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Back to Journal »Infection and Resistance» Volume 14

Analysis of serum exosomes microRNA in AIDS complicated with Talaromyces marneffei infection

Authors: Ning Qiyuan, Liu Nan, Wu Jizhong, Hu Dongfeng, Wei Qi, Zhou Jia, Zou Jun, Zang Nan, Li Guojie

Published on November 25, 2021, the 2021 volume: 14 pages 4931-4948

DOI https://doi.org/10.2147/IDR.S338321

Single anonymous peer review

Editor who approved for publication: Dr. Héctor M. Mora-Montes

Qiu Yuening,1,* Liu Na,2,* Wu Jizhou,1 Hu Diefei,1 Qi Wei,1 Zhou Jinai,3 Zou Jun,4 Zang Ning,5,6 Li Guojian1 1Department of Infectious Diseases, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi; 2 Institute of Life Sciences, Guangxi Medical University, Nanning, Guangxi; 3 Youjiang National Medical University, Baise, Guangxi; 4 Nanning Fourth People's Hospital, Nanning, Guangxi; 5 School of Basic Medicine, Guangxi Medical University, Nanning, Guangxi; 6 Guangxi Medical University, AIDS Key Laboratory of Prevention and Treatment, Nanning, Guangxi * Authors above have contributed equally to this research Corresponding Authors: Li Guojian; Ning Zang Phone 86 771-5356531 Email [Email Protection]; [email protected] Introduction: In order to find early diagnosis of AIDS combined TM infection For markers, we detected and analyzed serum exosomal miRNAs in AIDS patients with or without TM infection. Materials and methods: We analyzed the expression levels of miRNA in serum exosomes of 17 AIDS patients with TM-free infection and 15 AIDS patients with TM infection by RNA sequencing. For external validation, we used real-time quantitative polymerase chain reaction (qRT-PCR) to validate these results in an independent cohort of 35 AIDS patients without TM infection and 33 AIDS patients with TM infection. Finally, use bioinformatics to predict meaningful miRNA target genes and pathways. Results: A total of 131 serum exosomal miRNAs, including 73 up-regulated and 59 down-regulated miRNAs, were differentially expressed in the two groups (log2FC≥1 and FDR<0.01). Validation analysis showed that three miRNAs (miR-192-5p, miR-194-5p and miR-1246) were up-regulated in exosomes from AIDS patients with TM infection. ROC analysis showed that the AUC of the combined diagnosis of the three miRNAs was 0.742, and the diagnostic sensitivity and specificity were 0.568 and 0.861, respectively. In biological process analysis, all three miRNAs are involved in the positive regulation of transcription, DNA templating, and transcription of RNA polymerase II promoter. At the same time, related pathways involve TGF-β signaling pathway, AMPK signaling pathway, Wnt signaling pathway, MAPK signaling pathway, cGMP-PKG and cAMP signaling pathways. Conclusion: miR-192-5p, miR-194 serum exosomes -5p and miR-1246 may be potential biomarkers for AIDS patients with TM infection, and may be involved in TGF-β signaling pathway, AMPK signaling pathway, and Wnt signaling Pathways, MAPK signaling pathways, cGMP-PKG and cAMP signaling pathways. The biological functions and mechanisms of these miRNAs need to be further studied. Key words: exosomes, AIDS, Talaromyces marneffei, microRNA, biomarkers

The epidemic of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) is still a public health problem threatening the health of all human beings. According to the latest report of the World Health Organization, as of the end of 2019, there were 38 million human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) patients worldwide. 1 In 2019, there were approximately 71,204 newly infected patients with HIV/AIDS, and approximately 20,999 people in China died of AIDS2. In Guangxi3, one of the high-risk areas, the number of new HIV-1 infections exceeds 30,000.4.

After HIV-1 infection, it mainly attacks the human cellular immune system, resulting in a decrease in CD4 T lymphocytes, leading to immune function defects. When HIV-1 patients develop to the AIDS stage, they are prone to various opportunistic infections. Talaromyces marneffei (TM) is one of the common fungal infections. TM infections in China have obvious local epidemic characteristics, mainly concentrated in southern areas such as Guangxi, Guangdong, Hong Kong and Taiwan, 5 of which the incidence rate in Guangxi is 42.8%. 6 In addition, it is reported that in HIV-Guangxi 1 positive patients, when the CD4 T lymphocyte count is less than 50/μL, the TM infection rate is greater than 30%. 7 For patients with AIDS and TM infection, early diagnosis, timely antifungal combined with ART treatment can effectively control the disease progression. However, the mortality rate for those who are not diagnosed and treated in time is very high. According to statistics, the mortality rate of TM patients who received antifungal treatment was 24.3%, while the mortality rate of patients who did not receive antifungal treatment reached 50.6%. 6 It has been reported that the mortality rate of patients who did not receive antifungal treatment was promptly treated and even reached 91.3. %. 8 At present, the diagnosis of TM infection mainly relies on microscopic examination and culture of the pathogen, but the microscopic examination rate is low and the fungal culture time is long, so the early diagnosis of TM infection is very difficult.

