MMCs were isolated from 8-week-old male C57BL/6 mice (n=6) as previously described (15). The mice were euthanized by an intraperitoneal injection of sodium pentobarbital (100 mg/kg). All experiments were approved by the Animal Care and Use Committee of the Nanjing University of Chinese Medicine and complied with the Declaration of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. MMCs were obtained from the renal cortex of the 8-week-old male mice using sterile techniques. After washing with RPMI-1640 medium (Beyotime Biotechnology), the cortices were separated from the medullas, pooled, minced and the glomeruli were isolated. The glomeruli were then pre-treated with collagenase for 15 min and plated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 20 µg/ml insulin at 37°C with 5% CO₂. Cells at passages between 3 and 7 were used in all the experiments.

The MMCs were treated with normal glucose (5 mmol/l) plus mannitol (25 mmol/l) for the control group and high glucose (30 mmol/l) for the high glucose (HG) group. miR-379-5p mimics, inhibitors, agomir and siRNA against LIN28B and the negative controls were purchased from RiboBio (Guangzhou). All of the transient transfections were conducted using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific). 293T cells were acquired from the Chinese Academy of Sciences Cell Bank and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS.

The LIN28B transcript sequences were acquired from NCBI resources (gene ID, 380669). The 3′ UTR of LIN28B with predicted target sites for miR-379-5p seed sequence was amplified and cloned into the PGL-3 vector (Promega) using KpnI/HindIII sites. The primers used were as follows: Sense, 5′-GGTACCGCCTTTGATTCAGAAACGG-3′ and antisense, 5′-AAGCTTCTATAAAACATGACACCCGC-3′. The mutated 3′ UTR of LIN28B was generated using the QuikChange II Site-Directed Mutagenesis kit (Stratagene). At 48 h following the co-transfection of miR-379-5p mimics (100 nM) and the wild-type or mutated 3′ UTR of LIN28B, the luciferase activity of the 293T cells was measured using the dual-luciferase assay kit (Promega) with Renilla luciferase as the internal control.

Total RNA was extracted from the MMCs and renal tissues (see below) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific). cDNA was synthesized using the PrimeScript^® RT reagent kit (Takara). qPCR was performed using SYBR Premix ExTaq™ (Takara) on the platform of Applied Biosystems 7500. U6 was used as an endogenous normalization control. The total reaction system of 20 μl volume was as follows: 1 μl cDNA, 10 μl SYBR Premix EX Taq, 1 μl each of the primers (10 μM) and 7 μl ddH₂O. The PCR cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The primers used were as follows: miR-379-5p forward, 5′-GCGCTGGTAGACTATGGAA-3′ and reverse, 5′-GTG CAG GGT CCG AGG T-3′; U6 forward, 5′-CTC GCT TCG GCA GCA CAT ATA CT-3′ and reverse, 5′-ACG CTT CAC GAA TTT GCG TGT C-3′; Lin28B forward, 5′-GTC AAT ACG GGT AAC AGG AC-3′ and reverse, 5′-TTC TTT GGC TGA GGA GGT AG-3′; Let-7b forward, 5′-TGA GGT AGT AGG TTG TGT-3′ and reverse, 5′-GTC GTA TCC AGT GCA GGG TCC GAG GT-3′; Col-1 forward, 5′-CCA CCC CAG CCG CAA AGA GTC-3′ and reverse, 5′-GTC ATC GCA CAC AGC CGT GC-3′; Col-4 forward, 5′-ATC TCT GCC AGG ACC AAG TG-3′ and reverse, 5′-CGG GCT GAC ATT CCA CAA T-3′; GAPDH forward, 5′-GAA ATC CCA TCA CCA TCT TCC AGG-3′ and reverse, 5′-GAG CCC CAG CCT TCT CCA TG-3′. Data analyses were performed using the comparative Cq (ΔΔCq) method for calculating relative gene expression (16).

