Exosome miRNA closely related to SLE, target gene and application thereof

Abstract

The invention provides exosome miRNA closely related to SLE, a target gene and application of the exosome miRNA and the target gene, and belongs to the technical field of biological medicine. The exosome miRNA is let-7f-5p, and the RNA sequence table of the exosome miRNA is as shown in SEQ ID No. 1. The target genes of the exosome miRNA closely related to the SLE comprise Cd52, Gbp2, Gbp7 and Gatm, and the amino acid sequences of the coding proteins of the target genes are respectively shown as SEQ ID No.2-5. According to transcriptomic analysis, the biological function of an abnormally expressed gene is matched with that of an SLE related signal channel, let-7f-5p is abnormally expressed in lupus nephritis, and the exosome miRNA can be used as a biomarker for clinical diagnosis of SLE. The let-7f-5p can relieve the inflammatory state of an RAW264.7 cell inflammation model, and mesenchymal stem cell co-culture can relieve the inflammatory state of macrophages, so that the expression of the let-7f-5p in the macrophage inflammation model is increased, and the expression of a target gene Gbp7 of the let-7f-5p in the macrophage inflammation model is reduced, and further verification shows that the let-7f-5p can be used as the SLE biomarker.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically to exosomal miRNA, target genes and applications thereof that are closely related to SLE. Background Art

[0002] Systemic lupus erythematosus (SLE) is a highly destructive autoimmune disease. The intricate interactions between genetic susceptibility, adaptive and acquired immune responses lead to excessive production of type I interferon and other cytokines, which in turn trigger complement activation and immune complex deposition, ultimately leading to inflammatory responses and tissue damage. Lupus nephritis (LN) can occur in the early stages of the disease and is also the main type of organ damage in the late stages of SLE. Macrophage infiltration aggravates the inflammatory response and is significantly associated with the poor prognosis of LN. Advances in basic research have promoted the continuous optimization of treatment options, but due to the complex and diverse etiology of LN, the efficacy and prognosis are still not ideal.

[0003] Transcriptomics studies the sum of all RNA transcripts present in a biological sample. Over the past few decades, the explosive development of sequencing technology has provided a special impetus for transcriptomics research and gene expression measurement methods. For example, in 2003, the interferon gene associated with SLE was discovered through blood mononuclear cell microarray analysis, which was a landmark discovery in the early days of lupus. However, to this day, the detection objects of sequencing technology are mostly concentrated at the level of body fluid systems, tissues, and cell lines. More subtle biological structures, such as exosomes, can push biology and medicine to a new dimension with the valuable information they carry, relying on transcriptome analysis, which is conducive to our in-depth study of the pathogenesis of LN.

[0004] Exosomes are the smallest extracellular vesicles that carry a variety of proteins, lipids and genetic materials. They have a wide range of sources and can be released by almost all types of cells in the human body under various pathological and physiological conditions, and participate in local and systemic signal exchanges. So far, research on exosomes in LN has mainly focused on the cellular or body fluid level, and there is little exploration of tissue exosomes. Exosomes play an important immunoregulatory role in a variety of autoimmune diseases, especially the microRNAs (miRNAs) contained in them. MiRNAs are non-coding single-stranded RNA molecules that are stably present in exosomes. They can be transported to receptor cells with exosomes to regulate target gene expression, thereby affecting immune and inflammatory responses, and have the potential for diagnosis and treatment. Summary of the invention

[0005] The present invention provides an exosomal miRNA, a target gene and an application thereof that are closely related to SLE, and provides a biomarker for the clinical diagnosis of SLE, so as to facilitate subsequent better diagnosis and treatment.

[0006] The present invention adopts the following technical solution: In a first aspect, the present invention provides an exosomal miRNA closely related to SLE, wherein the exosomal miRNA is let-7f-5p, and its RNA sequence table is shown in SEQ ID No.1.

[0007] The above exosomal miRNAs are derived from renal tissue exosomes, and renal tissue exosomes are extracted by ultracentrifugation.

