Biomarker for diagnosis or auxiliary diagnosis of HIV (Human Immunodeficiency Virus) combined hyperlipemia and application of biomarker

Abstract

The invention belongs to the field of biomedicine, and particularly relates to biomarkers for diagnosis or auxiliary diagnosis of HIV (Human Immunodeficiency Virus) combined hyperlipemia and application of the biomarkers, and the biomarkers are PLIN5 and GPX3. Differential expression genes of HIV combined with hyperlipemia are screened through transcriptome sequencing, the diagnosis efficiency of the differential expression genes is verified in multiple layers through an internal queue, an external database and a clinical sample, and finally it is determined that PLIN5 and GPX3 can be used for diagnosing or assisting in diagnosing whether hyperlipemia exists in an HIV infected population or not. The method provides guidance for early intervention of hyperlipemia in HIV infected populations, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to biomarkers for diagnosing or assisting in the diagnosis of HIV-associated hyperlipidemia and applications thereof. Background Art

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

[0003] PLWH are at increased risk for metabolic disorders, including dyslipidemia and hyperlipidemia, due to the combined effects of viral effects and long-term antiretroviral therapy. Dyslipidemia is highly prevalent 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 of clinical diagnosis, these indicators only reflect static changes in blood lipid levels and are difficult to reveal the molecular mechanism or early pathological process of the disease.

[0005] With the completion of the Human Genome Project and the development of high-throughput sequencing technology, genetic screening has become a new method for disease diagnosis. The search for precise diagnostic markers will help guide the diagnosis and early intervention of hyperlipidemia in HIV-infected individuals. Summary of the Invention

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

[0007] The inventors performed RNA sequencing on peripheral blood mononuclear cells (PBMCs) from HIV-positive individuals with and without hyperlipidemia and discovered that PLIN5 and GPX3 were key downregulated genes in HIV-positive individuals with hyperlipidemia. Receiver operating characteristic (ROC) analysis revealed that GPX3 and PLIN5 exhibited excellent diagnostic performance, with high area under the curve (AUC) values. These ROC findings were subsequently independently validated using external datasets and clinical data. Based on this, the inventors completed the following invention.

[0008] The first aspect of the present invention is to provide a reagent for detecting the expression level of molecular markers in a biological sample for use in preparing a product for diagnosing or assisting in the diagnosis of HIV combined with hyperlipidemia, wherein the molecular markers are PLIN5 and GPX3.

[0009] In this application, the reagent can be used to perform quantitative or semi-quantitative analysis of PLIN5 and GPX3 mRNA transcripts based on real-time quantitative qRT-PCR, RT-PCR or gene chip methods; or to measure the concentration of PLIN5 and GPX3 proteins based on enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunoturbidimetry and other methods.

[0010] In this application, reagents used to detect the expression levels of PLIN5 and GPX3 in a sample include: oligonucleotide probes targeting PLIN5 and GPX3 mRNA sequences, such as those designed for specific fragments of PLIN5 and GPX3 mRNA, which can be used to detect mRNA through hybridization techniques such as Northern hybridization and in situ hybridization; PCR primers targeting PLIN5 and GPX3 mRNA, which can be used for PCR amplification techniques such as conventional PCR, real-time quantitative PCR, and nested PCR to analyze mRNA levels; monoclonal or polyclonal antibodies targeting PLIN5 and GPX3, which can be used to detect protein expression through immunological techniques such as Western Blot, immunohistochemistry, and immunofluorescence; PLIN5 and GPX3-specific nucleic acid aptamers, which can recognize PLIN5 and GPX3 molecules by using their specific binding ability; and PLIN5 and GPX3-targeted molecular imprinting polymers, which can be used 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 preparation, hybridoma technology, etc. For example, oligonucleotide probes can be directly prepared by chemical synthesis based on the known PLIN5 and GPX3 mRNA sequences.

[0012] In this application, the reagent may contain not only detection components such as probes, primers, and antibodies, but also auxiliary components such as buffers, reaction substrates, and standards, all of which fall within the scope of protection of the present invention.

