Adult SLE patients (n=52) were enrolled between January 2012 and December 2014 from the Department of Rheumatology and Immunology, Nanfang Hospital. The present study was approved by the Ethics Committee of Southern Medical University (Guangzhou, China). Written informed consent was obtained from all patients. Among them, 30 patients with consecutive SLE and active nephritis underwent kidney biopsy, and 22 patients were diagnosed as inactive SLE. Sections of the kidneys were surgically resected due to kidney biopsy, and subsequently fixed in 4% formaldehyde at room temperature overnight for in situ hybridization at 3–4 µm. The diagnosis was according to the American College of Rheumatology diagnostic criteria of SLE (11). The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score was determined at the time of the blood draw; scores <4 were considered as inactive disease, and scores ≥4 was categorized as active disease. Renal SLEDAI scores were determined as described (12). The mesangial cell proliferation was regarded as the main pathological change. A total of 10 paired renal tissue samples served as the control. Additionally, renal tissues were collected from 6 patients with Behcet's disease to compare SLE with other autoimmune disease. Patients with Behcet's disease were recruited from the Department of Rheumatology and Immunology, Nanfang Hospital. Sections of the kidneys were surgically resected due to pathological examination, which were subsequently fixed in 4% formaldehyde at room temperature overnight for in situ hybridization at 3–4 µm.

The MMC mouse mesangial cell line (cat. no. CRL-1927) and human embryonic kidney 293 (HEK-293) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The MMCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (3:1; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). HEK-293 cells were maintained in DMEM supplemented with L-glutamine and 10% FBS (Thermo Fisher Scientific, Inc.). Cells were transfected using the siPORTNeoFX agent with 50 nM mirVana (Invitrogen; Thermo Fisher Scientific, Inc.). hsa-miR-198 mimic, hsa-miR-198 inhibitor or their respective negative controls [(NC); miR-NC, as-miR-NC] were used for transfection (Ambion; Thermo Fisher Scientific, Inc.). Three independent experiments were conducted. The transfected cells were collected for total RNA or protein isolation at 48 h post-transfection.

Total RNA was extracted from mouse kidneys and culture cell lysates using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The concentration and purity of the RNA was confirmed using an Agilent 2100 system. RNA was reverse transcribed to cDNA using the High Capacity RNA-to-cDNA kit or a PrimeScript 1st strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed in 96-well plates on an ABI 7500 system using Taqman Gene Expression assay according to the manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). The data were normalized based on the expression of the endogenous control GAPDH (mRNA), and U6 small nuclear RNA was used for miRNA normalization. The primers used were as follows: GAPDH, forward 5′-GAGAAGTATGACAACAGCCTC-3′ and reverse 5′-ATGGACTGTGGTCATGAGTC-3′; PTEN, forward 5′-AAGTCCAGAGCCATTTCCAT-3′ and reverse 5′-CAAGTCTAAGTCGAATCCATCCT-3′; miR-198, forward 5′-TCATTGGTCCAGAGGGGAGATAG-3′ and reverse 5′-GCAGGGTCCGAGGTATTC−3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′, and reverse 5′-AACGCTTCACGAATTTGCGT-3′. The PCR amplification conditions were: 95°C for 10 min, followed by 35 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec, then 4°C for 5 min. Cq values were normalized to the endogenous control and presented as the fold-change relative to the control sample (2^ΔΔCq) (13).

TargetScan 6.2 (http://www.targetscan.org) was used to screen the potential targets of miR-198. PTEN was identified as one of the miR-198 candidate targets.

The phosphatase and tensin homology deleted on chromosome ten (PTEN) 3′-UTR luciferase reporter was cloned into the pGL3 luciferase reporter vector between the SpeI and HindIII sites. The mutant PTEN-3′UTR was generated as (TCTGGA to TCGGGA). The sequence was confirmed by DNA sequencing. MMCs and HEK-293 cells were cultured in a 24-well plate the day before transfection. Cells were co-transfected with the firefly luciferase-3-UTR (pGL3-PTEN or pGL3-PTEN mutant; 500 ng) and the pRL-TK vector (Promega Corporation, Madison, WI), along with the miR-198, miR-198 inhibitor or the control sequences. At 48 h after transfection, cell lysates were prepared and luciferase assays were performed using an HT microplate reader (BioTek China, Beijing, China). Luciferase activities were normalized to Renilla luciferase activity. All the experiments were repeated at least three times.

Cell proliferation was examined using a Cell Counting kit-8 (CCK-8; Beyotime Institute of Biotechnology, Haimen, China) at 0, 24, 48, 72, 96 and 120 h after inoculation by means of a cell proliferation assay according to the manufacturer's protocol. The optical density was measured at a wavelength of 450 nm using a microplate reader.