Renal tissue (see below) and MMCs were lysed in radioimmunoprecipitation buffer. Approximately 80 μg of total proteins were separated by 12% or 10% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore Corp.) by electroblotting. After being blocked for 60 min with 5% non-fat milk, the membranes were incubated at 4°C overnight with primary antibodies against Col4 (ab6586; 1:1,000), fibronectin (FN; ab2413; 1:1,000), p21 (ab109199; 1:1,000), cyclin D1 (ab134175; 1:1,000) (all from Abcam) and LIN28B (#5422; 1:1,000; Cell Signaling Technology). GAPDH (ab9485; 1:2,000; Abcam) was served as the loading control. The membranes were then incubated with the secondary antibodies anti-rabbit IgG, HRP-linked antibody (1:1,000, #7074, Cell Signaling Technology) for 1 h at room temperature. Subsequently, they were visualized with ECL detection reagent (Millipore), and the gray values of the bands were calculated automatically (ImageJ software, version 4.3).

Cell viability was assayed using the Cell Counting kit-8 (Dojindo) following the manufacturer's instructions. The cells were seeded in 96-well plates at a density of 2×10³ cells per well. CCK-8 solution (10 μl) was added to each well for 1 h following 48 h of incubation, and OD450 was determined using a microplate reader (ELx×808, BioTek). Moreover, 5-ethynyl-2′-deoxyuridine (EdU) assay (Beyotime Biotechnology) was performed to detect the proliferation of the MMCs. Following incubation with 10 μM EdU for 2 h at 37°C, the cells were fixed in 4% paraformaldehyde. Subsequently, Hoechst 33342 (Beyotime Biotechnology) was used to stain the nuclei at room temperature for 30 min. Finally, the cells were visualized under a fluorescence microscopy (Leica).

For cell cycle analysis, the MMCs were harvested, suspended in 500 μl of phosphate-buffered saline (PBS), fixed with 70% ethanol and subjected to PI/RNase (Beyotime Biotechnology) staining for 30 min at 4°C. The MMCs were then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

A total of 18 C57BL/KsJ type 2 diabetic db/db male mice (aged 4 weeks, weighing 18-22 g) and a total of 6 heterozygote db/m male mice (aged 4 weeks, weighing 18-22 g) used in this study were purchased from the Model Animal Research Center of Nanjing University. The mice were housed in a clean environment with 50-60% humidity, 20-22°C and a 12-h dark/light cycle, and fed a standard diet during a 1-week adaptation period. When the mice were 8 weeks old, they were randomly divided into the control group (untreated db/m mice, n=6); the DN group (untreated db/db mice, n=6); an agomir NC (agomir NC-treated db/db, n=6) group and an agomir-379-5p (agomir-379-5p-treated db/db, n=6) group. Every 2 days, the db/db mice were injected with miR-379-5p agomir or agomir NC (10 mg/kg) (GenePharma) via the tail vein. Four weeks later, the mice were sacrificed by an intraperitoneal injection of sodium pentobarbital (100 mg/kg) and the kidneys were harvested for analysis. During the experiments, animal health and behavior were monitored every 2 days. All experiments were approved by the Animal Care and Use Committee of the Nanjing University of Chinese Medicine and complied with the Declaration of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Metabolic cages were used to collect urine samples in a 24-h period, and urinary protein levels were evaluated using the mouse albumin ELISA quantitation set (Bethyl Laboratories).

Fresh renal tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded specimens were sectioned (4 μm thicknesses) and stained with PAS, Masson's trichrome staining (Beyotime Biotechnology) at room temperature for 15 min and immunohistochemistry. The antibodies included LIN28B (ab71415; 1:50), Ki-67 (ab15580; 1:500) and normal rabbit IgG (ab6728, 1:1,000) (all from Abcam) were incubated for 2 h at 37°C.

To detect the presence of miR-379-5p in the renal tissues, in situ hybridization was performed. The frozen renal tissues sections of mice from control group (untreated db/m mice), DN group (untreated db/db mice) and agomir-379-5p (agomir-379-5p-treated db/db mice) group were fixed in paraformaldehyde followed by rinsing in PBS. A digoxin-labeled oligonucleotide probe (5′-TGG TAG ACT ATG GAA CGT AGG-3′) (0.5 μg/ml) was synthesized by Dingguo BioTechnology Co. Ltd. In situ hybridization was performed with the ISH kit according to the protocol (Dingguo BioTech). The slides were treated with 3,3′-diaminobenzidine (DAB) for 5 min, and then counterstained with hematoxylin for 1-2 min at room temperature. All images were acquired by using a light microscope (Olympus) at the magnification of ×400.