Exosomes are nanovesicles released by different cell types, with a diameter of about 30-150nm, which are present in various body fluids, including blood, urine, saliva, tears, semen, breast milk, and ascites. Exosomes contain a variety of molecules such as proteins, lipids, nucleic acids, etc. that exhibit biological activity and reflect the state and type of their original cells. Fungi, bacteria, viruses, and other microorganisms can not only secrete exosomes that are used to spread infection and avoid the host's immune system, but they can also stimulate the production of exosomes in host cells. 9,10 Therefore, as a product of the interaction between the two. Host immune cells, pathogens, and exosomes simultaneously carry information from the host and pathogens, which has become a research hotspot. The content of exosomes varies with disease or treatment. They act as signal organelles between cells, participate in many physiological and pathological processes, and have potential roles in clinical diagnosis and treatment. 11

As one of the nucleic acid cargoes of exosomes, the microRNA (miRNA) 12 first discovered in Caenorhabditis elegans is an endogenous short-stranded non-coding RNA with regulatory functions, containing 18-25 nucleotides. It can affect the complementary sequence of mRNA and regulate gene expression after transcription. miRNA plays an important immunomodulatory role in TM infection13-15, and exosome-mediated miRNA transfer is also considered to be a genetic communication mechanism between cells. 16,17 In addition, the secretion and uptake of miRNA is not a random process but a controlled process, so it can provide new ideas for the pathogenesis of HIV-1, disease progression, related inflammation, the efficacy of ART, and therapies aimed at reducing immune activation. Research direction. 18 Therefore, this experiment aims to explore the study of serum exosomal AIDS and TM infection patients, in order to find potential biomarkers for the rapid diagnosis of TM infection, and provide new diagnostic methods for the early clinical diagnosis and early treatment of TM patients.

This study was reviewed and approved by the Ethical Review Committee of the First Affiliated Hospital of Guangxi Medical University. All research subjects signed the relevant informed consent in written form before enrollment, and conducted clinical research in accordance with the principles of the Declaration of Helsinki.

The research subjects are divided into experimental group and control group. The selected subjects were all HIV-positive patients from the First Affiliated Hospital of Guangxi Medical University and the Fourth People's Hospital with CD 4 T cell count ≤200 cells/μL. The diagnosis of all AIDS patients meets the diagnostic criteria of the "China AIDS Diagnosis and Treatment Guidelines (2018 Edition)". Laboratory testing of hepatitis B surface antigen (HBsAg), hepatitis B core antibody (HBc-Ab), hepatitis C antibody (HCV-Ab), cytomegalovirus (Cytomegalovirus, CMV) DNA, Treponema pallidum particle agglutinate (TPPA) test, Epstein-Barr Viral antibodies (EBV-Ab) and EBV-RNA were negative. Patients with Talaromyces marneffei (TM) cultured in clinical specimens were included in the experimental group, and patients with TM culture were included in the control group. All patients did not receive antiretroviral therapy, and malignant tumors and other deep fungal infections, such as cryptococcus, Candida, Aspergillus, Pneumocystis, and Mucor were excluded. Finally, the experimental cohort included 15 AIDS patients with TM infection as the experimental group and the control group. There were 17 AIDS patients with TM infection without TM. The verification cohort included 33 AIDS patients with TM infection in the experimental group and 35 AIDS patients with TM infection in the control group. The verification cohort includes all subjects in the test cohort.

The researchers’ blood samples were collected in biochemical tubes containing clotting agents, transported on ice, centrifuged at 4°C and 3000 rpm for 15 minutes, serum was collected and stored at -80°C until further use. All blood samples were processed within 12 hours after collection.

According to the manufacturer's recommendation (SBI, USA), use ExoQuick Exosome Precipitation Solution to isolate exosomes from serum. 19 Take the frozen serum sample from the -80°C refrigerator, thaw it at 25°C, and centrifuge at 3000 rpm at 4°C for 15 minutes to remove cell debris. Then, transfer about 500 μL of supernatant to a new tube, add 125 μL of ExoQuick exosome precipitation solution, vortex to mix, and incubate at 4°C for 30 minutes. After incubation, the samples were centrifuged at 4°C and 13000 rpm for 2 minutes. Without destroying the pellet, remove and discard the supernatant. Dissolve the exosomes in the pellet in a 200 µL volume of PBS and store at -80°C.