[0008] The method for obtaining let-7f-5p comprises the following steps: (1) Establish MRL / lpr spontaneous and pristane-induced lupus mouse models, evaluate the skin damage, urine protein and serum anti-dsDNA antibody levels of the models, and detect the levels of renal inflammatory factors; (2) Exosomes were extracted from renal tissues of MRL / lpr lupus mice and MRL / MpJ control mice, and the exosomes were identified by particle size analysis, transmission electron microscopy observation, and flow cytometry detection of surface markers; (3) Library construction and RNA sequencing were performed on the RNA extracted from renal tissue exosomes to obtain miRNA and mRNA expression profiles, and the differential expression analysis of miRNA and mRNA was performed using the DESeq algorithm; (4) Real-time fluorescence quantitative PCR technology was used to verify the expression level of let-7f-5p in renal tissue exosomes.

[0009] In a second aspect, the present invention provides target genes of exosomal miRNA closely related to SLE, including Cd52, Gbp2, Gbp7 and Gatm, and the amino acid sequences of proteins encoded by each target gene are shown in SEQ ID No. 2-5, respectively.

[0010] In a third aspect, the present invention provides an application of exosomal miRNA closely related to SLE, wherein mesenchymal stem cells increase the expression of let-7f-5p in the RAW264.7 macrophage inflammation model.

[0011] It can be seen from the above description of the present invention that, compared with the prior art, the present invention has the following advantages: 1. The exosomal miRNA of the present invention, through transcriptomic analysis, the biological function of abnormally expressed genes is consistent with the SLE-related signaling pathway. Let-7f-5p is abnormally expressed in lupus nephritis and can be used as a biomarker for the clinical diagnosis of SLE.

[0012] 2. let-7f-5p can alleviate the inflammatory state of the RAW264.7 cell inflammation model, while co-culture of mesenchymal stem cells can alleviate the inflammatory state of macrophages, increasing the expression of let-7f-5p and decreasing the expression of its target gene Gbp7 in the macrophage inflammation model, further verifying that let-7f-5p can be used as a biomarker for SLE. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The following are the general characteristics of MRL / lpr and MRL / MpJ mice (A. facial skin lesions; B. erythema on the back of the head; C. external ear ulceration; D. subcutaneous lymph node swelling; E. spleen enlargement; F. anti-dsDNA antibody level; G. urine protein level).

[0014] Figure 2 The following are the transcriptional status of inflammatory factors in renal tissue of MRL / lpr and MRL / MpJ mice (A. IL-1β; B. TNF-a; C. iNOS).

[0015] Figure 3 The following are the general characteristics of pristane-induced lupus mice and BALB / c control mice (A. facial skin lesions; B. joint swelling; C. spleen enlargement; D. abdominal fat granuloma; E. anti-dsDNA antibody level; F. urine protein level).

[0016] Figure 4 Schematic diagram of the transcription of inflammatory factors in renal tissue of pristane-induced lupus mice and BALB / c control mice (A. IL-1β; B. TNF-a; C. iNOS).

[0017] Figure 5 Schematic diagram of the electron microscopy results of exosomes in renal tissue of MRL / lpr and MRL / MpJ mice.

[0018] Figure 6 Schematic diagram of NTA of exosomes from MRL / lpr and MRL / MpJ mouse tissues (A, B: average particle size detection; C, D: particle concentration).

[0019] Figure 7 Schematic diagram of nanoflow cytometry analysis for detecting exosome surface protein expression.

[0020] Figure 8 Volcano plot and heat map of differentially expressed miRNAs (A. Volcano plot, B. Heat map).

[0021] Fig. 9 Venn diagram of predicted and validated target genes for differentially expressed miRNAs.

[0022] Fig.10 Enrichment analysis diagram of target genes predicted for differentially expressed miRNAs (A. GO analysis, B. KEGG analysis, C. Reactome analysis).

[0023] Fig.11 Enrichment analysis diagram of the validated target genes of differentially expressed miRNAs (A. GO analysis, B. KEGG analysis, C. Reactome analysis).

[0024] Fig.12 The distribution of mRNA on chromosomes and the principal component analysis diagram of samples (A, distribution of mRNA on chromosomes; B, distribution of differentially expressed mRNA on chromosomes; C, two-dimensional PCA analysis diagram).

