Biomarkers for diagnosing or aiding in the diagnosis of hiv combined hyperlipidemia and uses thereof

Abstract

The present application belongs to the field of biomedicine, and particularly relates to biomarkers for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia and application thereof, wherein the biomarkers are PLIN5 and GPX3. The present application screens differential expression genes of HIV combined with hyperlipidemia through transcriptome sequencing, verifies the diagnostic efficiency thereof through internal queue, external database and clinical sample multi-level verification, and finally determines that PLIN5 and GPX3 can be used for diagnosing or assisting in diagnosing whether hyperlipidemia exists in HIV infected population, provides guidance for early intervention of hyperlipidemia in HIV infected population, and has broad clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular, to biomarkers for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia and application thereof. BACKGROUND

[0002] The widespread use of antiretroviral therapy (ART) has transformed human immunodeficiency virus (HIV) infection into a manageable chronic disease. However, this success has been accompanied by an increase in the incidence of non-AIDS-related comorbidities, especially cardiovascular disease (CVD), which has become the leading cause of morbidity and mortality in people living with HIV (PLWH).

[0003] Due to the combined effects of viral effects and long-term antiretroviral therapy, PLWH have an increased risk of developing metabolic diseases, including dyslipidemia and hyperlipidemia. Dyslipidemia is very common in patients with chronic HIV infection and is associated with an increased risk of cardiovascular disease. In the era of effective antiretroviral therapy, HIV has become a chronic disease.

[0004] Although traditional blood lipid tests such as total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) are still the basis for clinical diagnosis, these indicators only reflect the static changes in blood lipid levels and are difficult to reveal the molecular mechanisms or early pathological processes of the disease.

[0005] With the completion of the Human Genome Project and the development of high-throughput sequencing technology, genetic screening technology has become a new method for disease diagnosis. Exploring precise diagnostic markers will help to provide guidance for the diagnosis and early intervention of hyperlipidemia in HIV-infected populations. SUMMARY

[0006] The purpose of the present application is to provide a biomarker for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia and application thereof.

[0007] The inventors performed RNA sequencing on peripheral blood mononuclear cells (PBMCs) of HIV-positive individuals with and without hyperlipidemia and found that PLIN5 and GPX3 are key down-regulated genes in HIV-positive individuals with hyperlipidemia, and performed receiver operating characteristic analysis and found that GPX3 and PLIN5 exhibit excellent diagnostic performance with high area under the curve (AUC) values; these ROC findings were then independently validated using external data sets and clinical data. Based on this, the inventors completed the following present application.

[0008] The first aspect of the present application provides the use of a reagent for detecting the expression level of a molecular marker in a biological sample in the preparation of a product for diagnosing or aiding in the diagnosis of HIV combined with hyperlipidemia, wherein the molecular marker is PLIN5 and GPX3.

[0009] In this application, the reagent can be used for quantitative or semi-quantitative analysis of the mRNA transcript of PLIN5 and GPX3 by real-time quantitative qRT-PCR, RT-PCR or gene chip method; or for measuring the concentration of PLIN5 and GPX3 protein by enzyme-linked immunosorbent assay ELISA, chemiluminescence immunoassay CLIA, immunoturbidimetry and other methods.

[0010] In this application, the reagent for detecting the expression level of PLIN5 and GPX3 in the sample includes: oligonucleotide probes targeting PLIN5 and GPX3 mRNA sequences, such as specific fragments designed for PLIN5 and GPX3 mRNA, which can detect mRNA by hybridization techniques such as Northern blotting and in situ hybridization; PCR primers targeting PLIN5 and GPX3 mRNA for PCR amplification techniques such as ordinary PCR, real-time quantitative PCR, and nested PCR to analyze mRNA levels; antibodies targeting PLIN5 and GPX3, such as monoclonal or polyclonal antibodies, which can detect protein expression by immunological techniques such as Western blotting, immunohistochemistry, and immunofluorescence; PLIN5 and GPX3 specific aptamers, which can recognize PLIN5 and GPX3 molecules using their specific binding ability; PLIN5 and GPX3 target molecularly imprinted polymers for specific recognition in affinity chromatography or sensor construction.

[0011] In this application, the above-mentioned probes, primers, antibodies or nucleic acid aptamers and other reagents can be prepared or obtained by conventional methods in the art, such as chemical synthesis, genetic engineering, hybridoma technology, etc. For example, oligonucleotide probes can be directly prepared by chemical synthesis according to known PLIN5 and GPX3 mRNA sequences.