At room temperature, drop a volume of 20 μL of exosomes on a copper mesh with a diameter of 2 nm for 10 minutes, dry the filter paper, stain with a 3% phosphotungstic acid solution for 2 minutes, and air dry. After installing the copper mesh in the sample slot of the transmission electron microscope, adjust the focus and brightness to observe and photograph the sample, and analyze the vesicle structure and the shape of the exosomes. All TEM experiments were performed in the facilities of the Transmission Electron Microscope Laboratory of Guangxi Medical University.

The Zetasizer Nano ZS system (Malvern Instruments, Malvern, UK) was used to detect the particle size distribution and polydispersity index of the separated serum exosomes. 20 In the Zetasizer software, after setting the parameters, clean the measuring cell with ultrapure water, filter the sample using a 0.22 μm syringe filter, and place 40 μL of exosomes in the measuring cell for measurement.

The exosomes isolated from the serum samples were lysed with Exosomes special lysis buffer (Umibio, China) at 4°C, and the protein content was determined using the BCA protein concentration determination kit (Solarbio, China). Add SDS loading buffer (5X) to the sample and incubate at 95°C for 6 minutes. 60 micrograms of exosomal proteins per well were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane (PVDF). Then, the PVDF membrane was washed with 1×TBST buffer (pH 7.6) and blocked with 4% milk for 1 hour at room temperature. After the PVDF membrane was washed again with TBST buffer, the PVDF membrane was incubated with primary antibodies CD9, CD63 and Calnexin (Abcam, USA) at 4°C overnight. Finally, the PVDF membrane was incubated with a fluorescent secondary antibody (Shanghai Zhennuo Biotech Co., Ltd., China) for 2 hours at room temperature, and visualized with the Odyssey dual-color infrared fluorescence imaging system (LI-COR, USA).

Follow the manufacturer's instructions to isolate exosomal RNA using the Total Exosomal RNA Isolation Kit (System Biosciences, CA, USA). 19 Use the NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA) to measure the total RNA concentration.

According to the manufacturer’s recommendation, use the TruseqTM Small RNA Sample Preparation Kit (Illumina, CA) to prepare a barcoded small RNA-Seq library from 1μg of serum exosomal RNA, and collect it on a 6% Novex TBE PAGE gel, 1.0 mm, 10 wells (Invitrogen, California). The recovered library was quantified by TBS380 (Picogreen), mixed according to the data ratio, and put on the machine. Thereafter, bridge PCR amplification was performed on cBot to generate clusters, and SE50 sequencing was performed on the HiSeq sequencing platform. The entire sequencing process was implemented by Major Biotech in Shanghai, China.

In order to verify candidate miRNAs from the sequencing data, we performed qRT- PCR analysis, miR-486-5p and miR-1246, while U6 was used as an internal control. The miRNA-specific primers were designed by Kedi Company, Nanning, Guangxi, China, and synthesized by TAKARA Company. U6 specific primers are provided by TAKARA. Use Mir-XTM miRNA First Strand Synthesis Kit (TAKARA) for reverse transcription at 37°C for 60 minutes, and then inactivate the enzyme at 85°C for 5 minutes. After denaturing the template DNA at 95°C for 10 seconds, use the TB Green® Premix Ex TaqTM II kit (TAKARA), 21 in the ABI StepOne/StepOnePlus real-time system. The Ct value is normalized by the global average (ΔCt), and 2-ΔΔCt is calculated to explain the difference between groups.

Continuous variables are expressed as mean ± standard deviation. The independent sample t test was used to compare the group means of clinical and qRT-PCR miRNA measurement data, and the X2 test was used for count data. If the data is not normally distributed, we use the Wilcoxon test. Individual evaluation or logistic binary regression model was used to integrate the variables, and the receiver operating characteristic curve (ROC) was drawn at the same time. All data analysis and graphical representation are executed and generated in GraphPad Prism 5.0 and software SPSS 20.0. Based on any |log2FC|≥1 and false discovery rate (FDR) <0.01, select the exosomal miRNA ratio analysis between the two groups, and use the DESeq analysis software to express all expression data in Log2 scale, and verify the candidate miRNA qRT- At the same time, PCR is used to analyze the bioinformatics analysis on the imageGP online software. In addition, the TargetScanHuman7.2 and starBase V2.0 databases were used to predict target genes, the online gene function annotation tool of the DAVID website was used to enrich the differential genes, and the relevant signal transduction pathways were analyzed through the KEGG database (considering the p value<0.05 Interaction).