[0025] Fig.13 Volcano plot and heat map of differentially expressed mRNA (A. Volcano plot of differential mRNA, B. Heat map of differential mRNA).

[0026] Fig.14 Enrichment analysis diagram of differentially expressed mRNA (A. GO analysis, B. KEGG analysis, C. Reactome analysis).

[0027] Fig.15 Gene joint analysis diagrams (A. Venn diagram; B. Bar graph of enriched entries; C. Web graph of enriched entries).

[0028] Fig.16 are the relative expression levels of miRNAs molecules in the renal tissue and exosomes of lupus group and control group mice; A, B, and C are the verification of the exosome levels in the renal tissue of MRL / lpr and MRL / MpJ mice; D and E are the verification of the renal tissue levels of pristane-induced mice and BALB / c mice.

[0029] Fig.17 Enrichment analysis of target genes of mmu-let-7f-5p (A, GO analysis: network diagram of key terms; B, GO analysis: statistical significance of key terms; C, KEGG enrichment analysis bar chart; D, Venn diagram of mmu-let-7f-5p target genes).

[0030] Fig.18 Schematic diagram of the expression levels of each target gene in the kidney tissue of lupus model mice and control group mice.

[0031] Fig.19 Schematic diagram of RAW264.7 mouse monocyte-macrophage culture and LPS intervention.

[0032] Fig. 20 Schematic diagram of the expression of various molecules after LPS intervention in RAW264.7 cells.

[0033] Fig.21 Schematic diagram for evaluating the efficiency of transfection with let-7f-5p mimics.

[0034] Fig. 22 Schematic diagram of the expression of various molecules in RAW264.7 cells after transient transfection of mmu-let-7f-5p mimics after LPS intervention.

[0035] Fig.23 Diagram of induced differentiation of mesenchymal stem cells (A, adipogenic differentiation; B, osteogenic differentiation; C, chondrogenic differentiation).

[0036] Fig.24 This is a flow chart of the surface markers of mesenchymal stem cells.

[0037] Fig.25 Schematic diagram of the effect of mesenchymal stem cells on the expression levels of various molecules in RAW264.7 cells. DETAILED DESCRIPTION

[0038] The specific implementation of the present invention is described below. In order to fully understand the present invention, many details are described below, but for those skilled in the art, the present invention can be implemented without these details. For well-known components, methods and processes, they are not described in detail below.

[0039] Example 1: Acquisition of exosome miRNA 1. Isolation and identification of exosomes from renal tissue of lupus mice.

[0040] 1.1 Establishment of spontaneous lupus (MRL / lpr) mouse model and induced lupus (MRL / MpJ) mouse model; Five 7-week-old female MRL / lpr mice and five 7-week-old female MRL / MpJ mice were divided into two groups and observed after one week of adaptive feeding to construct a spontaneous lupus mouse model. From 8 to 16 weeks of age, the MRL / MpJ control group mice showed smooth fur, good mental state, weight gain, flexible limbs, and no joint redness and swelling. MRL / lpr mice began to develop facial lesions, dry hair, and erythematous lesions on the back of the head from 12 to 15 weeks of age, which gradually worsened. Some mice developed external ear ulcers, swollen subcutaneous lymph nodes, mental depression or abnormal excitement, relatively slow weight gain, limited limb movement, and joint swelling, which could affect the entire paw. At 16 weeks of age, urine protein was elevated by CCB method (P<0.0001), and serum anti-dsDNA antibody levels were also significantly elevated by ELISA method (P<0.01) (Figure 1).

[0041] MRL / lpr lupus mice and MRL / MpJ control mice were killed after anesthesia. The spleen of MRL / lpr mice was enlarged after dissection. The kidney tissues of the two groups were taken to extract mRNA. The expression of inflammatory factors in the kidney tissues of mice was detected by RT-qPCR. It was found that the expression levels of IL-1β and TNF-α were significantly increased in MRL / lpr lupus mice (IL-1β, p<0.001; TNF-α, p<0.01). iNOS also showed an increasing trend in MRL / lpr mice, but it was not statistically significant (p>0.05) (Figure 2).