[0012] In this application, the reagent contains detection components such as probes, primers, antibodies, and auxiliary components such as buffers, reaction substrates, standards, etc., which are within the scope of the present application.

[0013] In this application, the product includes detection chips, test strips, kits and other detection tools, which rely on high-throughput sequencing platforms such as Illumina sequencers, fluorescent quantitative PCR instruments, chip signal readers and other equipment.

[0014] In the application, the detection chip comprises a protein chip and / or a gene chip, the chip is provided with probes for detecting the expression levels of PLIN5 and GPX3 and internal reference probes, and the internal reference probes can be selected from conventional internal reference genes or proteins such as GAPDH and β-Actin.

[0015] The second aspect of the present application provides a system for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia, and application of the system in preparation of products for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia. The system is configured to include an input device for inputting the expression levels of molecular markers of a subject; a calculation device for judging whether the subject is suffering from HIV combined with hyperlipidemia according to the expression levels; and an output device for outputting a diagnosis result.

[0016] In the application, the molecular markers are PLIN5 and GPX3.

[0017] In the application, the system further comprises a detection device for detecting the expression levels of the molecular markers; the detection device comprises a device for running a PCR program, a device for detecting the nucleic acid levels of the molecular markers through nucleic acid amplification technology, or a device for detecting the protein levels of the molecular markers through antigen-antibody reaction when performing immunodetection, such as an enzyme-labeled instrument, a chemiluminescence analysis device or an immunoturbidimetry device.

[0018] The third aspect of the present application provides a method for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia, which judges whether a subject is suffering from HIV combined with hyperlipidemia according to the expression levels of the molecular markers PLIN5 and GPX3.

[0019] The fourth aspect of the present application provides a computer readable medium, on which the method for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia is recorded or run.

[0020] In the application, the computer readable medium comprises various media capable of storing, transmitting or carrying computer programs and related data for implementing the aforementioned diagnosis method, such as hard disks, floppy disks, optical disks, U disks, magnetic tapes, memory cards, solid state disks and the like. Those skilled in the art can easily understand how to use any currently known computer readable medium to create a storage carrier containing programs and data for implementing the aforementioned diagnosis method.

[0021] The present application has the following advantages: the present application provides new biomarkers for the diagnosis of HIV combined with hyperlipidemia, detects the expression levels of PLIN5 and GPX3 of a subject, and can assist existing clinical detection means to achieve more sensitive and accurate identification of HIV combined with hyperlipidemia, thereby providing guidance for early intervention of hyperlipidemia in HIV infected population, and having a broad clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the accompanying drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and all other drawings obtained by those of ordinary skill in the art without creative effort based on these drawings also belong to the protection scope of the present application.

[0023] Figure 1 The violin plot of the expression levels of GPX3 and PLIN5 in the HIV group and the HIVHLP group provided for Embodiment 1 of the present application;

[0024] Figure 2 The ROC curve diagram drawn according to the expression levels of GPX3 and PLIN5 in the HIV group and the HIVHLP group for Embodiment 1 of the present application;

[0025] Figure 3 The violin plot of the expression levels of GPX3 and PLIN5 in the HIV group and the HIVHLP group provided for Embodiment 2 of the present application;

[0026] Figure 4 The ROC curve diagram drawn according to the expression levels of GPX3 and PLIN5 in the HIV group and the HIVHLP group for Embodiment 2 of the present application;

[0027] Figure 5 The scatter plot of the expression levels of GPX3 in the HIV group and the HIVHLP group provided for Embodiment 3 of the present application;

[0028] Figure 6 The scatter plot of the expression levels of PLIN5 in the HIV group and the HIVHLP group provided for Embodiment 3 of the present application;

[0029] Figure 7 The ROC curve diagram drawn according to the expression levels of GPX3 in the HIV group and the HIVHLP group provided for Embodiment 3 of the present application;

[0030] Figure 8 The ROC curve diagram drawn according to the expression levels of PLIN5 in the HIV group and the HIVHLP group provided for Embodiment 3 of the present application. DETAILED DESCRIPTION

[0031] The technical solutions of the present application will be described clearly and completely in combination with the embodiments. Obviously, the described embodiments are only some embodiments of the present application, and not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort also belong to the protection scope of the present application.

[0032] The terms "first", "second", "third" in the present application are only for descriptive purpose, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second", "third" can explicitly or implicitly include a plurality of the features. In the description of the present application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise explicitly and specifically limited. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present application are only used to explain the relative position relationship, movement condition, etc. between components in a certain posture (as shown in the drawings), and if the certain posture changes, the directional indications also change accordingly. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion. 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 can optionally include steps or modules that are not listed, or can optionally include other steps or modules inherent to the process, method, product or device.

