Application of lncRNA-Gm13561 as a new target in the diagnosis and treatment of diabetic nephropathy

Abstract

The present invention discloses the application of lncRNA‑Gm13561 as a new target in the diagnosis and treatment of diabetic nephropathy. The present invention constructs a DN animal model through db / db mice, and performs single-cell sequencing on the renal tissues of normal mice and DN mice to screen out lncRNAs specifically expressed in DN podocytes, and verifies and studies the mechanism of lncRNA specifically expressed in podocytes through cell biology and molecular chemistry and other technologies. The research of the present invention confirms that lncRNA‑Gm13561 in renal podocytes is significantly reduced in diabetic nephropathy, and it is found that it can slow down the occurrence and development of diabetic nephropathy by alleviating the apoptosis of podocytes and oxygen death caused by ROS. The research of the present invention proves that lncRNA‑Gm13561 can be used as a new drug target in the treatment of diabetic nephropathy, and can also be used as a new clinical diagnostic indicator.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of lncRNA-Gm13561 as a new target in the diagnosis and treatment of diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DN) has now become a major cause of chronic kidney disease in my country. It can induce end-stage renal failure and eventually lead to death. Studies have shown that under the environment of long-term hyperglycemia, the pathological characteristics of DN are mainly glomerular sclerosis and renal interstitial fibrosis, which lead to increased urinary protein excretion rate and renal dysfunction. Podocytes, i.e., epithelial cells in the visceral layer of the renal capsule, are located on the outside of the glomerular basement membrane and together with vascular endothelial cells and the glomerular basement membrane, constitute the glomerular blood filtration barrier. The damage and apoptosis of podocytes in DN patients are one of the main factors leading to massive proteinuria and glomerular sclerosis. Therefore, podocytes play an important role in the process of glomerular damage and repair. In-depth study of the relationship between podocytes and DN is an important topic in the prevention and treatment of DN, and is of great significance to the pathogenesis and clinical diagnosis and treatment of DN.

[0003] A large amount of research data shows that the vast majority of eukaryotic transcribed RNAs are non-coding RNAs. Among them, long non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 nt, lacking a specific and complete open reading frame, and without protein-coding function. Although lncRNA lacks the ability to encode proteins, it still has a wide range of biological functions, such as participating in chromosome modification and remodeling, regulating target gene transcription, regulating target gene translation as miRNA sponges (miRNAsponges) or competitive endogenous RNA (Competitive endogenous RNA, ceRNA), and binding to proteins to regulate protein activity or localization. Involved in cell proliferation, differentiation and metabolic processes. Existing studies have shown that many lncRNAs play an important role in podocyte damage and apoptosis in the occurrence and development of DN. However, the specific relationship between lncRNA and the occurrence and development of DN is still not fully clear. Summary of the invention

[0004] The purpose of the present invention is to address the above problems and provide an application of lncRNA-Gm13561 as a new target in the diagnosis and treatment of diabetic nephropathy.

[0005] In order to achieve its purpose, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the use of a reagent for detecting a biomarker in a sample in the preparation of a product for diagnosing diabetic nephropathy, wherein the biomarker is lncRNA-Gm13561.

[0007] In the above-mentioned application technology scheme, compared with the normal control, the expression level of lncRNA-Gm13561 in the patient is downregulated.

[0008] In the above-mentioned application technology scheme, the content of lncRNA-Gm13561 in the patient's test sample is detected by total RNA extraction, reverse transcription, and quantitative PCR.

[0009] The second aspect of the present invention provides the use of a lncRNA-Gm13561 expression promoter in the preparation of a drug for preventing and treating diabetic nephropathy.

[0010] The third aspect of the present invention provides the use of lncRNA-Gm13561 as a target in screening drugs for preventing and treating diabetic nephropathy.

[0011] In the above-mentioned application technology scheme, the drug promotes the expression of lncRNA-Gm13561.

[0012] The cDNA sequence of lncRNA-Gm13561 is shown in SEQ ID NO.1.