[0042] Pristane-induced lupus model was constructed simultaneously. 7-week-old clean-grade BALB / c female mice were used. After one week of adaptive feeding, the model was constructed by a single intraperitoneal injection of 0.5 ml of Pristane. The modeling process lasted for 4 months. During this period, the BALB / c control group had normal hair, no skin lesions, and free movement of the limbs and joints, while the Pristane group had slow weight gain, dull hair, and mental depression. In the fourth month, facial lesions, swollen limb joints, and increased urine protein levels appeared (P<0.0001). After anesthesia, blood was drawn from the medial canthus vein to check the serum anti-dsDNA antibody level, which was significantly increased (P<0.05). After anesthesia, the mice were killed and dissected to see that the spleen of the Pristane mice was enlarged and there were scattered fat granulomas in the abdominal cavity (Figure 3).

[0043] Renal tissues of the two groups were used to extract mRNA, and the expression of inflammatory factors in the renal tissues of mice was detected by RT-qPCR. It was found that the expression levels of IL-1β and TNF-α were significantly increased in the kidneys of pristane-induced lupus mice (IL-1β, p>0.05; TNF-α, p<0.001), and the expression level of iNOS did not change significantly (p>0.05) (Figure 4).

[0044] 1.2 Isolation and identification of exosomes from MRL / lpr mouse tissues Exosomes were extracted from the renal tissues of MRL / lpr and MRL / MpJ mice and observed under a transmission electron microscope (TEM). It can be seen that the exosomes in the renal tissues of both groups of mice are round vesicle-like complete membrane structures, with a double-layer lipid membrane with obvious boundaries on the outside and darker staining, while the inside is low-electron density material with lighter staining, showing a typical "tray" or "cup-shaped" morphology (Figure 5). Exosomes vary in size, with diameters ranging from 30-150nm. Nanoparticle tracer analysis (NTA) showed that the average particle size of exosomes in the renal tissues of MRL / lpr mice was 85.1nm, and the particle concentration was 9.77x1010 Particles / mL, and the average particle size of exosomes in the renal tissues of MRL / MpJ mice was 77.7nm, and the particle concentration was 7.02x1010 Particles / mL ( Figure 6 ). The expression rates of exosome surface markers CD9, CD63 and CD81 detected by nanoflow cytometry in the two groups of samples were significantly higher than those in the blank control (Figure 7).

[0045] 2. Bioinformatics Analysis of Differential Genes in Exosomes 2.1 Bioinformatics analysis of exosomal miRNA in renal tissue miRDeep2 was used to identify the expression levels of miRNA (annotated) in the exosomes of renal tissues of MRL / lpr and MRL / MpJ mice, and DESeq2 was used in R to normalize the expression levels and perform differential expression analysis. A total of 25 differentially expressed mature miRNAs were screened using the criteria of P<0.05 and |logFC|>1. Among them, 15 were upregulated molecules, accounting for 60.0%, and 10 were downregulated molecules, accounting for 40.0%, as shown in Figure 8. For these differentially expressed miRNAs, Rpackage multiMiR was used to retrieve their corresponding target genes, including predicted target genes and target genes with experimental verification results (experimental verification methodologies include qPCR, western blot, fluorescent labeling, northern blot or degradation group sequencing, etc.), and the 25 differential miRNA predictions and verified target genes were visualized using a Venn diagram (Figure 9). To further evaluate the possible functions of differentially expressed exosomal miRNAs between lupus mice and control groups, this study functionally annotated the predicted and validated target genes by GeneOntology (GO), KEGG, and Reactome enrichment analysis, respectively.

[0046] When the predicted target genes were enriched, it was found that they were enriched in the Wnt signaling pathway through both GO and KEGG. In KEGG analysis and Reactome analysis, the predicted target genes were also enriched in TGF-β-related signaling pathways, PI3K-Akt signaling pathways, MAPK signaling pathways, and synaptic guidance pathways (P<0.05). The scatter plot was used for visualization to specifically display the top 20 enriched entries (Figure 10).