[0033] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment can be included in a variety of embodiments of the present application. The appearance of the phrase in various places in the specification does not necessarily all refer to the same embodiment, nor is it necessarily independent or alternative embodiments to other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0034] Design process of the present application: differential expression genes of HIV combined with hyperlipidemia are screened by transcriptome sequencing, intersection analysis of lipid metabolism related gene set is combined, core molecular markers are screened by machine learning algorithm, and finally PLIN5 and GPX3 are determined as molecular markers for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia. The process will be described in detail through specific embodiments as follows.

[0035] Example 1, screening of molecular markers for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia:

[0036] Through RNA sequencing (RNA-seq) analysis, the expression differences of different genes in blood samples of HIV combined with hyperlipidemia patients and simple HIV infected patients are detected, and molecular markers for diagnosing or assisting in diagnosing HIV combined with hyperlipidemia are preliminarily screened, which specifically includes the following steps:

[0037] 1) Sample collection

[0038] A total of 16 peripheral blood samples were collected from the Infectious Disease Department and the Cardiovascular Medicine Department of the Second Affiliated Hospital of Chengdu Medical College・Siyili Nuclear Industry Hospital, including 8 HIV-infected patients with hyperlipidemia (HIVHLP group) and 8 HIV-infected patients without hyperlipidemia (HIV group). All participants provided informed consent, and the research protocol was approved by the institutional ethics committee. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient centrifugation for transcriptome analysis, and matched serum samples were stored at -80°C;

[0039] 2) RNA sequencing and preprocessing

[0040] Total RNA was extracted from PBMCs using TRIzol reagent (Invitrogen, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Library preparation was performed using the Illumina TruSeq RNA Sample Preparation Kit, and sequencing was performed on the Illumina HiSeq X Ten platform with 150 bp paired-end reads. Raw reads were quality-filtered and aligned to the human reference genome (GRCh38) using HISAT2. Transcript abundance was quantified using StringTie and normalized to FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Principal component analysis (PCA) was performed using the DESeq2 package in R software (v4.2.2) to visualize transcriptome variation;

[0041] 3) Differential gene expression and lipid metabolism gene intersection

[0042] DESeq2 was used to identify differentially expressed genes (DEGs) between the HIVHLP and HIV groups, with a screening criterion of adjusted P-value <0.05 and |log2FoldChange|>0.5 to determine genes that were significantly up- or down-regulated between the two groups; a total of 1041 DEGs were identified, with 416 genes up-regulated and 625 genes down-regulated in the HIVHLP group compared to the HIV group; lipid metabolism-related genes were obtained from MSigDB and relevant literature. The VennDiagram package was used to identify overlapping genes between DEGs and the lipid metabolism gene set, and it was found that there were 57 overlapping genes between DEGs and the lipid metabolism-related gene set.

[0043] 4) Core gene selection based on machine learning

[0044] Three machine learning methods were used to select core genes from the 57-gene panel: least absolute shrinkage and selection operator (LASSO), random forest, and support vector machine - recursive feature elimination (SVM-RFE). The LASSO regression model used cross-validation curves to determine the optimal lambda value for gene selection, and the coefficient path analysis determined the candidate genes. Meanwhile, the random forest algorithm assessed feature importance based on the mean decrease accuracy, which revealed the top-ranked genes, among which GPX3 and PLIN5 exhibited significant feature importance. SVM-RFE analysis also validated these findings, demonstrating peak classification performance and confirming the robustness of the selected features. The intersection analysis of the three algorithms ultimately identified GPX3 and PLIN5 as core genes.

[0045] 5) Data processing and analysis

[0046] See Figure 1 To validate the expression profiles of the identified core genes GPX3 and PLIN5, the RNA sequencing data of the sample cohort was re-analyzed. The expression analysis consistently showed that GPX3 and PLIN5 were significantly downregulated in the HIVHLP group compared to the HIV group.

[0047] See Figure 2 Taking whether having HIV combined with hyperlipidemia as a binary outcome and the expression levels of GPX3 and PLIN5 genes as predictors, the sensitivity and specificity at different thresholds were calculated by changing the threshold for judging the disease, and the ROC curve was drawn; the results showed that both GPX3 and PLIN5 exhibited excellent diagnostic performance with high area under the curve (AUC) values of 0.938 and 0.906, respectively.

