the Department of Infectious Diseases and the Department of Cardiovascular Medicine of the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital, including 8 patients with HIV and hyperlipidemia and 8 patients with simple HIV infection.

[0034] 2) Blood cell separation and RNA extraction: Use conventional methods to separate blood cells to obtain relatively pure white blood cell pellets, and use Invitrogen TRIzol reagent to extract RNA from the white blood cell pellets; after RNA extraction, use a spectrophotometer to measure the concentration and purity of RNA to ensure that the OD260 / OD280 ratio is between 1.8-2.2; then use agarose gel electrophoresis to detect the integrity of RNA, observe the clarity and brightness ratio of 28S and 18SrRNA bands, and ensure that the RNA quality meets the requirements of subsequent experiments;

[0035] 3) Library construction: Total RNA is fragmented using conventional methods and used as a template to synthesize cDNA using reverse transcriptase and random primers. Connectors containing PCR primer binding sites and sequencing identification tags are attached to both ends of the cDNA, and multiple rounds of PCR amplification with primers complementary to the adapters are performed to increase the cDNA concentration to meet sequencing requirements.

[0036] 4) RNA sequencing: Use the Illumina series high-throughput sequencing platform for sequencing. Before sequencing, calibrate and debug the sequencing platform to ensure that the instrument is in the best working condition. Prepare the reagents and consumables required for sequencing, and appropriately dilute the constructed library to make its concentration meet the requirements of the machine.

[0037] 5) Result analysis; see Figure 1 , compared the differences in gene expression in the blood of patients with HIV combined with hyperlipidemia and patients with simple HIV infection. Through statistical tests, the difference fold |log2 Fold Change|≥1 and the corrected P value Padj<0.05 were set as screening criteria to determine the genes that were significantly upregulated or downregulated between the two groups. After analysis, it was finally found that 156 genes were significantly upregulated and 102 genes were significantly downregulated.

[0038] Example 2, screening of differentially expressed gene ABCG1, is performed by RNA (RNA-seq) sequencing, specifically comprising the following steps:

[0039] 1) Sample collection: Blood samples were collected from 76 patients in the Department of Infectious Diseases and the Department of Cardiovascular Medicine of the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital, including a simple HIV group and a HIV combined with hyperlipidemia group. The simple HIV group included 33 patients with simple HIV infection, and the HIV combined with hyperlipidemia group included 33 patients with HIV combined with hyperlipidemia.

[0040] 2) Blood cell separation and RNA extraction: Use conventional methods to separate blood cells to obtain relatively pure white blood cell pellets, and use Invitrogen TRIzol reagent to extract RNA from the white blood cell pellets; after RNA extraction, use a spectrophotometer to measure the concentration and purity of RNA to ensure that the OD260 / OD280 ratio is between 1.8-2.2; then use agarose gel electrophoresis to detect the integrity of RNA, observe the clarity and brightness ratio of 28S and 18SrRNA bands, and ensure that the RNA quality meets the requirements of subsequent experiments;

[0041] 3) Library construction: Total RNA is fragmented using conventional methods, and used as a template to synthesize cDNA using reverse transcriptase and random primers. Connectors containing PCR primer binding sites and sequencing recognition tags are connected to both ends of the cDNA, and then multiple rounds of PCR amplification are performed using primers complementary to the adapter to increase the cDNA concentration to meet sequencing requirements.

[0042] 4) RNA sequencing: Use the Illumina series high-throughput sequencing platform for sequencing. Before sequencing, calibrate and debug the sequencing platform to ensure that the instrument is in the best working condition. Prepare the reagents and consumables required for sequencing, dilute the constructed library appropriately to make its concentration meet the requirements of the machine, and then perform sequencing to obtain a large number of sequencing fragments.

[0043] 5) Data processing and analysis: Calculate the number of ABCG1 gene sequencing reads for each sample in the HIV-only group and HIV-combined hyperlipidemia group. Figure 2 In the HIV combined with hyperlipidemia group, the expression level of ABCG1 was significantly downregulated, and Mann-Whitney test p < 0.05, the difference was statistically significant.

[0044] Whether or not the patient has HIV combined with hyperlipidemia is used as the binary classification result, and the expression level of the ABCG1 gene is used as the predictor. By changing the threshold for judging the disease, the sensitivity and specificity under different thresholds are calculated, and the ROC curve is drawn. Figure 3 As shown, the area under the ROC curve AUC = 0.9486, the sensitivity is 95.35%, and the specificity is 78.79%, indicating that the use of ABCG1 gene expression level as a diagnostic indicator can better distinguish HIV patients with hyperlipidemia and simple HIV infection patients, and has good diagnostic efficacy.