[0047] When enrichment analysis was performed on verified target genes, it was found that the results of GO enrichment analysis were mainly concentrated on the regulation of GTPase-activated proteins, while the enrichment results of KEGG analysis were more consistent with the KEGG enrichment results of predicted target genes, such as PI3K-Akt signaling pathway, MAPK signaling pathway, ErbB signaling pathway, endocytosis and tumor proteoglycans. The results enriched by the Reactome database were mainly related pathways mediated by GTPase-activated proteins and Toll-like receptors. The top 20 enriched entries were specifically displayed using a scatter plot visualization process (Figure 11).

[0048] 2.2 Bioinformatics analysis of exosome mRNA in renal tissue Using Hisat2 software, according to the data measured by the Illumina platform, a comparison analysis based on the reference genome was performed, and the protein coding gene was quantified and annotated by feature counts. The mRNA distribution on chromosomes 2, 7 and 11 was the largest, and the distribution of differentially expressed mRNA on chromosomes was similar. The principal component analysis (PCA) was performed using the R software DESeq2 package. The higher the similarity of expression between the samples to be tested, the closer the samples are in two-dimensional space. From the two-dimensional PCA scatter plot, it can be intuitively seen that the similarity within the MRL / lpr mouse kidney sample group is very high, and it is significantly distinguished from the MRL / MpJ control group mouse samples. This shows that the repeatability within the group is good, providing a basis for the reliability of subsequent differential gene expression analysis (Figure 12).

[0049] The R software DESeq2 package was used to screen differentially expressed genes. According to the threshold P<0.05,|LogFC|>1, a total of 1393 differentially expressed genes were obtained, including 551 up-regulated genes and 842 down-regulated genes. The gene expression volcano map was drawn with Log2 Fold Change as the horizontal axis and -Log10 P value as the vertical axis. At the same time, the cluster heat map was used to visualize the expression changes of these differentially expressed genes in different samples ( Fig.13 ).

[0050] The differentially expressed mRNAs were enriched, including Gene Ontology (GO), KEGG, and Reactome enrichment analysis. GO analysis revealed that biological functions related to viral processes, regulation of viral life cycles, and response to interferon were significantly enriched. KEGG analysis enriched Toll-like receptor signaling pathways, EB virus infection, and IL-17 signaling pathways. Reactome enrichment analysis focused on platelet-related biological activities and cell aging, which are consistent with the currently known pathogenesis of SLE. This study used a scatter plot to display the analysis results (Figure 14).

[0051] The GSE160488 dataset is based on the chip data of MRL / lpr and MRL / MpJ renal tissue biopsies, including 3 MRL / MpJ control mice and 3 MRL / lpr lupus mice. The GEO2R analysis tool was used for online differential analysis, and it was determined that there were 2092 differentially expressed genes in renal tissues between MRL / lpr and MRL / MpJ according to P<0.05 and |logFC|>1, of which 1358 were upregulated and 734 were downregulated. The differentially expressed genes in the GSE160488 dataset were intersected with the differentially expressed mRNA in the sequencing data of this study, and further determined by the Venn diagram that there were 108 genes upregulated in both the sequencing data of this study and the GSE160488 dataset, and 14 genes downregulated. The above 122 up- and down-regulated genes were subjected to GO and KEGG enrichment analysis in the Metascape online database, and the top 20 entries with the most significant differences were displayed. As shown in Figure 15, immune-related biological activities such as innate immune response, positive regulation of defense response, and inflammatory response were significantly enriched. According to the enriched signal pathways and biological processes, clusters were formed and term networks were constructed. Entries with similarity scores greater than 0.3 were linked by edges to better understand the relationship between pathways and biological processes. The visualization results are shown in Figure 15. Innate immune response is closely connected with other clusters and significantly enriched.

