Ethics statement

All participants completed an informed consent form prior to participation in the present research. Ethical approval for this study was provided by the Ethics Committee of the Gongli Hospital of Shanghai Pudong New Area (approval number: 2019-011). Also, the study was conducted in compliance with the Declaration of Helsinki.

Participants and data collection

Patients with type 2 DM (T2DM) admitted to Gongli Hospital of Shanghai Pudong New Area Hospital between January 2019 and December 2020 were included in this study. T2DM diagnosis adhered to the American Diabetes Association criteria [25]: (a) fasting blood glucose (FBG) ≥ 7.0 mmol/L; (b) blood glucose 2 h after glucose load ≥ 11.1 mmol/L or glycated haemoglobin A1c (HbA1c) ≥ 6.5%. Among them, patients with T2DM were divided into two groups: those with DN (n = 68) and those without DN (DM, n = 55). DN inclusion criteria were as follows: (a) history of T2DM; (b) persistent albuminuria (≥ 30 mg/24 h) or a random albuminuria to creatinine ratio of ≥ 30 mg/g or eGFR < 60 mL/min/1.73 m2; (c) age 18–70 years. The exclusion criteria were as follows: (a) type I DM or other diabetic complications; (b) concurrent chronic kidney diseases such as glomerulonephritis; (c) autoimmune diseases, malignant tumors, and haematologic diseases; (d) presence of cardiovascular diseases (myocardial infarction, unstable angina); (e) use application of glucocorticoids, or immunosuppressive drugs within the last 6 months. Additionally, 50 healthy volunteers (HVs) were included as controls, matched for age and gender with the patient groups. Individuals with DM, cardiovascular disease, renal disease, autoimmune disease, and those using antibiotics or corticosteroids were excluded. Demographic characteristics and clinical baseline information of the participants are presented in Table 1.

Table 1 Comparison of the baseline data of the subjectSpecimen collection and biochemical

Venous blood was obtained from the upper extremities of the participants after 8 h of fasting. A portion of the collected blood was stored in anticoagulation tubes for HbA1c levels. The remaining blood was allowed to stand at room temperature and then centrifuged at 3000 g for 10 min to obtain the upper serum. Using a biochemistry analyser (Instrumentation Laboratory, USA), various indicators such as FBG, total cholesterol (TC), and triglyceride (TG)were measured. Additionally, a part of the serum was stored at -80℃ for the analysis of DANCR and miRNA messenger RNA levels. Urine samples (10 mL) were collected and centrifuged at 400 g for the determination of urine albumin and other levels.

Cell culture and high glucose (HG) treatment

Human proximal tubular epithelial cells (human kidney 2 [HK-2]) were procured from the BeNa Culture Collection (cat: BNCC339833, China). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, cat: C11995500, Invitrogen, USA) supplemented with 10% fetal bovine serum (cat: 16-000-044, Invitrogen, USA), and 1% penicillin/streptomycin (cat: 1,510,122, Invitrogen, USA). The cells were maintained in a humidified incubator with 5% carbon dioxide at 37℃. HK-2 cells were treated with HG (30 mM glucose, cat: G-8769, Sigma-Aldrich, USA) for 12 h, 24 h, and 36 h. The control group was exposed to normal glucose (NG, 5.5 mM glucose + 24.5 mM mannitol, cat: M-1902, Sigma-Aldrich, USA).

Cell transfection

Transfection was initiated upon achieving a cell fusion rate reached of 70%. Small interfering (si) RNA targeting DANCR (si-DANCR) and its negative control (si-NC) or, as well as the miR-214-5p mimic, miR-214-5p inhibitor, and their negative control (mimic NC and inhibitor NC, obtained from GenePharma, China), were combined with the transfection reagent Lipofectamine 3000 (cat: 1,662,152, Invitrogen, USA) in DMEM. The mixture was then allowed to incubate for 20 min at room temperature. Subsequently, the prepared mixture was introduced into the cells, and the incubation continued for 6 h before replacing the DMEM.