There were 17 people in the control group and 15 people in the experimental group in the experimental cohort, and 35 people in the control group and 33 people in the experimental group in the verification cohort (including the experimental cohort). figure 1). A comparative analysis of all clinical data collected showed that there were no significant differences between the control group and the experimental group in terms of age, gender, white blood cells, neutrophils, lymphocytes, and platelets, while monocyte counts, CD4 T lymphocyte counts, and CD8 T lymphocyte count and CD4/CD8 ratio are significantly different between the control group and the experimental group. The number of monocytes, the number of CD4 T lymphocytes, the number of CD8 T lymphocytes and the CD4/CD8 ratio of the experimental group were significantly lower than those of the control group (Table 1). Table 1 The clinical characteristics of the participants in the validation cohort. Figure 1 summarizes the study design of the cohort, methods, and data analysis. Using the ExoQuick kit, 500 μL of serum from subjects in the test or validation group was used to isolate exosomes. Exosomes were measured by TEM and WB. Prepare a small RNA library and sequence it on HiSeq, and verify candidate miRNAs by qRT-PCR.

Table 1 The clinical characteristics of the participants in the validation cohort

Figure 1 summarizes the study design of the cohort, methods, and data analysis. Using the ExoQuick kit, 500 μL of serum from subjects in the test or validation group was used to isolate exosomes. Exosomes were measured by TEM and WB. Prepare a small RNA library and sequence it on HiSeq, and verify candidate miRNAs by qRT-PCR.

ExoQuick exosome precipitation solution was used to isolate exosomes from the subjects' serum and characterized by transmission electron microscopy (TEM) and western blotting of exosomal markers (Figure 2). TEM showed vesicles with a diameter of 50-150nm (Figure 2A), consistent with the size range of exosomes. The DLS chart shows that the main peak is around 80nm, and the impurity peak density is low, which is consistent with the size of the exosomes, indicating acceptable purity (Figure 2B). In Western blot detection, the serum exosomal markers CD9 and CD63 can be clearly shown, and there is no calnexin in the two groups (Figure 2C). Figure 2 Characterization of exosomes isolated from serum. (A) TEM image of exosomes isolated from human serum. (B) DLS image of exosomes isolated from human serum. (C) Western blot detection of antibodies CD9, CD63 and Calnexin in the mixed control (n = 17) and experimental (n = 15) samples.

Figure 2 Characterization of exosomes isolated from serum. (A) TEM image of exosomes isolated from human serum. (B) DLS image of exosomes isolated from human serum. (C) Western blot detection of antibodies CD9, CD63 and Calnexin in the mixed control (n = 17) and experimental (n = 15) samples.

In order to verify the serum exosomal miRNA expression profile of AIDS patients with TM infection, two small RNA libraries were constructed for each group of mixed samples (17 control samples and 15 experimental samples) for deep sequencing. After removing the low-quality raw readings, a completely clean reading for subsequent bioinformatics analysis was obtained. Table 2 shows that all samples produced sequence reads, and the single-base sequencing error rate was less than 0.1%. The Q20 and Q30 of each sample are both >80%. The size distribution, base preference and distribution between the mapped reads in each sample were further analyzed. Figures 3A and B show that most of the two samples are between 18 nt and 32 nt in length, most of which are concentrated between 18 nt and 24 nt, corresponding to miRNA and siRNA. No significant deviation of GC is found in Figure 3C and D. In order to check the diversity of serum exosomal RNA cargo, reads are mapped to miRNA, rRNA, tRNA, snRNA, etc. in the Rfam database. Figures 3E and F show the different distributions of RNA species identified in serum exosomes of the control and experimental groups, respectively. Table 2 Statistical results of the original sequencing data Figure 3 Characterization of exosomal miRNA. (A) The sequence length distribution of the control group. (B) The sequence length distribution of the experimental group. (C) MiRNA nucleotide deviation at each position of the control group. (D) The miRNA nucleotide deviation of each position in the experimental group. (E) The distribution of small RNA types in serum exosomes of the control group. (F) The distribution of small RNA types in serum exosomes of the experimental group.

Table 2 Sequencing raw data statistics results

Figure 3 Characterization of exosomal miRNA. (A) The sequence length distribution of the control group. (B) The sequence length distribution of the experimental group. (C) MiRNA nucleotide deviation at each position of the control group. (D) The miRNA nucleotide deviation of each position in the experimental group. (E) The distribution of small RNA types in serum exosomes of the control group. (F) The distribution of small RNA types in serum exosomes of the experimental group.

