MiR-183 delivery attenuates murine lupus nephritis-related injuries via targeting mTOR

2.1 Clinical information

From June 2017 to July 2018, a total of 20 patients who were diagnosed with LN in Liaocheng People's Hospital were recruited in this study. There were 9 males and 11 females in this cohort, aged at 37.6 ± 15.8 years. No immune suppressant, immune modulator or steroid hormone has been used within 3 months. No one was complicated with malignant tumour, acute/chronic infection or other autoimmune diseases. SLE disease activity index (SLEDAI) was employed based on clinical manifestation and laboratory tests. Those patients with higher than 5 points were regarded as in the active phase (average score = 10.7 ± 4.1). Pathological samples were collected from renal tissues of LN patients. Another 20 normal renal tissue samples were collected from age/sex-matched patients who received renal tumour resection, but without primary glomerulonephritis, diabetes and hypertension. Renal tissue samples were snap frozen with liquid nitrogen and stored at −80°C for subsequent experiments. Informed consent was derived from each participant before participation, and the procedures were approved by the human ethics committee of Liaocheng People's Hospital.

2.2 Reverse transcription and quantitative real-time PCR

The expression levels of miR-183-3p and mTOR mRNA were analysed using High-Capacity cDNA Reverse Transcription Kit and TaqMan® miRNA assays (Applied Biosystem) following the manufacturer's recommendation. The probe ID number for hsa-miR-183-3p in the TaqMan® assays is 002270. U6 was used as a reference transcript for normalization. The expression levels of mTOR mRNA were measured by qPCR with ABI Step One Plus Detection System. The probe ID number for mTOR in the TaqMan® assays is Hs00234508_m1. 18S was used as a reference transcript for normalization. All reactions were performed in duplicate.

2.3 Mice

Female MRL/lpr mice were purchased from SLAC (Shanghai, China). All experiments were approved by the ethical committee of Liaocheng People's Hospital and were performed in line with the National Institute of Health Guide for Care and Use of Animals. The sex- and age-matched C57BL/6 mice as normal control group (Ctrl) were used as normal control. Meanwhile, two additional groups of 12-week-old female MRL/lpr mice (15 mice per group) were used to compare the survival from 12 to 28 weeks of age.

2.4 Lymphocytes and CD4+ T cell isolation

Renal tissues of MRL/lpr mice were cut into 1-2-mm pieces and incubated in RPMI 1640 containing 1% foetal calf serum (Invitrogen), 1 mM, dithiothreitol and 5 mM EDTA for 20 minutes at 37°C. Tissue pieces were then digested with Liberase TL and DNase I (0.125 U/mL) in RPMI 1640 while shaking. The resulting tissue suspension was passed through a 100-μm cell strainer, centrifuged and resuspended in 40% Percoll and underlayed with 80% Percoll. After centrifugation at 600 g for 20 minutes, lymphocytes were recovered from the 40/80% interface and resuspended in complete medium.

The CD4+ T cell was isolated from lymphocytes using the Miltenyi MACs Kit according to manufacturer's instructions. CD4+ T purified using the Miltenyi system are ≥96% pure.

2.5 Lymphocytes staining

Total lymphocytes from mice were suspended in phosphate-buffered saline (PBS) containing anti-FcgRII/III mAb (BD Biosciences), stained at 4°C with peridinin-chlorophyll (PerCP)-conjugated anti-CD4 (BD Biosciences) and allophycocyanin (APC)-H7 conjugated anti-CD8 antibodies (BD Biosciences) for 30 minutes. After washed in PBS, cells were further stained for 30 minutes at 4°C with anti-IL-17A-FITC (Mabtech) or combinations of anti-CD25 and anti-Foxp3. Cell population were analysed by BD FACSCAlibur 2 Laser, 4 Color Flow Cytometer.

2.6 miR-183 mimic and antibodies

MiR-183 mimic (miR-183) and miRNA control (miRNA-control) were all purchased from GenePharma Co., Ltd. MRL/lpr mice received intraperitoneal injection with either miR-183 mimic (183 group) or miRNA-control (LN group) at a dose of 1 nmol per mouse twice per week from 12 to 28 weeks of age. Antibodies against C3, lgG, p-mTOR, mTOR, p-P70S6K and P70S6K were purchased from Cell Signaling Technology.

2.7 Blood urea nitrogen detection

Blood urea nitrogen (BUN) level was detected by a commercial auto-analyser (Beckman Coulter). Urine samples from experimental mice were collected every 2 weeks using metabolic cages. Urinary protein levels were semi-quantified by Multistix 10SG reagent strips (Bayer Healthcare).

2.8 Immune complex deposition detection

The frozen kidney sections were blocked with 10% foetal bovine serum and then stained with FITC-conjugated rabbit anti-mouse IgG (Santa Cruz) or FITC-conjugated goat IgG fraction to mouse complement C3 (Cedarlane). Fluorescence intensity was calculated and scored from 0 to 3 (0, no fluorescence; 0.5, trace, just detectable above background; 1, fluorescence scattered and light; 2, bright but not diffuse; and 3, bright and diffuse).

2.9 Anti-dsDNA ELISA

An anti-dsDNA ELISA Kit (Laibio) was used to quantitate serum antibodies that bind to double-stranded DNA. The assay was performed according to the manufacturer's instructions. The quantity of anti-dsDNA antibodies was determined by comparison against a standard curve.

2.10 Statistical analysis

The results were analysed using the SPSS 13 statistical software. Quantitative variables were analysed using Student's t test between two groups or one-way analysis among multiple groups. The differences were considered significant at P < .05. The correlations between miR-183 and mTOR mRNA expression levels were analysed using Spearman rank correlation test. The differences were considered significant at P < .05.