TargetScan (http://www.targetscan.org/) was used to predict the putative target genes for miR-379-5p.

GraphPad Prism (version 5.0; GraphPad Software, Inc.) was used to conduct all statistical analyses. Measurement data are presented as the means ± standard deviation. One-way ANOVA analysis followed by a Tukey's post hoc test were used to compare the difference between multiple groups. Values of P<0.05 and P<0.01 were considered to indicate statistically significant and highly statistically significant differences, respectively.

To explore the potential role of miR-379-5p in DN, we analyzed the expression of miR-379-5p in MMCs stimulated with HG. As shown in Fig. 1A and B, miR-379-5p expression was significantly decreased in the cells treated with high glucose in a dose- and time-dependent manner, and its expression was approximately one third of that of the control following stimulation of the cells with 30 mM glucose for 48 h.

Mesangial cell proliferation and the accumulation of ECM components are important pathological features of DN (17). In this study, to explore the effects of miR-379-5p on cell proliferation and the accumulation of ECM components, the MMCs were transfected with miR-379-5p mimics. The results revealed that miR-379-5p expression was markedly increased in the MMCs transfected with miR-379-5p mimics (Fig. 1C). The results of CCK-8 assay also revealed that HG stimulation markedly promoted the viability of the MMCs. However, the promoting effects of HG stimulation on cell viability were reversed following transfection of the MMCs with miR-379-5p mimics (Fig. 2A). The results of EdU assays yielded similar conclusions, in that transfection with miR-379-5p mimics attenuated the effects of HG stimulation on cell proliferation (Fig. 2B). Furthermore, cell cycle analysis indicated that there was a decrease in the percentage of cells in the G₀/G₁ phase, as well as an increase in the percentage of cells in the S phase in the HG group compared with the control group. MMCs transfected with miR-379-5p mimics exhibited an increase in the percentage of cells in the G₀/G₁ phase and a decrease in the percentage of cells in the S phase compared with the HG group (Fig. 2C). This suggested that the overexpression of miR-379-5p prevented MMCs cell cycle progression.

p21, a cyclin-dependent kinase inhibitor, almost inhibits all cyclin/CDK complexes. The expression level of p21 and cyclin D1 can reflect the proliferation of MMCs (18). In this study, the results of western blot analysis revealed that transfection with miR-379-5p mimics attenuated the decrease and increase in the expression level of p21 and cyclin D1, respectively induced by HG stimulation (Fig. 2D). The cell cycle analysis and the related protein experimental data suggested that the overexpression of miR-379-5p suppressed the proliferation of MMCs following HG stimulation. Tubulointerstitial fibrosis is always involved with the increased deposition of ECM proteins, such as collagens and FN (19,20). In this study, we detected Col4 and FN expression by western blot analysis. As shown in Fig. 2E, Col4 and FN expression levels were markedly promoted in the HG group and the mimics-NC group compared to the control group, and decreased in the mimics group compared to the mimics-NC group. Taken together, these results indicated that miR-379-5p suppressed the proliferation and accumulation of ECM components in MMCs.

The TargetScan database was used to identify the targets of miR-379-5p. It was suggested that miR-379-5p had a conserved binding site in the 3′ UTR of LIN28B (Fig. 3A). To verify the association between miR-379-5p and LIN28B, luciferase reporter assays were performed. The results revealed that the luciferase activity was significantly decreased in the MMCs co-transfected with miR-379-5p mimics and the wild-type 3′ UTR reporter gene other than the mutated 3′ UTR reporter gene, indicating that miR-379-5p directly binds to the 3′ UTR of LIN28B (Fig. 3B).