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

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

[0015] A second aspect of the present invention provides a system for diagnosing or assisting in the diagnosis of HIV-associated hyperlipidemia, and its use in the manufacture of products for diagnosing or assisting in the diagnosis of HIV-associated hyperlipidemia. The system comprises an input device for inputting the expression level of a subject's molecular marker; a calculation device for determining whether a subject has HIV-associated hyperlipidemia based on the expression level; and an output device for outputting the diagnosis result.

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

[0017] In this application, the system also includes a detection device for detecting the expression level of the molecular marker; the detection device includes a device for running a PCR program to detect the nucleic acid level of the molecular marker through nucleic acid amplification technology; or a device used for immunoassay to detect the protein level of the molecular marker through antigen-antibody reaction, such as an enzyme-linked microplate reader, a chemiluminescence analysis device, or an immunoturbidimetric device.

[0018] The third aspect of the present invention is to provide a method for diagnosing or assisting in the diagnosis of HIV-associated hyperlipidemia, wherein the expression levels of the molecular markers PLIN5 and GPX3 are used to determine whether a subject has HIV-associated hyperlipidemia.

[0019] A fourth aspect of the present invention is to provide a computer-readable medium on which the aforementioned method for diagnosing or assisting in the diagnosis of HIV-related hyperlipidemia is recorded or executed.

[0020] In this application, computer-readable media include various media capable of storing, transmitting, or carrying computer programs and related data for implementing the aforementioned diagnostic methods, such as hard disks, floppy disks, optical disks, USB flash drives, magnetic tapes, memory cards, solid-state drives, etc. Those skilled in the art can readily understand how to use any currently known computer-readable media to create a storage medium containing the program and data for implementing the aforementioned diagnostic methods.

[0021] The significant advantages of the present invention are that it provides a new biomarker for the diagnosis of HIV-associated hyperlipidemia. By detecting the expression levels of PLIN5 and GPX3 in subjects, it can assist existing clinical detection methods in achieving more sensitive and accurate identification of HIV-associated hyperlipidemia, and help provide guidance for early intervention of hyperlipidemia in HIV-infected populations. It has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Violin plot comparing the expression levels of GPX3 and PLIN5 in the HIV group and HIVHLP group provided in Example 1 of the present invention; Figure 2 This is a ROC curve diagram drawn according to the expression levels of GPX3 and PLIN5 in the HIV group and the HIVHLP group in Example 1 of the present invention; Figure 3 Violin plot comparing the expression levels of GPX3 and PLIN5 in the HIV group and HIVHLP group provided in Example 2 of the present invention; Figure 4 This is a ROC curve diagram drawn according to the expression levels of GPX3 and PLIN5 in the HIV group and the HIVHLP group in Example 2 of the present invention; Figure 5 This is a scatter plot of GPX3 expression levels in the HIV group and HIVHLP group provided in Example 3 of the present invention; Figure 6 This is a scatter plot of PLIN5 expression levels in the HIV group and HIVHLP group provided in Example 3 of the present invention; Figure 7 ROC curves plotted for GPX3 expression levels in the HIV group and HIVHLP group provided in Example 3 of the present invention; Figure 8 The ROC curve diagram of the PLIN5 expression levels in the HIV group and HIVHLP group provided in Example 3 of the present invention is drawn. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] The terms "first," "second," and "third" in this application are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features indicated. Therefore, features specified as "first," "second," or "third" may explicitly or implicitly include multiple such features. In the description of this application, "multiple" means at least two, for example, two, three, etc., unless otherwise specifically defined. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are intended only to illustrate the relative positional relationships and movement of components in a specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indications will also change accordingly. Furthermore, the terms "including," "having," and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or device comprising a series of steps or modules is not limited to the listed steps or modules and may optionally include steps or modules not listed, or may optionally include other steps or modules inherent to such process, method, product, or device.

[0026] References to "embodiments" herein mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in multiple embodiments of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0027] The design process of this invention involves screening for differentially expressed genes in HIV-associated hyperlipidemia through transcriptome sequencing, combining this with intersection analysis of lipid metabolism-related gene sets, and using machine learning algorithms to screen for core molecular markers. The diagnostic efficacy is then validated across multiple levels using internal cohorts, external databases, and clinical samples. Ultimately, PLIN5 and GPX3 were identified as molecular markers for the diagnosis or auxiliary diagnosis of HIV-associated hyperlipidemia. This process is detailed below through specific examples.