Mouse kidneys or HEK-293T cells were lysed using radioimmunoprecipitation assay buffer with complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Proteins (30 µg) were separated by 10% SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking with non-fat milk for 30 min at room temperature, the membrane was incubated overnight at 4°C with a PTEN primary antibody (MAB4037, 1:1,000 dilution, EMD Millipore, Billerica, MA, USA) or a mouse monoclonal anti-β-actin primary antibody (MABT825, 1:1,000 dilution, EMD Millipore). Horseradish peroxidase (HRP)-conjugated anti-rabbit (sc-2004, 1:3,000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or anti-mouse IgG antibodies (sc-2005, 1:3,000, Santa Cruz Biotechnology, Inc.) was used as the secondary antibody. At last, the membranes were visualized with the ECL Western Blotting Detection System (GE Healthcare Life Sciences).

Statistical analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). The results are expressed as the mean ± standard deviation. Comparison of parameters of the three groups (SLE, patients with Behcet's disease and normal controls) was carried out using one way analysis of variance/Kruskal-Wallis test. Multiple comparisons between the groups were performed using Student-Newman-Keuls method. The correlations between biomarker and the activity were calculated by Spearman's rank correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was used to examine miR-198 expression in the SLE patients and those with active nephritis. As presented in Fig. 1A, the expression of miR-198 was significantly higher in lupus patients compared with normal controls, and although the miR-198 expression in patients with Behcet's disease appeared higher than the controls, there was no statistical differences. Next, correlational analysis was performed to determine whether there was any correlation between the expression of miR-198 and clinical features. As presented in Fig. 1B, the expression of miR-198 was higher in patients with active SLE compared with those with inactive SLE and the normal controls. Furthermore, a direct positive correlation was observed between miR-198 levels and the SLEDAI scores, as well as between the miR-198 levels and the renal SLEDAI scores (Fig. 1C). Thus, it was hypothesized that higher miR-198 expression positively correlates with SLE disease activity.

As high expression levels of miT-198 were observed in the LN group, it was hypothesized that miR-198 may correlate with glomeruli cell proliferation. To validate this hypothesis, a hsa miR-198 mimic, or hsa-miR-198 inhibitor or their respective controls were transfected into MMCs or HEK-293 cells. Successful overexpression (Fig. 2A) or downregulation (Fig. 2B) of miR-198 in the two cells was confirmed by RT-qPCR. Furthermore, a CCK-8 assay was performed in the MMCs (Fig. 2C) or HEK-293 (Fig. 2D) cells transfected with miR-control, miR-198, miR-NC or miR-198 inhibitor, and the growth ratio was observed. Overexpression of miR-198 could significantly increase the cell growth rates, while the knockdown of miR-198 remarkably inhibited the growth level.

In order to elucidate the mechanism of miR-198 to stimulate cell proliferation, bioinformatics analysis was used to identify its target genes. TargetScan 6.2 (http://www.targetscan.org) was used to screen the potential target and PTEN was identified as one of the miR-198 candidate targets (Fig. 3A). To determine whether miR-198 can potentially bind to PTEN, a luciferase reporter assay was performed in the MMCs and HEK-293 cell lines. As presented in Fig. 3B, in cells transfected with miR-198 mimic, the luciferase activity of the PTEN-3′UTR was remarkably decreased compared with the control. Conversely, when cells were transfected with miR-198 inhibitor, the PTEN-3′UTR luciferase activity was obviously increased (Fig. 3C). However, when cells were transfected with mutant PTEN-3′UTR (TCTGGA to TCGGGA), the inhibition effect of miR-198 was almost abolished (Fig. 3D and E).

To investigate the possible mechanism underlying miR-198 in cell proliferation, the present study detected the function of miR-198 on the expression of PTEN using RT-qPCR. miR-198 reduced the expression of PTEN compared with the control cells (Fig. 4A) while the miR-198 inhibitor upregulated the expression of PTEN in MMCs and HEK-293 cells (Fig. 4B). Furthermore, western blot analysis was performed to analyze the function of miR-198 on PTEN protein expression. Forced expression of miR-198 mimics significantly reduced the expression of PTEN protein in MMCs and HEK-293 cells (Fig. 4C), whereas the expression of PTEN protein was upregulated after miR-198 was inhibited in the two cell lines (Fig. 4D).

In order to determine the importance of PTEN as a functional target of miR-198, it was hypothesized that forced expression of PTEN could circumvent the inhibitory effects of miR-198 on cell proliferation. A lentiviral vector expressing PTEN was constructed and transfected into the MMCs and HEK-293 cells, and RT-qPCR was used to demonstrate that PTEN expression was restored after PTEN lentiviral infection (Fig. 5A and B). Furthermore, CCK-8 analysis was performed in the above cell lines; compared with the miR-198 groups, forced expression of PTEN rescued the function of miR-198 on cell proliferation (Fig. 5C and D).

To better understand the functionality of miR-198 in SLE progression, the present study assessed the correlation between miR-198 and PTEN levels in the active SLE patients. As presented in Fig. 6A, there was a negative correlation between miR-198 and PTEN. Furthermore, the expression of PTEN was significantly lower in SLE patients compared with normal controls (Fig. 6B). Additionally, the expression of PTEN was lower in patients with active SLE compared with those with inactive SLE (Fig. 6C). These results further support the hypothesis of the present study.