[0052] 3. Verification of differential miRNAs and target genes 3.1 Verification of differential miRNA levels in exosomes From the 25 differentially expressed miRNAs, mmu-let-7f-5p, mmu-miR-483-5P and mmu-miR-155-5p were selected for differential expression verification. The relative expression of mmu-let-7f-5p in the exosomes of renal tissue of MRL / lpr mice was detected by RT-qPCR, and it was found that the expression of mmu-let-7f-5p decreased (P<0.05), which was consistent with the sequencing results, while the expression level of mmu-miR-155-5p tended to increase, but there was no statistical significance. The expression level of mmu-miR-483-5p was not statistically significant in the two groups. This study further evaluated the expression levels of mmu-let-7f-5p, mmu-miR-483-5p and mmu-miR-155-5p in the renal tissues of Pristane-induced lupus mice. The expression of mmu-let-7f-5p in Pristane-induced lupus mice showed a downward trend, although the difference was not statistically significant (P>0.05), while mmu-miR-483-5p increased significantly in the renal tissues of Pristane-induced lupus mice (P<0.05) (Figure 16). In addition, the transcription level detection of mmu-miR-155-5p was not completed in Pristane-induced mice, and RT-qPCR showed that the number of cycles required for the target sequence was too high, and the results were not statistically significant.

[0053] 3.2 Prediction of let-7f-5p target genes The target genes of mmu-let-7f-5p were retrieved using the R software multiMiR, including predicted target genes and target genes that have been experimentally verified (qPCR, western blot, fluorescent labeling, northern blot or sequencing, etc.). Fig.17As shown in the figure, there are a total of 2100 target genes predicted based on whether there are reliable binding sites with the 3'UTR of mRNA and experimentally verified target genes. Among them, there are 13 molecules predicted as mmu-let-7f-5p target genes in 6 or more relevant databases, including Adrb3, Cdc34, Gatm, Hand1, Hmga2, Igf2bp1, Lin28b, Map4k3, Nme6, Nr6a1, Pbx3, Sall4 and Trim71. Enrichment analysis was performed on these 2100 molecules to evaluate the important regulatory role of mmu-let-7f-5p in life activities. KEGG analysis suggests that the genes targeted by mmu-let-7f-5p are enriched in MAPK signaling pathway, FoxO signaling pathway, stem cell pluripotency regulation and tumor-related pathways. GO analysis suggested that the genes targeted by mmu-let-7f-5p were enriched in amide metabolism, cellular response to stress, and cellular catabolic processes ( Figure 17 ).

[0054] The intersection of all target genes predicted and verified by mmu-let-7f-5p, the up-regulated genes obtained by sequencing in this study, and the up-regulated genes in the GSE160488 dataset was taken. The specific molecules are shown in Table 1. It was found that among the 108 differentially expressed genes up-regulated in both the GSE160488 dataset and this sequencing, 8 genes were target genes of mmu-let-7f-5p, namely Gbp7, Cd52, Gbp2, Rgs16, Nrros, Fasl, Klrd1, and Ms4a6b. Combined with literature retrieval, Gbp7, Gbp2, and CD52 were selected to verify the expression level in kidney tissue. At the same time, combined with the literature, the molecule Gatm, which was predicted as a target gene of mmu-let-7f-5p in 6 databases, was tested for expression level in kidney tissue, and the results are shown in Figure 18. Compared with MRL / MpJ control mice, the expression of Cd52, Gbp2, Gbp7 and Gatm in the kidney tissue of MRL / lpr lupus mice was significantly increased (Cd52, p<0.01; Gbp2, p<0.001; Gbp7, p<0.001; Gatm, p<0.05). To explore whether the transcriptional abnormalities of the four molecules are universal in lupus mice, the pristane-induced lupus mice were also tested. Compared with the healthy BALB / c group, the expression of Cd52, Gbp2, Gbp7 and Gatm in the kidney tissue of pristane-induced lupus mice was also significantly increased (Cd52, p<0.05; Gbp2, p<0.05; Gbp7, p<0.001; Gatm, p<0.01).

