A total of 21 patients with DN without other complications were selected in Shengli Oilfield Central Hospital (Dongying, China) between March 2017 and June 2018. The patients included 11 males and 10 females (age range, 46-64 years; mean age, 54.6±6.2 years). These patients had not received treatment within 3 months before admission. Pathological kidney and adjacent normal tissues were obtained by biopsy. Each patient provided written informed consent and agreed to the study being published. The present study was approved by the Ethics Committee of Shengli Oilfield Central Hospital (approval no. Q/ZXYY-ZY-YWB-LL202037).

HMCs were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was used to culture the cells at 37˚C in 5% CO₂. Small interfering (siRNA)-negative control (si-NC, 5'-UUCUCCGAACGUGUCACGU-3') and siRNA-NORAD-1/-2 (si-NORAD-1, 5'-AAGCCACCUUUGUGAACAGUA-3'; si-NORAD-2, 5'-GAGAAAUGGUAGAAUGACA-3') were obtained from Sangon Biotech Co., Ltd. NORAD overexpression (ov-NORAD), NRF1 overexpression (ov-NRF1), miR-485 mimics (5'-AGAGGCUGGCCGUGAUGAAUUC-3'), miR-NC (5'-UUCUCCGAACGUGUCACGUTT-3'), miR-485 inhibitor (5'-GUCAUACACGGCUCUCCUCUCU-3'), and inhibitor NC (5'-CAGUACUUUUGUAGUACAAA-3') were procured from Guangzhou RiboBio Co., Ltd. HMCs were transfected with the aforementioned agents (all 50 nM) using a Lipofectamine RNAiMAX kit (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37˚C. In addition, the HMCs (6x10⁵ cells/well) were further divided into high glucose (HG; 30 mM) and normal glucose (NG; 5.5 mM) groups. At 48 h after treatment, the cells were harvested to perform the following experiments.

Total RNA was extracted from HMCs using a TRIzol^® reagent kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. In accordance with the manufacturer's protocols of GoScript™ reverse transcription system (Promega Corporation), the extracted RNA was initially reverse transcribed into cDNA at 37˚C for 60 min, followed by 85˚C for 5 min and then subjected to qPCR analyses with the Applied Biosystems SYBR™ Green PCR Master mix (Thermo Fisher Scientific, Inc.). The thermocycling conditions were: Initial denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 20 sec and 72˚C for 34 sec. U6 or GAPDH was used as the internal reference standard. The primers were designed as follows: NORAD forward: 5'-GGAGAATCGCTTGAACT-3' and reverse, 5'-CAAACACCCAATGAATAG-3'; miR-485 forward, 5'-CCAAGCTTCACCCATTCCTAACAGGAC-3' and reverse, 5'-CGGGATCCGTAGGTCAGTTACATGCATC-3'; NRF1 forward, 5'-TTACTCTGCTGTGGCTGATGG-3' and reverse, 5'-CCTCTGATGCTTGCGTGGTCT-3'; U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3' and reverse, 5'-CGCTTCACGAATTTGCGTGTCAT-3' and GAPDH forward, 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse, 5'-GAAGATGGTGATGGGATTTC-3'. Gene expression was quantified using the 2^-ΔΔCq method (29).

Radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing protease inhibitors was used to extract proteins from cells. The protein concentrations were determined using a bicinchoninic acid Protein assay kit (Thermo Fisher Scientific, Inc.). A total of 50 µg of protein/lane was separated via 10% SDS-PAGE (Boster Biological Technology). The resolved proteins were then transferred onto polyvinylidene fluoride membranes. Blocking was performed using 5% bovine serum albumin (Thermo Fisher Scientific, Inc.) at room temperature for 2 h. Following blocking, the membranes were incubated overnight at 4˚C with primary antibodies against NRF1 (1:1,000; cat. no. ab175932; Abcam) and GAPDH (1:1,000; cat. no. ab8245; Abcam). Next, the membranes were washed three times in TBS-Tween-20 (0.05%). The secondary antibody horseradish peroxidase-conjugated anti-mice immunoglobulin G (1:2,000; cat. no. ab6728; Abcam) was added and incubated for 1 h at room temperature. GADPH was used as the internal reference. The membranes were developed using an ECL reagent (Thermo Fisher Scientific, Inc.) under Gel-Pro analyzer (version 4.0; Media Cybernetics, Inc.).

MTT assays were used to determine HMC viability. Cells were seeded onto a 96-well plate with 2x10⁵ cells/well and cultured in serum-free medium overnight at 37˚C. Subsequently, cells were incubated under the designated glucose conditions for 24, 48, 72 and 96 h at 37˚C. Next, 20 µl MTT (Merck KGaA) was added to each well and the cells were incubated for another 2 h at 37˚C. The supernatant was removed and the formazan crystals were dissolved using DMSO (150 µl/well). The absorbance at 450 nm was analysed using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific, Inc.).