[0013] In the above-mentioned application technology scheme, lncRNA-Gm13561 regulates podocyte apoptosis and ROS accumulation, alleviating podocyte apoptosis and ROS-induced oxygen death.

[0014] In the above-mentioned application technology scheme, Gm13561 regulates the oxygen death pathway KEAP1-PGAM5-AIFM1.

[0015] In the above-mentioned application technology scheme, the upregulation of Gm13561 increased the expression of Bcl-2 and podocin in podocytes, decreased the expression of Bax and cleaved-caspase-3, reduced the expression of Keap1, PGAM5 and AIFM1, and increased the phosphorylation level of AIFM1.

[0016] The beneficial effects of the present invention are:

[0017] The DN animal model was constructed by db / db mice, and single-cell sequencing was performed on the renal tissues of normal mice and DN mice to screen out lncRNAs specifically expressed in DN podocytes. The lncRNAs specifically expressed in podocytes were verified and studied by cell biology and molecular chemistry. Through the research of this invention, the specific mechanism of lncRNAs related to podocyte injury and apoptosis in the development of DN was explored, and new targets were found for the clinical diagnosis of DN.

[0018] The present invention study confirmed that lncRNA-Gm13561 in renal podocytes was significantly reduced in diabetic nephropathy, and it was found that it could slow down the development of diabetic nephropathy by alleviating podocyte apoptosis and oxygen death caused by ROS. The present invention study proved that lncRNA-Gm13561 can be used as a new drug target in the treatment of diabetic nephropathy, and can also be used as a new clinical diagnostic indicator. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The experimental results of establishing the podocyte-specific lncRNA expression profile of DN based on single-cell nuclear sequencing: A: body weight, kidney weight, blood sugar and urine albumin-creatinine ratio between DN mice and normal mice; B, C: kidney cells were grouped based on single-cell nuclear sequencing; D: expression levels of 24 podocyte-specific lncRNAs in each cell subpopulation based on single-cell nuclear sequencing (P<0.05); E: TOP20 lncRNAs differentially expressed in the disease group, 4 lncRNAs were upregulated; 16 lncRNAs were downregulated (P<0.05); F: 6 candidate genes were screened based on differentially expressed genes and genes specifically expressed in podocytes; G: differential expression multiples of the 6 candidate genes in the disease group and the normal group; H: expression levels of the 6 candidate genes in each cell subpopulation.

[0020] Figure 2 PCR identified the cellular specificity of lncRNA Gm13561 in glomerular podocytes in high and low glucose renal intrinsic cells and the biological characteristics of lncRNA Gm13561. Experimental results: A: qRT-PCR was used to verify the specificity of the expression of 6 candidate genes in podocytes in podocytes, mesangial cells and epithelial cells; B: qRT-PCR was used to verify the differential expression of 6 candidate genes in podocytes cultured in normal glucose and high glucose; C: qRT-PCR was used to detect the expression of Gm13561 in the nucleus and cytoplasm of podocytes; D: FISH was used to detect the localization of Gm13561 in podocytes.

[0021] Figure 3The experimental results of overexpression and knockdown of Gm13561 regulating podocyte injury and apoptosis: A: qRT-PCR detection of Gm13561 expression after transfection of Gm13561 overexpression plasmid; B: qRT-PCR detection of Gm13561 expression after transfection of three siRNAs for Gm13561; C: Genes that were specifically highly expressed in podocytes in the single-cell sequencing results and differentially expressed after Gm13561 overexpression (48 in total) were selected and functional classification enrichment analysis was performed using KOBAS 3.0; D: Flow cytometry was used to examine the apoptosis of podocytes after Gm13561 overexpression and knockdown; E: Flow cytometry was used to detect fluorescence intensity to evaluate the level of ROS in podocytes after Gm13561 overexpression and knockdown; F: WB was used to detect the expression of podocyte apoptosis and oxygen death markers after Gm13561 overexpression and knockdown. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified; the materials and reagents used are all conventional materials and reagents in the art and are commercially available unless otherwise specified.