[0055] Table 1 Screening of key target genes of mmu-let-7f-5p in kidney tissue of MRL / lpr mice Data Source Number of genes Gene name GSE160488(up) ∩RNA-Seq(up) 108 Itgb7; Gbp7; C1qa; Ccl12; Rgs1; Cd52; Aif1; Ciita; Ccl3; Rasal3; H2-T22; Ctss; Klrk1; Lcn2; Clec4d; Gbp2; Psmb8; Dennd1c; Iigp1; Vmn1r124; Clec12a; Zic4; Rgs16; Uhrf1; Tnfrsf13c; Cxcl9; Jchain ;Tap1;C1qb;H2-DMb2;Stat1;Grap2;Clec4a3;Stfa2l1;Plek;Ccr5;Irgm1;Gm4841; Xlr4a; Cyp11a1; Cd300c; Saa2; Syt6; H2-K1; Gm572; Chrna1; Lhx8; Pld4; Cd3g; Nrros ;Cst7; Slc37a2; H2-M2; Tcf7; Ubd; Nkg7; Apol10b; Pigr; Il1f6; Lyz2; H2-Ab1; Tyrob p;Plac8;Ccl5;Fasl;Klrd1;Irgm2;Gpr83;Klra2;S100a9;Wfdc17;C1qc;Fcrl1;Sel plg; Ccl9; Sit1; Lilra5; Gm42372; Saa3; Fcer1g; Oas1g; Ms4a6b; Traf3ip3; Rab39; C d74;Alox5ap;Chia1;Ly86;Unc13d;Ccl8;Tlr1;Pilra;S100a8;Isg15;Cd3d;Casp4; H2-Eb1; H2-Aa; Psmb9; Ccdc88b; Gpr68; Il18rap; Irf7; Ms4a4b; Cd68; Gbp5; Clec4a1; Lat GSE160488(down)∩RNA-Seq(down) 14 Spint4; Gm1553; Arhgef25; Aldh1a1; Igfbp5; Trim5; Apoa2; Car3; Gpr85; Ren1; Slc10a2; Prss35; Tmem263; Itgb1bp2 3.3 Construction of macrophage inflammation model Based on the extensive macrophage inflammatory infiltration in renal tissues in lupus nephritis, this study constructed an in vitro macrophage inflammation model. RAW264.7 cells were treated with 500ng / ml lipopolysaccharide (LPS) for 12 hours. Under a 20x microscope, RAW264.7 cells that were not stimulated by LPS grew in a round / elliptical shape without obvious pseudopodia. After LPS stimulation, the macrophages were elongated, with pseudopodia, and the cells were not easy to adhere to the wall (Figure 19).

[0056] The mRNA levels of iNOS, IL-1β, TNF-α and IL-6 were detected to identify the construction of the RAW264.7 cell inflammation model. As shown in Figure 20, the transcription levels of iNOS, IL-1β, TNF-α and IL-6 in macrophages after LPS intervention were significantly increased (iNOS, p<0.0001; IL-1β, p<0.001; TNF-α, p<0.0001; IL-6, p<0.05), and the inflammation model was completed. This study also evaluated the expression levels of let-7f-5p, Cd52, Gatm, Gbp7 and Gbp2 in the inflammatory cell model. The results showed that after LPS intervention, the transcription level of let-7f-5p in RAW264.7 cells was significantly decreased (p<0.0001), and the transcription levels of Cd52, Gbp7 and Gbp2 were significantly increased (Cd52, p<0.001; Gbp7, p<0.05; Gbp2, p<0.01). The transcription level of Gatm was downregulated after LPS intervention (p<0.05).

[0057] 3.4 Effects of regulating let-7f-5p levels on RAW264.7 cells In order to explore the effect of let-7f-5p on RAW264.7 cells, the transfection efficiency of let-7f-5p was first evaluated. As shown in Figure 21, the level of let-7f-5p in the mimics transfection group was significantly increased compared with the control group (P<0.01), while there was no statistical difference between the mimics NC transfection group and the control group (P>0.05). After the transfection efficiency was verified, a mouse macrophage inflammation model was constructed to evaluate the effect of regulating the level of mmu-let-7f-5p on the inflammatory factors of RAW264.7 macrophages under LPS intervention. Therefore, RAW264.7 cells were intervened with 500ng / ml lipopolysaccharide (LPS) for 12 hours. After the medium was changed, the RAW264.7 macrophages were further transfected with different artificial synthetic products of mmu-let-7f-5p according to the grouping. The results showed that after transfection of mmu-let-7f-5p in the macrophage inflammation model, the elevated levels of TNF-α, iNOS, IL-1β and Gbp7 were reversed to varying degrees. Although IL-6, Gbp2 and Cd52 showed the same trend, they were not statistically significant. The level of Gatm decreased significantly after transfection of let-7f-5p mimics, but no effect of let-7f-5p on the expression level of Gatm was found in the presence of LPS (Figure 22).