Excluding non-miRNAs, the expression of identified miRNAs is normalized using TPM (transcripts per million; miRNAs are standardized by TPM). The miRNAs of the two groups were sorted according to their expression levels. Among the top ten miRNAs, miR-126-5p is the most abundant miRNA, followed by miR-486-5p and let-7a-5p in the control group (Figure 4A), and miR-122-5p is the most abundant miRNA miRNA, followed by let-7a-5p and miR-126-5p in the experimental group (Figure 4B). The types and abundances of miRNAs expressed in different tissues are different. Therefore, miRNAs were screened according to certain criteria (TMP≥20) to identify specific and co-expressed miRNAs in each group of samples. The results show that 245 known miRNAs are common between the control and experimental groups, and 58 and 63 are unique to the control and experimental groups (Figure 4C). Figure 4 Small RNA-seq miRNA profiles in the control and experimental groups. (A) The top 10 miRNAs in the control group. (B) The top 10 miRNAs in the experimental group. (C) Venn diagram shows common and unique miRNAs identified in the control and experimental groups.

Figure 4 Small RNA-seq miRNA profiles in the control and experimental groups. (A) The top 10 miRNAs in the control group. (B) The top 10 miRNAs in the experimental group. (C) Venn diagram shows common and unique miRNAs identified in the control and experimental groups.

Next, we studied the miRNAs associated with AIDS and TM infection. Cluster analysis and principal component analysis (PCA) were performed on the miRNAs of the two groups. The results showed that the miRNAs of the experimental group and the control group were clustered on both sides (Figure 5A), and the principal components of the two groups were quite different (Figure 5B). Further differential expression analysis using DEGseq screening revealed 73 known up-regulated miRNAs and 59 known down-regulated miRNAs (absolute value of log2FC ≥ 1 and FDR <0.01) (Figure 5E and Table 3). These differentially expressed miRNAs are ranked according to log2FC. The top ten up-regulated miRNAs are miR-5589-3p, miR-194-3p, miR-378c, miR-556-3p, miR-4271, miR-141-3p, miR-148a-5p, miR-548h-3p , MiR-548z and miR-497-5p (Figure 5C); at the same time, the top ten down-regulated miRNAs are miR-205-5p, miR-18a-5p, miR-335-3p, miR-942-5p, miR- 3605-5p, miR-379-3p, let-7e-3p, miR-4433b-3p, miR-636 and miR-935 (Figure 5D). Table 3 Summary of differentially expressed miRNAs Figure 5 Differentially expressed miRNAs of exosomes. (A) Cluster analysis of the two groups of miRNAs. (B) Principal component analysis of miRNA. (C) The top 10 up-regulated miRNAs in the experimental group. (D) The top 10 down-regulated miRNAs in the experimental group. (E) The volcano plot shows the overall differentially expressed miRNA (DEmiRNA) in the two groups. Each dot represents a miRNA. Green dots indicate down-regulated miRNAs (log2FC≥1 and FDR<0.01), while red dots indicate up-regulated miRNAs (log2FC≥1 and FDR<0.01). Gray dots indicate miRNA (log2FC<1 and FDR≥0.01).

Table 3 Summary of differentially expressed miRNAs

Figure 5 Differentially expressed exosomal miRNAs. (A) Cluster analysis of the two groups of miRNAs. (B) Principal component analysis of miRNA. (C) The top 10 up-regulated miRNAs in the experimental group. (D) The top 10 down-regulated miRNAs in the experimental group. (E) The volcano plot shows the overall differentially expressed miRNA (DEmiRNA) in the two groups. Each dot represents a miRNA. Green dots indicate down-regulated miRNAs (log2FC≥1 and FDR<0.01), while red dots indicate up-regulated miRNAs (log2FC≥1 and FDR<0.01). Gray dots indicate miRNA (log2FC<1 and FDR≥0.01).

According to the expression level of miRNAs, qRT-PCR was used to analyze 8 candidate miRNAs in a smaller test cohort of 17 subjects in the control group and 15 subjects in the experimental group, including 5 up-regulated and 3 down-regulated miRNAs. The qRT-PCR results showed that the expression levels of miR-192-5p, miR-194-5p, miR-455-3p and miR-1246 were more than 1.5 times higher, while miR-199a-3p and miR-486-5p were combined in AIDS TM infection was less than 0.67 times in patients (Table 4, test cohort). For further verification, the expression levels of the four high-expressing miRNAs in 35 subjects in the control group and 33 subjects in the experimental group (including patients in the previously smaller test cohort) were further analyzed. Based on the verification results, four miRNAs (miR-192-5p, miR-194-5p and miR-1246) were significantly up-regulated in serum exosomes of patients with AIDS co-infection with TM (fold change ≥ 2 and p value <0.05) (Figure 6, Table 4, verification queue). Table 4 Relative expression of candidate miRNAs Figure 6 The use of qRT-PCR analysis to verify 4 highly expressed miRNAs. (A) The relative expression level of miR-192-5p. (B) The relative expression level of miR-194-5p. (C) The relative expression level of miR-1246.