Quantitative reverse transcription polymerase chain reaction (PCR) (RT-qPCR)

The TRIzol LS Reagent (cat: 15,596,026, Invitrogen, USA) was used for pre-incubation, followed by purification using the miRNeasy Serum/Plasma Kit (cat: Q217184, Qiagen, Germany) to extract total RNA from 500 μL of serum. Meanwhile, for the cells, the miRNeasy Mini Kit (cat: Q217004, Qiagen, Germany) was used to purify total RNA from the cells. The concentration and purity of the extracted RNA were determined using the NanoDrop2000 micro ultraviolet spectrophotometer (NanoDrop Technologies, USA). The reverse transcription of RNA into complementary deoxyribonucleic acid (cDNA) was performed using M-MLV Reverse Transcriptase (cat: M170B, Promega, USA) or the miRcute Plus miRNA First-Strand cDNA Kit (cat: KR211, TIANGEN Biotech, China). Amplification reactions were performed by combining SuperReal PreMix Plus (SYBR Green) (cat: FP205, TIANGEN Biotech, China) or the miRcute Plus miRNA qPCR Kit (SYBR Green) (cat: KR411, TIANGEN Biotech, China) with primers using cDNA as a template. Where β-actin and U6 were normalized separately, and the quantification was performed using the 2−ΔΔCt method.

Western blot assay

Transfected and HG-induced HK-2 cells were spiked with RIPA lysis buffer (cat: P0013B, Beyotime, China), and the supernatant was collected after centrifugation to extract total protein. The protein concentration was analyzed with a BCA protein quantitation assay kit (cat: P0012, Beyotime, China), followed by denaturation by mixing the protein with 10% alkyl sodium sulfate buffer in a certain ratio and boiling for 5 min in a 95℃-water bath. Electrophoretic separation was performed using SDS-PAGE gels and transferred to 0.22 μm PVDF membranes (cat: ISEQ00010, Millipore, USA). Then, the membranes were blocked for 2 h at room temperature in 5% bovine serum albumin (BSA)(cat: A9647, Millipore, USA), followed by incubation with primary antibodies (KFL5 antibody, cat: 668,501-Ig, Proteintech, China; β-actin antibody, cat: 81115-1-RR, Proteintech, China) at 1:1000 dilutions overnight at 4℃. After washing with 0.05% tris buffered saline/Tween (TBST), the membrane was incubated with enzyme-labeled secondary antibody (Proteintech, China, 1:5000 dilution) for 2 h. Finally, proteins were displayed using the enhanced chemiluminescent luminescence (ECL) kit (cat: NEL103001EA, Perkin Elmer, USA) and protein band images were analyzed using the Bio-Rad ChemiDOC XRS system (Bio-Rad Corporation, USA). The original gel chart has been presented in the supplementary material.

Cell proliferation assay

Transfected and HG-induced HK-2 cells were converted into cell suspensions, and 1 × 104 cells were inoculated into 96-well plates for incubation. Before the assessment, 10 μL of cell count kit-8 solution (cat: KB491, Dojindo Laboratories, Japan) was added to the cells, and the incubation process was continued for 1 h. The cell proliferation rate was determined by measuring the optical density value at 450 nm.

Cell apoptosis assay

Cells were inoculated into six-well plates, and 24 h later, HG was introduced, followed by the transfection of plasmids. After 72 h, the cells were collected and washed with pre-cooled phosphate-buffered saline (PBS) and the binding buffer was added. Annexin V and propidium iodide from an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide cell apoptosis kit (cat: 556,547, BD, USA) kit was then stained in a darkroom for 10 min. The quantification of apoptotic cells was performed using flow cytometry (BD-Biosciences, USA).

Enzyme-linked immunosorbent assay (ELISA)

Serum from the participants and the supernatant from HG-inducted and transfected HK-2 cells were collected. The expression levels of interleukin (IL) -1β (cat: E-EL-H0149c, Elabscience Biotech, China), IL-6 (cat: E-EL-H6156, Elabscience Biotech, China), and tumor necrosis factor α (TNF-α, Cat: E-EL-H0109c, Elabscience Biotech, China) were measured according to the manufacturer’s instructions.