[0058] Example 2: Effect of MSCs on let-7f-5p in RAW264.7 macrophages 1. Cultivation and identification of MSCs Subcutaneous fat from surgical patients with no underlying diseases and autoimmune diseases was isolated, and primary adipose-derived mesenchymal stem cells were extracted and cultured by enzyme digestion. After 24 hours, small spindle cells were observed to grow scattered on the wall. In this study, mesenchymal stem cells were identified based on their unique ability to differentiate into three lines of adipogenesis, osteogenesis, and chondrogenesis. The cells of the third passage were induced and cultured using adipogenic, osteogenic, and chondrogenic induction fluids, respectively. The results showed that adipogenic, osteogenic, and chondrogenic induction were all positive (Figure 23). Flow cytometry analysis of cell surface markers was further performed, and it was found that the cells expressed CD90, CD105, and CD73, but did not express CD45, CD34, and HLA-DR compared with the isotype control (Figure 24).

[0059] 2. Effects of MSCs on RAW264.7 macrophages The results of co-culture of mesenchymal stem cells and RAW264.7 macrophages showed that compared with the blank control group, the transcription of let-7f-5p decreased in RAW264.7 cells after LPS intervention, while the transcription levels of iNOS, IL-1β, TNF-α, IL-6, Gbp2, CD52 and Gbp7 increased. After 12 hours of LPS intervention, the medium was changed and the chamber containing MSCs was placed in the RAW264.7 cells and cultured for another 12 hours. It was found that compared with the RAW264.7 cells after LPS intervention, the transcription level of let-7f-5p increased in RAW264.7 cells after MSCs co-culture, while the transcription levels of iNOS, IL-1β, TNF-α, IL-6, Gbp2 and Gbp7 decreased. However, LPS and MSCs had little effect on the transcription level of Gatm ( Fig.25 ).

[0060] The above is only a specific implementation of the present invention, but the design concept of the present invention is not limited to this. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. Exosomal miRNA closely related to SLE, characterized by: The exosomal miRNA is let-7f-5p, and its RNA sequence table is shown in SEQ ID No.

1.

2. The exosomal miRNA closely related to SLE according to claim 1, characterized in that: The exosomal miRNA is derived from renal tissue exosomes, and the renal tissue exosomes are extracted by ultracentrifugation.

3. The exosomal miRNA closely related to SLE according to claim 1, characterized in that The method for obtaining let-7f-5p comprises the following steps: (1) Establish MRL / lpr spontaneous and pristane-induced lupus mouse models, evaluate the skin damage, urine protein and serum anti-dsDNA antibody levels of the models, and detect the levels of renal inflammatory factors; (2) Exosomes were extracted from renal tissues of MRL / lpr lupus mice and MRL / MpJ control mice, and the exosomes were identified by particle size analysis, transmission electron microscopy observation, and flow cytometry detection of surface markers; (3) RNA from renal tissue exosomes was extracted for library construction and RNA sequencing to obtain miRNA and mRNA expression profiles, and the DESeq algorithm was used to perform differential expression analysis of miRNA and mRNA; (4) Real-time fluorescence quantitative PCR technology was used to verify the expression level of let-7f-5p in renal tissue exosomes.

4. A target gene of exosomal miRNA closely related to SLE as claimed in claim 1, characterized in that: Including Cd52, Gbp2, Gbp7 and Gatm, the amino acid sequence of the protein encoded by each target gene is shown in SEQ ID No.2-5 respectively.

5. A use of the exosomal miRNA closely related to SLE as claimed in claim 1, characterized in that: Mesenchymal stem cells upregulate let-7f-5p expression in the RAW264.7 macrophage inflammation model.

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