[0028] Example 1, Screening of molecular markers for diagnosis or auxiliary diagnosis of HIV combined with hyperlipidemia: RNA sequencing (RNA-seq) analysis was used to detect differences in the expression of different genes in blood samples from patients with HIV-positive hyperlipidemia and those with simple HIV infection. The initial screening of molecular markers for diagnosis or auxiliary diagnosis of HIV-positive hyperlipidemia was conducted. The specific steps involved were: 1) Sample collection A total of 16 peripheral blood samples were collected from the Department of Infectious Diseases and the Department of Cardiology at the Second Affiliated Hospital of Chengdu Medical College and the 416th Nuclear Industry Hospital. These samples included 8 HIV-infected individuals with hyperlipidemia (HIVHLP group) and 8 HIV-infected individuals without hyperlipidemia (HIV group). All participants provided informed consent, and the study 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. 2) RNA sequencing and preprocessing Total RNA was extracted from PBMCs using TRIzol reagent (Invitrogen, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit and sequenced on the Illumina HiSeqXTen platform using 150-bp paired-end reads. Raw reads were quality-filtered using HISAT2 and aligned to the human reference genome (GRCh38). Transcript abundance was quantified using StringTie and normalized to per kilobase of transcript per million mapped reads (FPKM). Principal component analysis (PCA) was performed using the DESeq2 package in R software (v4.2.2) to visualize transcriptome variation. 3) Differential gene expression and lipid metabolism gene intersection DESeq2 was used to identify differentially expressed genes (DEGs) between the HIV HLP and HIV groups. The screening criteria were adjusted P value < 0.05 and |log2FoldChange| > 0.5. Genes that were significantly upregulated or downregulated between the two groups were identified. A total of 1041 differentially expressed genes (DEGs) were identified, of which 416 were upregulated and 625 were downregulated in the HIV HLP group compared with the HIV group. Lipid metabolism-related genes were obtained from MSigDB and relevant literature. The VennDiagram package was used to identify overlapping genes between the DEGs and the lipid metabolism gene set, revealing 57 genes that overlapped between the differentially expressed genes (DEGs) and the lipid metabolism-related gene set.

[0029] 4) Core gene selection based on machine learning Core genes were selected from the intersection of 57 genes using three machine learning methods: 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 candidate genes were identified through coefficient path analysis. Meanwhile, the random forest algorithm assessed feature importance based on mean reduced accuracy, revealing top-ranked genes, with GPX3 and PLIN5 demonstrating significant feature importance. SVM-RFE analysis also validated these findings, demonstrating peak classification performance and confirming the robustness of the selected features. Intersection analysis of the three algorithms ultimately identified GPX3 and PLIN5 as core genes.

[0030] 5) Data processing and analysis See Figure 1 To validate the expression profiles of the identified core genes GPX3 and PLIN5, we reanalyzed the RNA sequencing data of the sample cohort. Expression analysis consistently demonstrated that GPX3 and PLIN5 were significantly downregulated in the HIV HLP group compared with the HIV group.

[0031] See Figure 2 , taking whether or not the patient has HIV combined with hyperlipidemia as the result of binary classification, and the expression levels of GPX3 and PLIN5 genes as predictors, by changing the threshold for judging the disease, calculating the sensitivity and specificity under different thresholds, and drawing the ROC curve; the results showed that both GPX3 and PLIN5 showed excellent diagnostic performance, with high area under the curve (AUC) values ​​of 0.938 and 0.906, respectively.

[0032] Example 2: External database verification of the diagnostic efficacy of molecular markers: The diagnostic performance of GPX3 and PLIN5 was validated using the GSE198339 dataset from the GEO database. The HIV group included four patients with HIV alone, and the HIV HLP group included four patients with HIV and hyperlipidemia. Raw expression data were downloaded according to standard protocols and processed using the R software environment (version 4.3.1). Differential gene expression analysis of GPX3 and PLIN5 was performed using the limma package. Violin plots with inline boxplots were generated using the ggplot2 package to visualize expression distributions between groups. Statistical significance was assessed using an unpaired two-tailed t-test, and p-values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method.