Table 4 Relative expression levels of candidate miRNAs

Figure 6 Use qRT-PCR analysis to verify 4 highly expressed miRNAs. (A) The relative expression level of miR-192-5p. (B) The relative expression level of miR-194-5p. (C) The relative expression level of miR-1246.

In addition, the ROC curve was drawn to evaluate the diagnostic ability of these four miRNAs to identify AIDS patients with TM infection. The area under the curve (AUC) values ​​of miR-192-5p, miR-194-5p and miR-1246 are 0.658, 0.703 and 0.721, respectively, indicating that it is possible to distinguish AIDS patients with TM infection from AIDS patients without TM infection (Figure 7A-C). The diagnostic sensitivity and specificity of miR-192-5p, miR-194-5p and miR-1246 for AIDSTM infected patients were 0.676 and 0.611, 0.649 and 0.694, 0.703 and 0.75, respectively (Table 5). Then, joint diagnosis and analysis of the three miRNAs were carried out. The AUC of the combined diagnosis was 0.742, and the sensitivity and specificity of the diagnosis were 0.568 and 0.861, respectively. The AUC and specificity of the combined diagnosis of the three miRNAs were significantly improved (Figure 7D and Table 5). Table 5 Diagnostic sensitivity and specificity of the four miRNAs Figure 7 ROC curves of the 4 highly expressed miRNAs. (A) ROC curve of miR-192-5p. (B) ROC curve of miR-194-5p. (C) ROC curve of miR-1246. (D) ROC curves of the three miRNAs used for combined diagnosis.

Table 5 Diagnostic sensitivity and specificity of four miRNAs

Figure 7 ROC curves of 4 highly expressed miRNAs. (A) ROC curve of miR-192-5p. (B) ROC curve of miR-194-5p. (C) ROC curve of miR-1246. (D) ROC curves of the three miRNAs used for combined diagnosis.

In this experiment, Gene Ontology enrichment analysis was performed on the 3 miRNA target genes, and the top 10 enrichment results were analyzed. The results show that in the analysis of biological processes, all three miRNAs are involved in the transcription of RNA polymerase II promoter, DNA templating, and the positive regulation of transcription. In addition, most of them are also involved in the negative regulation of RNA polymerase II promoter transcription, negative regulation of transcription, DNA templated and positive regulation of transcription, and DNA templated (Figure 8A). This is consistent with the enrichment results of molecular functions that mainly involve protein binding, DNA binding, transcription factor activity, sequence-specific DNA binding, transcription co-inhibitory activity, and DNA binding in transcription regulatory regions (Figure 8B). At the same time, through the analysis of the KEGG pathway of predicted targets, important pathways rich in predicted miRNA targets were identified (Table 6). Figure 9 lists the top 10 pathways of the three miRNAs. Specifically, AIDS-related miRNA target TM infections belong to multiple pathways such as TGF-β signaling pathway, AMPK signaling pathway, cAMP signaling pathway, Wnt signaling pathway, and MAPK signaling pathway (Table 6). Table 6 Three miRNA enrichment pathways Figure 8 Three miRNA gene ontology enrichment analysis. (A) Target-related gene ontology (GO) enrichment leads to the biological process of three miRNAs. (B) Target-related gene ontology (GO) enrichment leads to the molecular functions of three miRNAs. Figure 9 The first 10 pathways of the three miRNAs enriched by KEGG.

Table 6 Three miRNAs enrichment pathways

Figure 8 Gene Ontology enrichment analysis of three miRNAs. (A) Target-related gene ontology (GO) enrichment leads to the biological process of three miRNAs. (B) Target-related gene ontology (GO) enrichment leads to the molecular functions of three miRNAs.

Figure 9 The first 10 pathways of the three miRNAs enriched by KEGG.

HIV-1 is known to cause dramatic changes in the expression profile of circulating miRNA22 and milk-derived exosomal miRNA23, but its effect on serum exosomal miRNA from TM-infected patients is still unknown. Here, we characterized the miRNA expression profile of serum exosomes from AIDS patients infected with TM and showed that HIV-1 infection with TM significantly changed the expression level of exosomal miRNA. The analysis of differentially expressed miRNAs based on GO and KEGG pathways revealed that several biological processes are affected by AIDS combined with TM infection. In addition, we identified four up-regulated miRNAs, which can potentially distinguish AIDS and TM infection from uninfected TM, and have good predictive power. Overall, these data provide for the first time a comprehensive understanding of the miRNA profile of serum exosomes involved during AIDS co-infection.