Reactive oxygen species (ROS) and malondialdehyde (MDA) measurement

The fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA) kit (cat: E004-1-1, Nanjing Jiancheng Bioengineering Institute, China) was used to detect ROS level according to the manufacturer’s instructions. Transfected and HG-induced HK-2 cells were incubated with 40 μL of DCFH-DA buffer for 30 min at 37℃. Following the incubation, the cells were washed, and the fluorescence intensity was quantified at wavelengths of 488 nm and 525 nm to assess ROS levels.

The MDA levels were analyzed using the thiobarbituric acid (TBA) method by an MDA kit (cat: A003-1-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s recommendations. In short, MDA powder was added to 32 mL of hot distilled water at 90–100℃ and fully dissolved. After cooling, 30 mL of glacial acetic acid was added and mixed. A 2:1 dilution was made with 50% glacial acetic acid and frozen in a 4℃-refrigerator protected from light as a working solution. The cells of Transfected and HG-induced HK-2 cells were crushed, shaken well, and then taken and added to the mixture of reagents containing the working solution. 95℃ water bath for 40 min, removed and cooled with running water, 3500  rpm, centrifuged for 10 min, then the supernatant was taken and detected the absorbance value at 532 nm.

Subcellular fractional location

The PARIS kit (cat: AM1921, Thermo Scientific, USA) was employed to explore the localization of DANCR on the HK-2 cells. In brief, cells were washed with PBS, centrifuged at 1000 rpm for 5 min, and harvested by removing PBS. Pre-cooled cell lysate was added, and the mixture was incubated for 1 min. The supernatant, obtained by centrifugation at 1200 rpm for 5 min, was used for cytoplasmic fragment RNA assay. The remaining precipitate was treated with the NER reagent from the kit, incubated for 10 min, and then used for collecting nuclear RNA. U6 and β-actin served as positive controls for the nucleus and cytoplasm. RT-qPCR was designed to assess DANCR levels in the cytoplasm and nucleus fractions.

Dual luciferase reporter (DLR) assay

The promoter sequences of DANCR or Krüppel-like factor 5 (KLF5) were cloned into pmirGLO Vectors to construct a wild-type (WT) DANCR or KLF5 reporter plasmid (DANCR-WT or KLF5-WT). Additionally, the binding site was mutated to construct mutant (Mut) DANCR or KLF5 reporter plasmid (DANCR-Mut or KLF5-Mut). These recombinant plasmids were co-transfected with miR-214-5p mimic or miR-214-5p inhibitor using Lipofectamine 3000 (Invitrogen, USA). HK-2 cells for 48 h. Subsequently, luciferase activity was evaluated using a commercial DLR kit (cat: E1960, Promega, USA).

RNA immunoprecipitation (RIP) assay

The RIP assay was performed to analyse the interaction between miR-214-5p, DANCR, and KLF5 by the RIP Assay Kit (cat: 17–704, Millipore, USA). HK-2 cells transfected with miR-214-5p mimic or mimic NC were lysed in RIP buffer and incubated overnight at 4℃ with magnetic beads coupled with human Ago2 or immunoglobulin G (IgG) antibodies. After the purification of co-precipitated RNA, RT-qPCR was employed to quantify the enrichment levels of DANCR and KLF5.

Statistical analysis

The experimental data from three biological replicates are presented as the mean ± standard deviation. Statistical analysis and figure generation were performed using GraphPad Prism 7.0 and SPSS 23.0. Student’s t-test was employed to assess the differences between the two groups. Analysis of variance and Turkey’s analysis were used to compare the differences between multiple groups. Receiver operating characteristic (ROC) curve analysis was conducted for diagnostic prospect assessment. Pearson’s correlation coefficient was calculated for correlation detection. Statistical significance was set at P < 0.05.