[0024] Example 1

[0025] 1 Experimental methods

[0026] 1.1 Cell culture

[0027] In this laboratory, we preserved glomerular podocytes (pod), tubular epithelial cells (TC), and mesangial cells (MCs) of DKD mice. 10% fetal bovine serum and 1% penicillin-streptomycin solution were added to DMEM medium (Gibco, Carlsbad, CA, USA) and cultured in an incubator at 37°C with 5% CO2. Many previous studies have shown that high-glucose conditions simulate the growth environment of mouse MPC5 in the DN state, while low-glucose conditions simulate the normal growth state. Accordingly, cells were stimulated with 5.5mmol / L glucose (low-glucose group) or 25mmol / L glucose (high-glucose group).

[0028] 1.2 Single-cell RNA sequencing

[0029] Renal tissues were extracted from db / db DKD (diabetic nephropathy, DKD) mice (n=3) and control mice (n=3) to prepare single-cell suspensions. The constructed libraries were subjected to SnRNAseq detection using the paired-end sequencing mode of the Illumina sequencing platform (Omicsmart, Guangzhou, China). The source data were integrated, normalized, and t-SNE dimension reduction were performed using the normalize data function, RunTSNE function, and search clustering function, and cell clustering analysis was performed to reveal differentially expressed genes at the single-cell level.

[0030] 1.3 RNA extraction, cytoplasm / nucleus separation and qRT-PCR detection

[0031] Total RNA from cells or tissues was extracted using the SteadyPure Mag Tissue&Cells RNA Extraction Kit (Accurate Biology, Hunan, China) according to the manufacturer's instructions, followed by reverse transcription and qRT-PCR. Cytoplasm and nucleus were isolated using PARIS TM Kit (Thermo Fisher scientific, USA). RNA from each part was separated according to the kit instructions, and then reverse transcription and qRT-PCR were performed. Total RNA was synthesized into cDNA using PrimeScript RT Reagent Kit (Takara, Japan) according to the kit instructions. cDNA was amplified on the Bio-Rad CFX96 system (Bio-Rad, CA, USA) using TB Green Premix Ex Taq (Takara, Japan). mRNA expression was measured by 2-ΔΔCT and normalized to β-actin. The primer sequences are shown in Table 1:

[0032] Table 1

[0033]

[0034] 1.4 Overexpression and knockdown of Gm13561

[0035] The podocyte genomic DNA was extracted, the full-length Gm13561 was amplified, and the PCR product was cloned into the pcDNA 3.1 expression vector. The plasmid was extracted by the endotoxin removal plasmid extraction kit (Omega, GA, USA) according to the manufacturer's protocol, digested by enzymes (the restriction sites of the Gm13561 overexpression plasmid are BamHI and EcoR V), PCR and sequencing were identified. siRNA targeting Gm13561 was synthesized. The overexpression plasmid and siRNA were specifically constructed and provided by Beijing Qingke Biotechnology Co., Ltd. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or HighGene plus transfection reagent (abClonal, Wuhan, China) according to the manufacturer's instructions. The siRNA sequences are shown in Table 2.

[0036] Table 2

[0037]

[0038] 1.5 RNA-sequencing

[0039] Illumina HiSeq sequencing (Novogene, Beijing, China) was used to analyze the differential expression of Gm13561 upregulated mRNA in HG-podocytes and compared with the NC group. Data analysis was performed by Novogene (Beijing, China).

[0040] 1.6 Western blot analysis

[0041] Total protein of cells or tissues was extracted with RIPA lysis buffer containing PMSF, and then subjected to SDS-PAGE and transferred to PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 10% skim milk, incubated with primary antibody at 4°C overnight, and incubated with enzyme-labeled secondary antibody (1:10000, Proteintech, USA) at room temperature for 2 h. Finally, SuperSignal TMWestFemto Maximum Sensitivity Substrate (Termo Fisher Scientific) and ECL Western Blotting Substrate Kit (Millipore, USA) were used to visualize the bands. The specific antibody dilutions were as follows: anti-cleaved-caspase-3 (1:5000, Immunoway, USA), anti-bax (1:1000, Proteintech, USA), anti-bcl-2 (1:50, Abmart, Shanghai, China), anti-podocin (1:50, Bioss, Beijing, China), anti-p-aifm1 (1:50, ECM biosciences, USA), anti-aifm1 (1:50, Immunoway, USA), anti-keap1 (1:50, Immunoway, USA), anti-pgam5 (1:50, Proteintech, USA).