According to manufacturer protocols, the levels of inflammatory [tumour necrosis factor (TNF)-α (cat. no. 70-EK182HS-96), interleukin (IL)-1β (cat. no. 70-EK101BHS-96) and IL-6 (cat. no. 70-EK106/2-96) and fibrotic [type IV collagen (Col. IV) (cat. no. RK-009-001-106), fibronectin (FN; cat. no. RK-KOA0169) and plasminogen activator inhibitor 1 (PAI-1) (cat. no. 70-EK1136-96)] factors in HMCs were measured using specific ELISA kits purchased from Multisciences Biotech, Ltd. The absorbance at 450 nm was determined using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific, Inc.).

The miRNA targets of NORAD were predicted using StarBase software version 2.0 (http://starbase.sysu.edu.cn/), and 272 targets were predicted. Among these miRNA targets, miR-485 was selected for the following assays due to its important role in DN (25) and unknown regulatory association with NORAD. In addition, the mRNA targets of miR-485 were predicted using miRDB software version 3.0 (http://mirdb.org/), and 1,646 targets were predicted. NRF1 was selected for the following assays due to its important role in renal diseases (30,31).

To verify the direct interactions between miR-485 and NORAD/NRF1, a dual-luciferase reporter assay was performed. Briefly, the mutant type (MUT) and wild type (WT) of NORAD/NRF1 binding sequences were cloned into the pGL3-promoter (Promega Corporation) to generate the recombinant vectors pGL3-NORAD-WT/-MUT or pGL3-NRF1-WT/-MUT. Cells were co-transfected with the aforementioned recombinant vectors and miR-485 mimic/miR-NC (all 50 nM) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 48 h. The supernatant was used to measure relative luciferase activity on a Dual-Luciferase Reporter assay system (Promega Corporation). The activity of firefly luciferase was normalized to that of Renilla luciferase.

SPSS version 20.0 (IBM Corp.) and GraphPad Prism version 5.01 (GraphPad Software, Inc.) were used to perform the statistical analyses. Data are expressed as the mean ± standard deviation. Student's t-tests were used to assess the differences between groups (paired, Figs. 1A, 3D and 5C; unpaired, Figs. 1B, 3E and 5D). One-way analysis of variance (ANOVA) was used to investigate the differences among multiple groups. Following ANOVA, pairwise comparisons were performed using Tukey's multiple comparisons tests. P<0.05 was considered to indicate a statistically significant difference. All experiments were conducted in triplicate in at least three independent experiments.

The NORAD expression in DN and normal tissues was detected by RT-qPCR. The results demonstrated higher expression in DN tissues compared with in normal tissues (P<0.001; Fig. 1A). Meanwhile, NORAD expression was significantly increased in HG-stimulated HMCs compared with in the NG group (P<0.05; Fig. 1B).

To investigate the possible role of NORAD in DN pathogenesis in vitro, si-NORAD-1/-2 was initially transfected into HMCs to detect the silencing efficiency. As illustrated in Fig. 2A, RT-qPCR demonstrated significantly decreased NORAD expression following transfection of si-NORAD-1 and si-NORAD-2 (P<0.01). Following treatment with HG, NORAD expression was also significantly decreased in the HG + si-NORAD-1 and HG + si-NORAD-2 groups (P<0.01; Fig. 2B). si-NORAD-1 was selected in the following experiments due to its greater silencing efficiency. The results of the MTT assay demonstrated increased HMC viability in the HG + si-NC group compared with in the NG group, whilst cell activity was partially inhibited in the HG + si-NORAD group compared with in the HG + si-NC group (P<0.05; Fig. 2C). Similarly, the ELISA results demonstrated highly increased levels of inflammatory factors (IL-6, IL-1β and TNF-α) in the HG + si-NC group compared with those in the NG group, which was attenuated in the HG + si-NORAD group (P<0.01; Fig. 2D-F). The levels of PAI-1, Col. IV and FN were higher in the HG + si-NC group compared with in the NG group, which was partially reversed in the HG + si-NORAD group (P<0.01; Fig. 2G-I). These results indicated that NORAD knockdown suppressed HMC proliferation, inflammation and fibrosis in vitro.

Analysis using StarBase software demonstrated potential binding sequences between lncRNA NORAD and miR-485 (Fig. 3A). The luciferase activity in the NORAD WT/miR-485 mimics group was decreased compared with that in the NORAD WT/miR-NC group (P<0.01; Fig. 3B). miR-485 expression was significantly downregulated in the HG + si-NC group compared with in the NG group, which was attenuated in the HG + si-NORAD group (P<0.01; Fig. 3C). RT-qPCR assays demonstrated decreased miR-485 expression in DN tissues compared with in normal tissues (P<0.001; Fig. 3D). The expression of miR-485 was also decreased in the HG group compared with in the NG group (P<0.01; Fig. 3E). These data indicated a negative regulatory association between NORAD and miR-485.