3.1 MiR-183 was downregulated in LN patients and mice

Previous studies have shown that mTOR, a key regulator of metabolic activity, plays a very important role in SLE disease pathogenesis and mTOR is a direct target of miR-183 in neuroblastic cells.24 To investigate the association of the expression levels of mTOR and miR-183, a total of 40 renal tissues with 20 from LN patients and 20 from healthy donors were collected for this study. Total RNA from each sample was extracted and the expression levels of mTOR mRNA and miR-183 were analysed by TaqMan real-time PCR. The results demonstrated that the mTOR mRNA levels were significantly upregulated; while miR-183 levels were markedly downregulated, in renal tissues from LN patients compared with that from healthy donors (Figure 1A,B). Spearman's rank correlation analysis showed that the expression levels of mTOR and miR-183 in 20 renal tissues from LN patients were inversely correlated, suggesting that miR-183 may target mTOR expression in LN patients (Figure 1C). Furthermore, downregulation of miR-183 and upregulation of mTOR were identified in kidney samples from MRL/lpr mice when compared to normal control mice (Ctrl) (Figure 1D,E), and the expression levels of mTOR and miR-183 in 15 renal tissues from MRL/lpr mice were inversely correlated (Figure 1F).

3.2 Urinary albumin levels and blood urea nitrogen (BUN) levels

Next, we aimed to compare the difference of urinary albumin levels and BUN levels between MRL/lpr mice and Ctrl. There were no significant differences in the urinary albumin levels among all the mice at the start point of the study. When the interaction between treatment and time was evaluated within groups, we observed that the urinary albumin values were significantly higher in MRL/lpr mice than that in Ctrl. The urinary albumin values were decreased in MRL/lpr mice receiving miR-183 injection treatment compared with that in MRL/lpr mice receiving miRNA-control injection treatment (LN group) (Figure 2A). Similarly, the BUN levels in MRL/lpr mice were about 3.5-folds higher than that in Ctrl. The BUN levels were decreased by almost 50% in MRL/lpr mice after receiving miR-183 injection treatment when compared with that in MRL/lpr mice receiving miRNA-control injection treatment (LN group) (Figure 2B). The results clearly showed that miR-183 injection decreased urinary albumin levels and BUN levels in MRL/lpr mice.

3.3 Anti-dsDNA antibodies and renal deposition of immune complex

As shown in Figure 3A,B, anti-DNA antibody (IgG) concentration and immune complex component levels (anti-C3 and anti-IgG) were significantly decreased in MRL/lpr mice after miR-183 injection treatment compared to miRNA-control treatment (LN group) (Figure 3A). The fluorescence intensity of C3 and IgG was also decreased in miR-183 treatment groups compared to LN group (Figure 3B). These results demonstrated that miR-183 injection can effectively reduce anti-dsDNA antibodies and renal deposition of immune complex in MRL/lpr mice.

3.4 Survival evaluation

We observed that the miRNA-control-injection MRL/lpr mice (n = 15) started to die at the age of 17 weeks and only 40% of them were still alive at age of 30 weeks. However, miR-183-injection MRL/lpr mice (n = 15) started to die at age of 22 weeks. At the age of 30 weeks, 40% of miRNA-control-injection mice (LN group) were alive and 60% of miR-183-injection mice were alive (Figure 4). These data strongly suggested that miR-183 injection treatment significantly prolonged the survival rate of MRL/lpr mice compared with miRNA-control LN groups.

3.5 Tregs and Th17 cell population

It was reported that impaired function of Tregs and/or increased Th17 cell population leads to a failure in immune tolerance and triggers autoimmunity. Total lymphocytes from kidneys of Ctrl, miRNA-control-injection MRL/lpr mice (LN group) and miR-183 injection MRL/lpr mice were isolated at the end of experiments, and the Tregs and Th17 cell population were analysed by flow cytometry assay. Gating strategy for all three groups could be found in Figure S1. Treg cells were evaluated as CD4+CD25+ lymphocytes expressing FoxP3, and Th17 cells were defined as CD4+IL-17A+ lymphocytes. The results showed that the CD4+CD25+Foxp3+ Treg cell population were significantly decreased, whereas CD4+IL-17+ Th17 cell population were increased, in miRNA-control-injection MRL/lpr mice (LN group) compared with that in Ctrl (Figure 5A,B). MiR-183 injection treatment resulted in upregulation of the number of Treg cells and downregulation of the number of Th17 cells, leading to partial restoration of Tregs and Th17 cell population.

3.6 MiR-183 targets mTOR

P70S6 kinase (p70S6K), a mitogen-activated serine/threonine protein kinase that is required for cell growth and G1 cell cycle progression, is a downstream target of mTOR signalling pathway. Overactivation of mTOR/p70s6k signalling pathway has been reported in murine LN.25, 26 Next, we aimed to confirm that miR-183 regulates mTOR/p70s6k signalling pathway through targeting mTOR expression. To reach this goal, CD 4+ T cells were isolated from renal tissues of MRL/lpr mice with miR-183 mimics or miRNA-control (LN group) treatment as well as Ctrl group. The CD 4+ T cells from these three groups were subjected to Western blotting assay. Indeed, we observed that both p-mTOR and total mTOR protein levels were significantly decreased upon miR-183 mimic transfection compared to miRNA-control (LN group). The p-p70s6k and total-p70s6k protein levels also decreased in miR-183 overexpression cells (Figure 6A,C). These data suggested that miR-183 inhibited mTOR and p70s6k expression and further inhibits mTOR/p70s6k signalling pathway activation (Figure S2).