LIN28B, a RNA-binding protein belongs to LIN28 family, mediates diverse biological functions (21). In this study, to examine the regulatory association between miR-379-5p and LIN28B, the expression levels of LIN28B and let-7b were detected in MMCs transfected with miR-379-5p mimics. As shown in Fig. 3C, the overexpression of miR-379-5p markedly suppressed the LIN28B expression level, which was induced by HG. The results of western blot analysis also revealed that miR-379-5p inactivated LIN28B expression in a HG-independent manner (Fig. 3D).

Previously, Kim et al demonstrated that LIN28B increased the expression of ECM proteins, such as Col1a2 and Col4a1, by decreasing let-7 levels (22). In this study, firstly, the transfection efficiency of miR-379-5p inhibitor was evaluated by RT-qPCR. The results revealed that the cells transfected with miR-379-5p inhibitor exhibited lower levels of miR-379-5p expression compared with the negative control group, indicating that miR-379-5p inhibitor was transfected into the cells successfully (Fig. 4A). To explore whether miR-379-5p suppresses the proliferation and accumulation of ECM components by regulating the LIN28/let-7 axis, a si-LIN28B vector was constructed and transfected into the MMCs (Fig. 4B and C). Transfection with si-LIN28B decreased LIN28B expression; however, miR-379-5p inhibitor partly reversed the decrease in LIN28B induced by si-LIN28B in the cells treated with HG (Fig. 4B and C). CCK-8 assay was performed in the HG group, si-NC group, inhibitors-NC group, si-LIN28B group and si-LIN28B + miR-379-5p inhibitors group to examine the effects of LIN28B on MMC viability. The results revealed that the knockdown of LIN28B suppressed MMC viability (Fig. 4D). Transfection with si-LIN28B decreased the mRNA expression levels of Col1 and Col4, and increased the let-7 expression level, as shown by RT-qPCR (Fig. 4E). Western blot analysis for Col-1 and Col-4 revealed that si-LIN28B decreased the accumulation of the ECM components, Col-1 and Col-4, compared to si-NC group (Fig. 4F). However, MMCs co-transfected with miR-379-5p inhibitors and si-LIN28B exhibited a higher proliferation rate and a greater accumulation of ECM components compared to the corresponding si-LIN28B group (Fig. 4D-F). Taken together, these results suggested that miR-379-5p suppressed the proliferation and accumulation of ECM components by regulating the LIN28/let-7 axis.

To examine the effects of miR-379-5p on renal fibrosis in DN, we used type 2 diabetic db/db mice for research in vivo with db/m mice as a normal control. miR-379-5p expression levels in the kidney tissues were significantly downregulated in the db/db mice, as detected by RT-qPCR and in situ hybridization assays. miR-379-5p agomir injection markedly promoted miR-379-5p expression (Fig. 5A and B). Consequently, LIN28B expression was upregulated in the kidney tissues of the db/db mice and downregulated after the injection of miR-379-5p agomir (Fig. 5C-E). Moreover, glomerular hypertrophy with an increased glomerular area was observed in the db/db mice. However, miR-379-5p agomir attenuated glomerular hypertrophy. As is well-known, the earliest pathological characteristic of DN is glomerular hypertrophy, which is mainly caused by glomerular MC proliferation and ECM accumulation. In this study, we wished to determine the role of the miR-379-5p/LIN28/let-7 axis in glomerular MCs during DN. Physiological markers, such as urine protein and blood glucose were also measured. It was found that urine protein levels were significantly increased in the db/db mice, and urine protein levels were decreased in the miR-379-5p agomir group (Fig. 5F).

In order to examine the effects of miR-379-5p on renal fibrosis in DN, some pathological markers, such as PAS and Masson's trichrome staining were used (Fig. 6A). The results indicated that miR-379-5p alleviated pathological changes, such as glomerular hypertrophy, mesangial amplification and renal fibrosis in the diabetic mice. Furthermore, mesangial cell proliferation detected by Ki-67 staining is presented in Fig. 6B, and ECM accumulation was measured by RT-qPCR and western blot analysis for let-7b, Col-4 and Col-1 expression (Fig. 6C and D). The results revealed that miR-379-5p agomir decreased mesangial cell proliferation and accumulation of ECM components that were increased in the db/db mice, which was consistent with the data obtained from the in vitro assays.