Next, miR-485 mimics, miR-485 inhibitor and miR-485 mimics + ov-NORAD were transfected into HMCs to detect the transfection efficiency. The expression of miR-485 was upregulated in the miR-485 mimics group compared with in the miR-NC group, whilst the inverse was observed in the miR-485 inhibitor group compared with the inhibitor NC group (P<0.01; Fig. 4A). Meanwhile, compared with the miR-485 mimics + ov-NC group, miR-485 expression in the miR-485 mimics + ov-NORAD group was partially inhibited (P<0.01; Fig. 4A). NORAD expression was then determined following transfection. The results of RT-qPCR analysis demonstrated that the expression of NORAD was upregulated by ov-NORAD, but was partially suppressed by ov-NORAD + miR-485 mimics (P<0.01; Fig. 4B). In addition, it was also discovered that miR-485 expression was significantly decreased in the HG group; its expression was upregulated in the HG + miR-485 mimics group and downregulated in the HG + miR-485 inhibitor group (P<0.01; Fig. 4C). All the results suggested that miR-485 mimics, miR-485 inhibitor, ov-NORAD and miR-485 mimics + ov-NORAD were successfully transfected into HMCs. The results of the MTT assay demonstrated significantly decreased HG-induced HMC viability in the miR-485 mimics group compared with in the miR-NC group; however, the viability of HG-induced HMCs was partially promoted in the miR-485 mimics + ov-NORAD group compared with in the miR-485 mimics group (P<0.05; Fig. 4D). Similarly, the ELISA results demonstrated significantly decreased levels of inflammatory (IL-6, IL-1β and TNF-α) and fibrotic (PAI-1, Col. IV and FN) factors in the miR-485 mimics group compared with in the miR-NC group. Furthermore, overexpression of NORAD attenuated the effects of miR-485 on inflammation and fibrosis in HG-induced HMCs (P<0.05; Fig. 4E and F). Therefore, the results suggested that NORAD may affect the occurrence and development of DN in vitro by regulating miR-485 expression.

Using miRDB software, the potential binding sites between NRF1 and miR-485 were predicted (Fig. 5A). Dual-luciferase reporter assays demonstrated significantly decreased luciferase activity in the NRF1 WT/miR-485 mimics group compared with in the control group. These results indicated that NRF1 was a direct target gene of miR-485 (P<0.01; Fig. 5B). The RT-qPCR results suggested that the expression of NRF1 was higher in DN tissues compared with in normal tissues (P<0.001; Fig. 5C). Similar to the mRNA levels, the results of the western blot analysis demonstrated downregulation of the NRF1 protein level in the HG + miR-485 mimics group compared with that in the HG + miR-NC group (P<0.01; Fig. 5D). These results suggested that miR-485 inhibited NRF1 expression.

The expression of NORAD and miR-485 following co-transfection was detected. The results of RT-qPCR demonstrated that NORAD expression was significantly increased in the si-NORAD + miR-485 inhibitor and si-NORAD + ov-NRF1 groups compared with their controls (si-NORAD + miR-NC and si-NORAD + ov-NC, respectively; P<0.05; Fig. 6A). Furthermore, the expression of miR-485 was inhibited by co-transfection with si-NORAD + miR-485 inhibitor compared with si-NORAD + inhibitor NC (P<0.01; Fig. 6B). The transfection efficiency of ov-NRF1 was subsequently determined using RT-qPCR, which demonstrated that NRF1 expression was increased by ov-NRF1 (P<0.01; Fig. 6C). The aforementioned results indicated that si-NORAD + miR-485 inhibitor or ov-NRF1 was successfully transfected into HMCs. Western blot analysis demonstrated downregulated NRF1 protein expression following transfection of si-NORAD, while co-transfection with miR-485 inhibitor or ov-NRF1 reversed this inhibitory effect (P<0.05; Fig. 6D). MTT assays demonstrated significantly inhibited HG-induced HMC viability in the si-NORAD group compared with that in the si-NC group. Meanwhile, cell viability was partially promoted in the si-NORAD + miR-485 inhibitor and si-NORAD + ov-NRF1 groups compared with in the si-NORAD group (P<0.05; Fig. 6E). Similarly, the ELISA results indicated that NORAD knockdown downregulated the levels of inflammatory (IL-6, IL-1β and TNF-α) and fibrotic (PAI-1, Col. IV and FN) factors. However, downregulation of miR-485 and upregulation of NRF1 reversed the effects of NORAD knockdown on inflammation and fibrosis in HG-induced HMCs (P<0.05; Fig. 6F and G). These results indicated that knockdown of NORAD may suppress HG-induced HMC proliferation, inflammation and fibrosis by regulating miR-485/NRF1.