In this experiment, the samples were selected strictly in accordance with the selection criteria. The final test cohort includes 15 people in the experimental group and 17 people in the control group. The verification cohort includes 33 people in the experimental group and 35 people in the control group. The number of people in the test cohort is included in the verification cohort. There were no significant differences between the experimental group and the control group in terms of age, gender, white blood cell, neutrophil, lymphocyte, and platelet counts. The monocyte count, CD4 T lymphocyte count, CD8 T lymphocyte count, and CD4/CD8 ratio of the experimental group were lower than those of the control group, but this is related to the clinical characteristics of the experimental group 24, 25 and this feature is in the TM infection It is inevitable among AIDS patients. In addition, once TM invades the human body and causes a systemic disseminated infection, it will first cause a mononuclear macrophage system response.26 This may be the reason for the decrease in the number of monocytes when AIDS is combined with TM infection. The decrease of T lymphocyte count and monocyte count in AIDS patients with TM may affect the abundance, size and miRNA expression of exosomes. 18 This is also the reason that we will explore the early markers of AIDS and TM serum exosomes.

The current traditional TM diagnosis methods cannot perform early and rapid diagnosis of TM, and there is an urgent need to find new potential biomarkers. In order to better understand the regulation of AIDS combined TM infection on serum exosomal miRNAs, we conducted a serum exosomal miRNA analysis. The results showed that serum exosomes from AIDS combined TM infection disrupted the expression levels of 131 miRNAs (FC > 2, P <0.01, Table 3). The combination of three miRNAs (miR-192-5p, miR-194-5p and miR-1246) has an AUC of 0.742, a sensitivity of 56.8%, and a specificity of 86.1% in predicting AIDS combined TM infection. And in previous literature reports, these three miRNAs are closely related to the body's inflammatory response, immune response, and disease progression. 27-30 In addition to being an intracellular pathogenic fungus, the immune clearance mechanism of TM infecting the host involves innate immunity and specificity. Therefore, this experiment verified that the up-regulated miR-192-5p, miR-194-5p and miR-1246 may be combined with AIDS TM The patient has a certain diagnostic potential and may become a biomarker for potential non-invasive AIDS patients with TM infection.

Furthermore, the pathway enrichment results showed that the pathways involved in 4 miRNAs ranged from 8 to 30, and pathways related to cancer, diabetes, and nervous system were all eliminated. The results indicate that the regulation of TGF-β signaling pathway is related to miR-192-5p and miR-194-5p. In addition, miR-192-5p is also involved in AMPK signaling pathway, miR-1246 is significantly related to cGMP-PKG and cAMP signaling pathway, miR-194-5p may regulate Wnt and MAPK signaling pathway. Among them, the AMPK signaling pathway was found to be related to infection in previous reports. 31,32 As far as HIV-1 infection is concerned, it is reported that HIV-1 infection leads to a drastic decrease in the cellular peroxidase pool, and Wnt signaling plays an important role in peroxisome homeostasis by regulating the production of biogenesis factors 33 indicates that Wnt signaling is closely related to HIV-1, and miR-194-5p, as a candidate marker for AIDS combined with TM infection, may be involved in the Wnt signaling pathway.

In addition, miR-1246 enriched pathways that may be related to AIDS and TM infection include gap junctions, cAMP signaling pathways, and cGMP-PKG signaling pathways. It is well known that miRNAs can transfer from original cells to neighboring cells and play an active role in recipient cells. 17 At the same time, the transfer of miRNA involves gap junctions between cells. Therefore, it can be inferred that the changes of serum exosomes miR-1246 in AIDS patients with TM infection may be closely related to gap junctions. In addition, the immune response is an important physiological response to infection. A recent report revealed the potential regulatory effect of exosomal miRNA in the plasma of patients with sepsis on the immune system. 34 On this basis, some scholars35 confirmed that sepsis-derived exosomes change the mRNA and protein level-related genes of apoptosis through miR-7 -5p, thereby regulating the apoptosis of T lymphocytes. The dual luciferase reporter gene detection method confirmed that miR-7-5p has a negative regulatory effect on bad genes (well-known pro-apoptotic genes) in the cGMP-PKG signaling pathway. It can be seen that the cGMP-PKG signaling pathway may be involved in the inflammatory regulation process of miRNA. In addition, as a cyclic nucleotide system, the cAMP signaling pathway is also known as the protein kinase A (PKA) system. In this system, extracellular signals can be combined with the corresponding receptors to cause a response by regulating the level of the second messenger cAMP in the cell. Signal molecules can be inflammatory factors, pathogens and their products, hormones, etc. The regulation of cAMP is carried out by adenylate cyclase. cAMP regulates key physiological processes, including metabolism, secretion, cell fate and gene transcription. In some studies, 36 caffeine has been shown to reduce the expression of miR-301b through the negative regulation of the cAMP/PKA/NF-κB axis, thereby enhancing the respiratory tract immunity and suppressing the inflammatory response during lung infection. In addition, studies have also found that miR-150 can inhibit the activity of PI3K and AKT through the cAMP-PKA-Csk signaling pathway, thereby effectively preventing CD28/B7 costimulatory signal transduction, reducing inflammatory cytokines (such as IL-2 and TNF) and Induce immune tolerance.