[0042] 1.7 Fluorescence in situ hybridization (FISH) detection

[0043] FISH probe mixture targeting the Gm13561 junction site was used using a fluorescent in situ hybridization kit (Genetic Pharmaceuticals, Shanghai, China) according to the manufacturer's protocol, followed by counterstaining with DAPI. U6 probe was used as a positive control to detect nuclear distribution. After sealing, samples were imaged using a laser scanning confocal microscope (Leica).

[0044] 1.8 Flow cytometry

[0045] 72 h after transfection, cells were harvested and stained with Annexin v-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Finally, a flow cytometer FACS Calibur (Becon Dickinson, Franklin Lakes, NJ, USA) was used to detect cell apoptosis. Hoechst 33342 and Tdt-mediated UTP nickel end labeling (TUNEL) staining (Beyotime) were used to detect cell apoptosis and observed under a fluorescence microscope (Leica).

[0046] 2 Results

[0047] 2.1 Establishment of DN podocyte-specific lncRNA expression profile based on single-cell nuclear sequencing

[0048] First, single-cell RNA-seq analysis revealed specific lncRNAs in podocytes of DN mice, and snRNA-seq was used to detect single-cell expression profiles in kidney tissues of db / db DKD mice (n=3) and normal control mice (n=3). The body weight, kidney weight, blood glucose, and urine albumin / urine creatinine ratio (ACR) of db / db mice were significantly higher than those of normal mice ( Figure 1 A) shows that our diabetic nephropathy mouse model was successfully established.

[0049] We then performed single-cell RNA-seq analysis of isolated renal tissues from db / db DKD mice and non-diabetic control mice. The obtained single cells were classified into 18 major renal cell types through the identification of cell marker genes, including mesangial cells, endothelial cells, epithelial cells, podocytes, proximal tubule cells, macrophages, proximal convoluted tubule cells, collecting duct chief cells, CD4+ embedded cells, CD4+ transient cells, Henley circulating cells, monocytes, B lymphocytes, and T lymphocytes ( Figure 1 B). Our transcriptome analysis revealed specific lncRNAs in 18 cell clusters ( Figure 1 C). The expression profiles of the top 20 specific lncRNAs and 24 DN-related lncRNAs were established ( Figure 1 D, E): Gm4128, Gm3772, Wt1os, Gm13814, 9130410C08Rik, Gm29266, Gm44129, Gm15866, Tmem150cos, Gm49969, Gm16083, 4921504A21Rik, Pard3bos1, Gm13561, Pard3bos3, Gm16337, Gm32880, Gm50469, Gm32036, Gm13944, 2310014F06Rik, Gm37229, E130310104Rik, Sox5os4. We found that there were 6 specific lncRNAs associated with DN in podocytes ( Figure 1 F): Gm13561, Gm44129, Gm16337, Gm15866, Gm32036, Gm3772; one of them (Gm3772) was up-regulated in DN, and five (Gm13561, Gm44129, Gm16337, Gm15866, Gm32036) were down-regulated in DN ( Figure 1 G). Compared with other cell clusters, these six candidate lncRNAs showed specific high expression in podocytes ( Figure 1 H). In conclusion, we obtained candidate lncRNAs through gene expression profiling at the single-cell level and investigated the cell-specific expression of lncRNAs involved in DN.

[0050] 2.2 Single cell nuclear sequencing analysis combined with PCR to identify the glomerular podocyte cell specificity of lncRNA Gm13561 in high and low glucose renal intrinsic cells and the biological characteristics of lncRNA Gm13561

[0051] We selected lncRNAs that were most relevant to the occurrence of DN and relatively highly expressed in podocytes. We first cultured cells in high glucose to simulate the disease state, and then used qRT-PCR to detect the expression of these candidate lncRNAs in podocytes cultured in high or low glucose, as well as the expression of these candidate lncRNAs in podocytes, mesangial cells, and epithelial cells.

