Patients and samples
Seventeen patients, one male and sixteen females, with LNs who were diagnosed and treated at the Department of Internal Medicine and Rheumatology, Juntendo Urayasu Hospital between June 2012 and June 2019 were included in this study. The mean age of the patients was 33 ± 15 years. All the patients met both the 1997 ACR classification criteria and the 2019 EULAR/ACR classification criteria. None of the patients had malignancies or acute or chronic infection. The SLE Disease Activity Index (SLEDAI) was scored based on clinical symptoms and laboratory tests. The mean pretreatment SLEDAI score was 18.5 ± 3.8 and the mean post-treatment SLEDAI score was 4.5 ± 4.0. The clinical characteristics of the patients are summarized in Table 1. The treatment of SLE was effective in all patients, as evidenced by a decrease in anti-double-stranded DNA antibodies, increase in CH50 (total activity of inactive C1-C9), decrease in urinary protein levels, and decrease in SLEDAI scores before and after treatment (Supplementary Fig. 1).
miRNA array analysis
Total RNA was extracted from PBMCs before and after treatment of No.1–6 LNs patients (Table 1) using the miRNeasy mini kit (Qiagen, Hilden, Germany) and analyzed comprehensively using a miRNA array. The extracted RNA was labeled using a 3D-Gene™ miRNA labeling kit (TORAY, Tokyo, Japan), and the labeled targets were hybridized to a 3D-Gene Human miRNA 4-plex chip (V21_V1.0.0; TORAY). Hybridization images were scanned using a GenePix4400A device (Molecular Devices, Sunnyvale, CA, USA), and the miRNA levels were assessed based on the signal intensity, which was calculated as the median of the foreground signal minus the mean of the negative control signals + 2 standard deviations. In the absence of any definite internal control for miRNAs in PBMCs for standardization, the global normalization method was used for calculations.
Cells
Normal human glomerular mesangial cells (NHMCs), human umbilical vein endothelial cells (HUVECs), and human renal proximal tubule epithelial cells (RPTECs) were obtained from Lonza (Basel, Switzerland). THP-1 cells were obtained from the ATCC (Manassas, VA, USA). NHMCs were maintained in Dulbecco’s modified Eagle medium (DMEM)/Ham’s F-12 (Wako, Osaka, Japan), supplemented with 10% fetal bovine serum (FBS). HUVECs were maintained in EGM™-2 BulletKit (Lonza) and RPTECs were maintained in REGM (Lonza) according to the manufacturer’s instructions. THP-1 was maintained in RPMI-1640 (Sigma-Aldrich Japan, Tokyo, Japan), supplemented with 10% FBS. THP1-derived macrophages (THP1-Mφ) were differentiated by adding phorbol 12-myristate 13-acetate (PMA) (50 ng/mL; Sigma-Aldrich Japan) to THP-1 for two overnight cultures and used for subsequent experiments.
Transfection
miRNA mimics and S-TuDs (Synthetic Tough Decoy: miRNA inhibitor) were purchased from GeneDesign (Osaka, Japan). NHMCs were transfected with miRNA mimics (20 nM), S-TuDs (20 nM), or negative control (NC) miRNA/S-TuDs (20 nM) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After one overnight incubation, they were stimulated with TNF-α (10 ng/mL: R&D Systems, Minneapolis, MN, USA) for 24 h and were then subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis or western blotting, as described in a later section.
Induction of miR-6516-3p by inflammatory stimuli
NHMCs and RPTECs were stimulated with TNF-α (10 ng/mL), IL-1β (10 ng/mL; R&D Systems), and IFN-α (50 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) for 24 h. HUVECs were stimulated with TNF-α (10 ng/mL), IL-1β (10 ng/mL), and IFN-α (50 ng/mL) for 6 h. THP1-Mφ were stimulated with lipopolysaccharide (LPS) (Escherichia coli 0111; B4, 1 µg/mL; Sigma-Aldrich Japan), TNF-α (10 ng/mL), IL-1β (10 ng/mL), or IFN-α (50 ng/mL) for 6 h. These cells were then subjected to qRT-PCR analysis.
RNA extraction and qRT-PCR
Total RNA was extracted from the cells using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific).
Real-time PCR for miRNA measurement was performed using THUNDERBIRD Probe qPCR Mix (TOYOBO, Osaka, Japan) and specific primers (hsa-miR-4657, Assay ID: 462809_mat; hsa-miR-6894-5p, Assay ID: 466432_mat; hsa-miR-3607-3p, Assay ID: 463814_mat; hsa-miR-6516-3p, Assay ID: 467192_mat; hsa-miR-6126, Assay ID: 475618_mat; hsa-miR-769-5p, Assay ID: 001998; hsa-miR-150-5p, Assay ID: 000473; hsa-miR-4689, Assay ID: 463337_mat; U6 small nuclear RNA (U6 snRNA), Assay ID: 001973; Thermo Fisher Scientific) and QuantStudio 5 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cycle threshold (Ct) values were calculated using the ∆∆Ct method (Schmittgen and Livak 2008). The values for miRNAs extracted from each cell were normalized to that for U6 snRNA.
For measuring mRNA expression, total RNA was reverse-transcribed using the Prime-Script RT Reagent kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Real-time PCR was performed using TB Green Premix Ex Taq (Takara Bio) and QuantStudio 5 according to the manufacturer’s instructions. The primer sequences for IL-1β, IL-6, IL-8, MMP-9, RECK, and β-actin are available on request.
Western blotting
Cells were lysed in RIPA buffer (BioDynamics Laboratory, Tokyo, Japan) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was determined using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). The RECK (D8C7) rabbit monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin (AC-15) was purchased from Sigma-Aldrich. Horseradish peroxidase-conjugated anti-IgG secondary antibodies against rabbit IgG (Dako, Glostrup, Denmark) or mouse IgG (Cell Signaling Technology) were used with the Chemi-Lumi One substrate (Nacalai Tesque, Kyoto, Japan). Densitometric analysis was performed using the ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/).
Statistical analysis
Data from the miRNA arrays were analyzed using Microsoft Excel (Microsoft, Redmond, WA, USA). Statistical analyses of in vitro studies were performed using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA). Paired t test was used to compare the results for samples collected from patients before and after treatment. Statistical analysis of other in vitro studies was performed using the nonparametric Mann–Whitney U test to compare data from different groups. A P value < 0.05 was considered to indicate a statistically significant difference.