Finally, the TGF-β signaling pathway related to miR-192-5p and miR-194-5p plays an important role in the development, homeostasis, and repair of most tissues in the organism. All immune cell lineages, including B cells, T lymphocytes, dendritic cells, and macrophages, can secrete TGF-β. It can negatively regulate cell proliferation, differentiation and activation of other cytokines. Related reports indicate that the miRNA-rich pathway associated with HIV-1 infection involves the TGF-β signaling pathway. 22,23 In terms of inflammation, some scholars38 compared exosomes derived from human amniotic epithelial cells with human lungs. Comparing the protein and miRNA content of fibroblast exosomes, it is found that exosomes derived from human amniotic epithelial cells can increase the phagocytosis of macrophages, reduce neutrophil myeloperoxidase, and directly inhibit the proliferation of T lymphocytes. , Reduce lung inflammation, and exosomal miRNA derived from human amniotic epithelial cells can enhance the phagocytic ability of macrophages, reduce neutrophil myeloperoxidase, and epithelial cells are rich in TGF-β and other pathways. In addition, in many fungal diseases, dendritic cells (DC) that promote inflammation and tolerance are involved in the regulation of CD4 T lymphocyte response, and TM infection also leads to increased expression of CD80 and CD86 on DC. This also resulted in a significant increase in the level of TGF-β in the cell supernatant, 39 indicating that TM infection is closely related to TGF-β signal transduction. It can be seen that TGF-β signal transduction is an inflammatory signal pathway that participates in the regulation of exosomal miRNAs derived from bacteria and fungi infected cells. 39 In our study, the differentially expressed exosomes miR-192-5p and miR-194-5p from AIDS patients with TM infection may be involved in the TGF-β signal transduction pathway.

In summary, the multiple pathways enriched by miR-192-5p, miR-194-5p and miR-1246, especially the TGF-β signaling pathway enriched by miR-192-5p and miR-194-5p may be involved This process. Such as inflammation and immune response, which are consistent with the biological functions of exosomes, also indicate that this study was carried out in the serum exosomes of patients with AIDS and TM infection. The proven miRNA alteration mechanism may involve these infected signaling pathways, and the specific targeted pathways need to be further studied.

In conclusion, our research shows that serum exosomal miRNAs of patients with AIDS and TM infection have undergone significant changes. Through miRNA sequencing and qRT-PCR verification studies, the results showed that three miRNAs (miR-192-5p, miR-194-5p and miR-1246) were significantly elevated in serum exosomes of AIDSTM infected patients, and these miRNAs Related inflammation, immune response and disease progression. These up-regulated serum exosomal miRNAs are expected to be functionally related to the TGF-β signaling pathway, AMPK signaling pathway, Wnt signaling pathway, MAPK signaling pathway, cGMP-PKG signaling pathway, and cAMP signaling pathway. It reflects the continuous pathophysiological process of AIDS combined with TM infection, and can be used as a potential biomarker of AIDS combined with TM infection and a targeted mechanism involved in the pathogenesis of the disease.

This study is the first to explore the serum exosomes of patients with AIDS and TM infection. In the experiment, we combined with the analysis method of bioinformatics, and unearthed the miRNAs that are differentially expressed in the serum of AIDS and TM infected persons, which have certain diagnostic value. Some of the limitations in our study include sample size, lack of healthy controls, and the fact that this is a simple cross-sectional study design. Further study of miR-192-5p, miR-194-5p, miR-1246 and the relationship between these three miRNAs may help the early diagnosis of AIDS patients with TM infection. In addition, a large number of samples, the pathways and target genes of miRNAs deduced by GO and KEGG, and other miRNAs in known pathways need to be further studied.

We thank all the participants for making this research possible and the experimental platform provided by the Guangxi Key Laboratory of AIDS Prevention and Control.

This research was supported by the Guangxi Graduate Education Innovation Project (YCBZ2020046), the Guangxi Basic Medical Science First-Class Subject Project (GXFCDP-BMS-2019), the Nanning Key R&D Project (20193008-1), and the National Natural Science Foundation of China (31560050).

The author declares that there is no conflict of interest in this work.

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