[0052] The results showed the same trend as the snRNA-seq results. Among them, Gm13561 was most specifically overexpressed in podocytes, and its expression level was significantly reduced in podocytes cultured in high glucose ( Figure 2 A, B). In order to understand the biological characteristics of Gm13561, we evaluated its possibility of encoding proteins by searching its open reading frame. Gm13561 showed low protein coding potential (Table 3). In addition, FISH and qRT-PCR were used to understand the subcellular localization of Gm13561. The results showed that Gm13561 was expressed in both the nucleus and cytoplasm of podocytes, but the expression level of Gm13561 in the nucleus was significantly higher than that in the cytoplasm ( Figure 2 C). In summary, Gm13561 may be a podocyte-specific and DN-related lncRNA, which is significantly downregulated in podocytes cultured in high glucose and is mainly located in the nucleus ( Figure 2 D).

[0053] The cDNA sequence of Gm13561 is shown in SEQ ID NO.1.

[0054] Table 3. ORF protein encoding potential

[0055]

[0056] 2.3 Overexpression and knockdown of Gm13561 regulate podocyte injury and apoptosis

[0057] To clarify whether Gm13561 plays a role in DN, we cloned the full-length Gm13561 into the pcDNA3.1 plasmid and synthesized siRNA specific for Gm13561. Then, qPCR analysis was performed to evaluate the overexpression and interference efficiency of Gm13561 ( Figure 3 A, B), successful overexpression and knockdown of Gm13561 were found.

[0058] We determined the genome-wide transcriptomic consequences of Gm13561 overexpression in podocytes by RNA-seq and then combined the specific genes highly expressed in podocytes screened by snRNA-seq to find 48 podocyte-specific genes affected by Gm13561. We performed GO analysis on them and the results showed that the biological processes enriched by these 48 genes included 35 pathways. Among them, there were 6 ROS (Reactive oxygen species, reactive oxygen species) related signaling pathways and cell apoptosis ( Figure 3 C), suggesting that Gm13561 may regulate podocyte apoptosis and ROS accumulation. Therefore, we examined the effect of Gm13561 on podocyte apoptosis and ROS levels. Flow cytometry analysis showed that after transfection with Gm13561, the number of apoptotic cells and ROS levels decreased. Gm13561 gene knockout reversed the results ( Figure 3 D, E). Many studies have shown that ROS are associated with a variety of cell death, such as necrosis, apoptosis (caspase-dependent), pyroptosis, and necroptosis. Inflammation-induced and caspase-independent cell death includes necrotic cell death, ferroptosis, autophagic cell death, and oxidative cell death. Oxygen-induced cell death is a new apoptosis-like cell death method proposed in 2018. Studies have confirmed that oxygen-induced cell death affects the development of some tumors, but its effect on DN is still unclear.

[0059] Therefore, we investigated whether Gm13561 affects apoptosis-related proteins and the specific oxygen death pathway KEAP1-PGAM5-AIFM1. Western blot analysis showed that knockdown of Gm13561 induced decreased expression of Bcl-2 and podocin, increased expression of Bax and cleaved-caspase-3, increased expression of Keap1, PGAM5, and AIFM1 in podocytes cultured in low glucose, but significantly decreased phosphorylation level of AIFM1. Decreased phosphorylation level of AIFM1 is a milestone event in oxygen death ( Figure 3 F). These data suggest that Gm13561 protects podocytes from high glucose-induced apoptosis and oxidative death.

[0060] These experiments inhibited / promoted podocyte apoptosis and oxytocin death by overexpressing / knockdown this target Gm13561, proving that lncRNA-Gm13561 can serve as a new target for DKD.

Claims

1. Application of lncRNA-Gm13561 as a target in screening drugs for preventing and treating diabetic nephropathy.

2. The use according to claim 1, characterized in that: The drug promotes the expression of lncRNA-Gm13561.

3. The use according to claim 1 or 2, characterized in that: The cDNA sequence of lncRNA-Gm13561 is shown as SEQ ID NO.1.

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