[0033] Result analysis: See Figure 3In the differential analysis of the GSE198339 dataset, the expression levels of GPX3 and PLIN5 were also significantly reduced in the HIVHLP group compared with the HIV control group; see Figure 4 ,In the GSE198339 dataset, the AUC value of GPX3 for diagnosing HIV with hyperlipidemia was 0.812, and the AUC value of PLIN5 for diagnosing HIV with hyperlipidemia was 0.812, which further verified that GPX3 and PLIN5 have good diagnostic efficiency.

[0034] Example 3: Clinical Dataset Verification of the Diagnostic Efficacy of Molecular Markers A total of 70 peripheral blood samples were collected from the Department of Infectious Diseases and the Department of Cardiology of the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital, including 35 HIV-infected patients with hyperlipidemia (HIVHLP group) and 35 HIV-infected patients without hyperlipidemia (HIV group).

[0035] The expression levels of GPX3 and PLIN5 were detected using enzyme-linked immunosorbent assay kits. The GPX3 kit was purchased from antibodies.com with the product number ABIN6958473, and the PLIN5 kit was purchased from CUSABIO with the product number CSB-E081398h. Samples were processed according to the instructions of the corresponding kits. After incubation, the relative optical density (OD) values ​​of GPX3 and PLIN5 proteins were measured at a specific wavelength.

[0036] The relative optical density values ​​of GPX3 and PLIN5 proteins in the HIV group and HIVHLP group were compared and statistically analyzed. Figure 5 and Figure 6 In the HIVHLP group, the expression levels of GPX3 and PLIN5 were significantly downregulated, and the Mann-Whitney test was less than 0.05, which was statistically significant.

[0037] The presence or absence of HIV combined with hyperlipidemia was used as a binary classification result, and the expression levels of GPX3 and PLIN5 proteins were used as predictors. By changing the threshold for judging the disease, the sensitivity and specificity at different thresholds were calculated, and the ROC curve was drawn. Figure 7 and Figure 8The area under the ROC curve (AUC) for GPX3 was 0.8049. When the threshold was <0.4311, the sensitivity was 62.86% (95% CI: 46.34% to 76.83%) and the specificity was 94.29% (95% CI: 81.39% to 98.98%). The area under the ROC curve (AUC) for PLIN5 was 0.8192. When the threshold was <0.7367, the sensitivity was 85.71% (95% CI: 70.62% to 93.74%) and the specificity was 85.71% (95% CI: 70.62% to 93.74%). This indicates that the GPX3 and PLIN5 protein detection results are generally consistent with the GPX3 and PLIN5 mRNA detection results in Examples 1 and 2, further validating that GPX3 and PLIN5 can serve as predictive indicators for patients with HIV and hyperlipidemia.

[0038] In summary, from the perspective of protein level, the present invention verifies through examples that GPX3 and PLIN5 can be used as easily measurable biomarkers for the diagnosis or auxiliary diagnosis of HIV-associated hyperlipidemia, providing guidance for early intervention of hyperlipidemia in HIV-infected populations.

[0039] 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 principles of the present invention should be included in the scope of protection of the present invention.

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.

5. The use according to claim 1, characterized in that The article of manufacture includes a kit, a system, a device, or a computer-readable medium.

6. The use according to claim 5, characterized in that The device includes a PCR instrument, a high-throughput sequencing platform, a detection chip or a chip signal reader.

7. The use according to claim 6, characterized in that The detection chip includes probes for detecting the expression levels of PLIN5 and GPX3, and the chip includes a PLIN5 and GPX3 protein chip and / or a gene chip.

8. The use according to claim 6, characterized in that The system includes an input device for inputting the expression level of a subject's molecular marker; a calculation device for determining whether the subject suffers from HIV combined with hyperlipidemia based on the expression level; and an output device for outputting the diagnosis result.

9. The use according to claim 8, characterized in that: The system also includes a detection device for detecting the expression level of molecular markers, and the detection device includes a device for running a PCR program or an immune detection device.

10. The use according to claim 5, characterized in that: The computer-readable medium records or runs a method for diagnosing HIV combined with hyperlipidemia, which includes determining whether a subject has HIV combined with hyperlipidemia based on the expression level of a molecular marker.

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