[0048] Example 2, external database validation of diagnostic efficacy of molecular markers:

[0049] The diagnostic efficacy of GPX3 and PLIN5 was verified using the GSE198339 dataset in the GEO database, where the HIV group included 4 HIV patients and the HIVHLP group included 4 HIV patients combined with hyperlipidemia. The original expression data was downloaded according to the standard process and processed using the R software environment (version 4.3.1). The limma package was used for differential gene expression analysis of GPX3 and PLIN5. The ggplot2 package was used to generate violin plots with embedded box plots to visualize the expression distribution between groups. The unpaired two-tailed t-test was used to evaluate statistical significance, and the Benjamini-Hochberg method was used for multiple comparison correction of p-values.

[0050] Results analysis: see Figure 3In the differential analysis in the GSE198339 dataset, the expression levels of GPX3 and PLIN5 were also significantly reduced in the HIVHLP group compared with the HIV group control; see Figure 4 In the GSE198339 dataset, the AUC value of GPX3 for diagnosing HIV combined with hyperlipidemia was 0.812, and the AUC value of PLIN5 for diagnosing HIV combined with hyperlipidemia was 0.812, further verifying that GPX3 and PLIN5 have good diagnostic efficiency.

[0051] Example Three, Verification of Diagnostic Efficiency of Molecular Markers by Clinical Dataset

[0052] A total of 70 peripheral blood samples were collected from the Infectious Disease Department and Cardiovascular Medicine Department of the Second Affiliated Hospital of Chengdu Medical College・Siyili Nuclear Industry Hospital, including 35 HIV-infected persons with hyperlipidemia (HIVHLP group) and 35 HIV-infected persons without hyperlipidemia (HIV group).

[0053] The expression levels of GPX3 and PLIN5 were detected by enzyme-linked immunoassay kit, wherein the GPX3 kit was purchased from antibodies.com, product number ABIN6958473; the PLIN5 kit was purchased from CUSABIO, product number CSB-E081398h; the samples were processed according to the corresponding kit instructions, and after incubation, the relative optical density value (OD value) of GPX3 and PLIN5 protein was determined at a specific wavelength.

[0054] The relative optical density values of GPX3 and PLIN5 protein in the HIV group and the HIVHLP group were compared and statistically analyzed. The results are shown in Figure 5 and Figure 6 In the HIVHLP group, the expression levels of GPX3 and PLIN5 were significantly down-regulated, and Mann-Whitney test <0.05, with statistical significance.

[0055] Taking whether suffering from HIV combined with hyperlipidemia as a binary classification result, using the expression levels of GPX3 and PLIN5 protein as a predictor, by changing the threshold for judging the disease, the sensitivity and specificity at different thresholds were calculated, and the ROC curve was drawn. The results are shown in Figure 7 and Figure 8The area AUC under the ROC curve of GPX3 is 0.8049, and when the threshold is <0.4311, the sensitivity is 62.86% (95%CI: 46.34% to 76.83%) and the specificity is 94.29% (95%CI: 81.39% to 98.98%). The area AUC under the ROC curve of PLIN5 is 0.8192, and when the threshold is <0.7367, the sensitivity is 85.71% (95%CI: 70.62% to 93.74%) and the specificity is 85.71% (95%CI: 70.62% to 93.74%). It is shown that the detection of GPX3 and PLIN5 proteins corresponds to the detection of GPX3 and PLIN5 mRNA in Example 1 and Example 2, and it is further verified that GPX3 and PLIN5 can be used as a prediction index for HIV patients with hyperlipidemia.

[0056] In summary, from the protein level, it is verified by the examples that GPX3 and PLIN5 can be used as biomarkers which are easy to measure, for diagnosing or assisting in diagnosing HIV patients with hyperlipidemia, and providing guidance for early intervention of hyperlipidemia in HIV infected population.

[0057] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a reagent for detecting the expression level of a molecular marker in a biological sample in the preparation of a product for diagnosing or assisting in the diagnosis of HIV combined with hyperlipidemia, characterized in that: The molecular markers are PLIN5 and GPX3.

2. The use according to claim 1, characterized in that The reagents include reagents for detecting the mRNA expression levels and / or protein expression levels of PLIN5 and GPX3.

3. The use according to claim 2, characterized in that The reagents are RNA-seq analysis reagents and / or enzyme-linked immunosorbent assay reagents.

4. The use according to claim 2, characterized in that The reagents include at least one of oligonucleotide probes targeting PLIN5 and GPX3 coding sequences, PCR primers targeting PLIN5 and GPX3 coding sequences, PLIN5 and GPX3 specific nucleic acid aptamers, and PLIN5 and GPX3 targeting molecular imprinting polymers.

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