[0045] Example 3, further verifying the diagnostic effect of ABCG1 by enzyme-linked immunosorbent assay (ELISA), comprising the following steps:

[0046] 1) Sample collection: A total of 87 blood samples were collected from the Department of Infectious Diseases and the Department of Cardiovascular Medicine of the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital, including a simple HIV group and a HIV combined with hyperlipidemia group. The simple HIV group included 41 patients with simple HIV infection, and the HIV combined with hyperlipidemia group included 46 patients with HIV combined with hyperlipidemia. It should be noted that in order to ensure the scientific and objective verification results, all patients in this example are completely independent of the patients in Example 2 and there is no intersection.

[0047] 2) Antibody and sample processing: Antibodies specific to ABCG1 protein were coated on the microplate and incubated at 4°C overnight to allow the antibodies to adsorb to the surface of the plate wells; the microplate was filled with a blocking solution containing BSA and incubated at room temperature for 1-2 hours; protein samples from the HIV-only group and HIV combined with hyperlipidemia group were added to the coated microplate, and standard samples and blank controls were set up at the same time. The plates were incubated at room temperature for 1-2 hours to allow the ABCG1 proteins in the samples to bind to the coated antibodies.

[0048] 3) Incubation: Primary antibody incubation: discard the liquid in the wells, add the primary antibody specific to the ABCG1 protein after washing, and incubate at room temperature for 1-2 hours to allow the primary antibody to bind to the ABCG1 protein bound to the coated antibody; Secondary antibody incubation: add the secondary antibody labeled with the enzyme after washing, and incubate at room temperature for 1-2 hours to allow the secondary antibody to bind to the primary antibody.

[0049] 4) Add substrate for color development and determination: Add enzyme substrate, the enzyme catalyzes the substrate to develop color, and the depth of color is proportional to the content of ABCG1 protein in the sample; use a microplate reader to measure the optical density value (OD value) of each well at a specific wavelength, and calculate the relative content of ABCG1 protein in the sample, i.e. the relative optical density value, based on the standard curve drawn by the standard.

[0050] 5) Data processing and analysis: The relative optical density values ​​of ABCG1 protein in the HIV-only group and the HIV-combined hyperlipidemia group were compared and statistically analyzed. Figure 4 In the HIV combined with hyperlipidemia group, the expression level of ABCG1 was significantly downregulated, and the Mann-Whitney test p < 0.05, which was statistically significant.

[0051] Whether or not the patient has HIV combined with hyperlipidemia is used as the binary classification result, and the expression level of ABCG1 protein is used as the predictor. By changing the threshold for judging the disease, the sensitivity and specificity under different thresholds are calculated, and the ROC curve is drawn. Figure 5 As shown, the area under the ROC curve AUC=0.9324, the sensitivity is 91.3, and the specificity is 78.05, indicating that the ABCG1 protein detection result corresponds to the ABCG1 gene detection result of Example 2, further verifying that ABCG1 can be used as a predictive indicator for patients with HIV and hyperlipidemia.

[0052] In summary, the present invention verifies the use of ABCG1 as a biomarker in the diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia through examples, and it has the two characteristics of high sensitivity and good specificity. It can be used as a biomarker for the early diagnosis of HIV combined with hyperlipidemia, and provide guidance for the early intervention of hyperlipidemia in HIV-infected populations.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of ABCG1 as a biomarker in the preparation of products for diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia.

2. The use according to claim 1, characterized in that The product includes a reagent for detecting the expression level of ABCG1 in a sample.

3. The use according to claim 2, characterized in that: The detecting the expression level of ABCG1 in the sample includes detecting the expression level of the ABCG1 gene and / or detecting the expression level of the ABCG1 protein in the sample.

4. The use according to claim 2, characterized in that: The reagent is at least one of an oligonucleic acid probe targeting an ABCG1 encoding DNA sequence, a PCR primer targeting an ABCG1 encoding DNA sequence, an antibody targeting ABCG1, an ABCG1-specific nucleic acid aptamer, or an ABCG1-targeted molecular imprinting polymer.

5. The use according to any one of claims 2 to 4, characterized in that: The sample is derived from any one of blood, cerebrospinal fluid, and tissue fluid.

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