A total of 32 4-week-old male C57BL/KsJ-db/db mice with type II DM and an additional 32 4-week-old male db/m mice were purchased from the Animal Center of Nanjing Medical University (Nanjing, China) and included in the present study. Mice were maintained under conventional conditions with 12 h light/dark cycle at 20–22°C and were provided with standard chow and water ad libitum. Prior to sacrifice, mice underwent fasting for 12 h. The bilateral kidneys were then collected following laparotomy in each mouse; the connective tissues and blood vessels on the renal hilum were removed. Next, the renal specimens of one kidney from each bilateral pair were fixed in 10% neutral formalin at room temperature for 48 h, embedded in paraffin and sliced into 2-µm sections. Then, the tissue sections underwent H&E staining and were analyzed under a light microscope (magnification, ×200) to determine pathological changes within the renal tissues. Simultaneously, renal specimens of the other kidney of each bilateral pair were preserved in liquid nitrogen for RNA extraction. The present study as approved by the Ethical Committee of Taixing City Second People's Hospital.

Mouse renal mesangial cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China; cat. no., GNM21). The renal mesangial cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), and glucose at a concentration of 20 mmol/l, in an atmosphere containing 5% CO₂. After 24 h, the cells were collected for subsequent analysis.

The mmu-let-7a-5p inhibitors and mmu-let-7a-5p mimics oligonucleotides were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Renal mesangial cells treated with 0, 20 or 30 mmol/l glucose were transfected with 50 nM mmu-let-7a-5p inhibitors or mmu-let-7a-5p mimics mixed with Lipofectamine RNAi Max (Thermo Fisher Scientific, Inc.). Cells were then cultured at 37°C in DMEM supplemented with 10% FBS in an atmosphere containing 5% CO₂ for 48 h. Cells were harvested at 48 h for the subsequent analysis. The sequences of the oligonucleotides were: mmu-let-7a-5p mimics, 5′-UGAGGUAGUAGGUUGUAUAGUU-3′; mmu-let-7a-5p mimics NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; mmu-let-7a-5p inhibitors, 5′-ACUAUACAACCUACUACCUCA-3′; mmu-let-7a-5p inhibitors NC, 5′-CAGUACUUUUGUGUAGUACAA-3′.

Total RNA was isolated from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the reverse transcription was performed with the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) with the temperature of 37°C for 15 min and 85°C for 5 sec. RT-qPCR was conducted with a SYBR ExScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) on an ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were: Initial denaturation, 95°C for 30 sec; followed by 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 30 sec. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The relative expression of high-mobility group AT-hook 2 (HMGA2) in each sample was normalized to that of GAPDH using the 2^−ΔΔCq method (17). The expression of let-7a-5p was determined using the Hairpin-it™ miRNAs qPCR Quantitation kit (Shanghai GenePharma Co., Ltd.). U6 was applied for the normalization of miRNA expression. The sequences of the primers used were: let-7a-5p, forward 5′-GCCGCTGAGGTAGTAGGTTGTA-3′, reverse 5′-GTGCAGGGTCCGAGGT-3′; HMGA2, forward 5′-CAGCAGCAAGAACCAACCG-3′, reverse 5′-TGTTGTGGCCATTTCCTAGGT-3′, PI3K, forward 5′-GAAATCTCCTGGGATGTGTCGT-3′, reverse 5′-ATCTGGTGGCTCTCGGAGTAA-3′; AKT, forward 5′-GATGGAGGCCAGGGTACAAA-3′, reverse, 5′-GCAGCGACACCACAAAAATGA-3′; GAPDH, forward 5′-TCAACGGATTTGGTCGTATTG-3′, reverse, 5′-TGGGTGGAATCATATTGGAAC-3′; U6, forward 5′-AACGCTTCACGAATTTGCGT-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′.

Renal mesangial cells were plated at a density of 5,000 cells/well in 96-well plates. A Cell Counting Kit-8 (CCK-8) assay was performed at 48 h after transfection to determine cell viability using a CCK-8 proliferation assay kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocol.

For the analysis of apoptosis, 48 h after transfection or following treatment with the protein kinase B (AKT) inhibitor, MK2206 (10 µmol/l), cells were stained using a propidium iodide/Annexin V-fluorescein isothiocyanate apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Briefly, cells were collected, and 5 µl Annexin V-FITC and propidium iodide (PI) solutions were mixed with the cells at room temperature, and incubated on ice in the dark for 30 min. Then the apoptotic rate and cell cycle in each sample were analyzed with a BD FACSVerse flow cytometer (BD Biosciences) according to the manufacturer's protocol. The data was analyzed by FlowJo version 8.7 (FlowJo LLC, Ashland, OR, USA).

Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) and then concentration of the proteins were quantified by Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). Then SDS-PAGE was performed to separate the extracted proteins using 8% gel, and 30 µg protein loaded into each lane. Then, the proteins were transferred onto polyvinylidene fluoride membranes, which were then blocked with 5% non-fat milk at room temperature for 1 h and incubated with primary antibodies [anti-phosphoinositide 3-kinase (PI3K, ab151549; 1:1,000), anti-AKT (ab8805; 1:1,000), anti-phosphorylated (p)-AKT (ab38449; 1:1,000), anti-HMGA2 (ab97276; 1:500) and anti-GAPDH (ab9485; 1:2,000) all from Abcam, Cambridge, MA, USA] overnight at 4°C. On day 2, the membranes were washed and incubated with a HRP-conjugated secondary antibody (A0208; 1:5,000; Beyotime Institute of Biotechnology) at room temperature for 45 min, and then washed and incubated with BeyoECL Plus (Beyotime Institute of Biotechnology) at room temperature for 1 min. The bands were visualized using a ChemiDoc™ XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was applied as the internal control. Densitometric analysis was performed with Image Lab software version 3.1.1 (Bio-Rad Laboratories, Inc.).

The prediction of target gene of mmu-let-7a-5p was performed using the online bioinformatic tool Target Scan (http://www.targetscan.org) (18) and miRanda (http://www.microrna.org/) (19) as previously described (18,19). HMGA2 revealed a potential binding site for mmu-let-7a-5p in the 3′-UTR of HMGA2.

The wild-type HMGA2 3′UTR (HMGA2-3′UTR) and mutant HMGA2 3′UTR (HMGA2-MUT) regions containing the mmu-let-7a-5p binding site were synthesized and cloned into the p-MIR-reporter plasmid (Thermo Fisher Scientific, Inc.). 293 cells (cat. no., GNHu43; Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) were then seeded onto 12-well plates at a density of 1×10⁵ cells/well and transfected with HMGA2-3′UTR or HMGA2-MUT, in addition to either let-7a-5p mimics or NC using the Lipofectamine RNAi Max kit. Cells were harvested 48 h after transfection and the luciferase activities of the experimental groups were detected using a dual-luciferase reporter system (Dual Luciferase Reporter Gene Assay kit, Beyotime Institute of Biotechnology). Renilla luciferase was used as the internal control.

All statistical analyses were performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation; a two-tailed independent samples t-test was performed for the comparison between two groups and one-way analysis of variance was applied for the comparison of multiple groups followed by a Dunnett's post-hoc test. Pearson correlation coefficient was used for the correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

Firstly, to explore the roles of let-7a-5p in DN, DN mice models were established. The pathological features of the kidney tissues of DN and the control mice are presented in Fig. 1A. The expression levels of let-7a-5p were examined using RT-qPCR. As demonstrated in Fig. 1B, let-7a-5p expression was significantly downregulated in the kidneys of DN mice at the mRNA level (P<0.05). In addition, online computational prediction tools (TargetScan and miRanda) were applied, which predicted HMGA2 as a target gene of let-7a-5p. It has been suggested that the overexpression of HMGA2 may promote the epithelial-to-mesenchymal transition of tubular epithelial cells (20), suggesting that HMGA2 may be involved in the pathogenesis of DN. Therefore, in the present study, the expression of HMGA2 in DN tissue and control was examined. It was observed that the expression of HMGA2 was significantly increased at the mRNA and protein levels, as detected by RT-qPCR and western blot analysis (Fig. 1B and C). Finally, the present study identified that the expression levels of let-7a-5p in DN mice were negatively correlated with that of HMGA2 (Fig. 1D; P=0.0088; r=−0.4555).

Renal mesangial cells were cultured under high-glucose conditions for 24 h in the present study; the effects of high-glucose conditions on the expression of let-7a-5p and HMGA2 were then examined by RT-qPCR. It was observed that high-glucose conditions induced significant decreases in the expression levels of let-7a-5p (Fig. 2A) and markedly increased the expression of HMGA2 (Fig. 2B), in a dose-dependent manner.

To additionally explore the roles of let-7a-5p in DN, renal mesangial cells were transfected with let-7a-5p mimics or inhibitors; the effects of let-7a-5p on the proliferation and apoptosis of renal mesangial cells under high-glucose conditions were examined using CCK-8 and flow cytometry analyses. As indicated in Fig. 3A, the expression of let-7a-5p was significantly upregulated in let-7a-5p mimic-transfected cells, and markedly downregulated in let-7a-5p inhibitor-transfected cells; in addition, transfection with let-7a-5p mimics induced a significant decrease in the proliferation and an increase in the apoptosis of renal mesangial cells cultured under high-glucose conditions; transfection with let-7a-5p inhibitors exhibited opposing effects (Fig. 3B-D; P<0.05).

To additionally explore the underlying mechanism of let-7a-5p in DN, the effects of let-7a-5p mimics or inhibitors on the expression of HMGA2, PI3K, AKT and p-AKT were examined. As demonstrated in Fig. 4, transfection with let-7a-5p mimics resulted in decreased expression levels of HMGA2 and inhibition of the PI3K/AKT signaling pathway, which was indicated by decreased expression levels of PI3K, AKT and p-AKT. Conversely, transfection with let-7a-5p inhibitors appeared to activate the PI3K-AKT signaling pathway.

As aforementioned, using the online computational prediction tools TargetScan and miRanda, HMGA2 was predicted as a target gene of let-7a-5p (Fig. 5A). Finally, a dual luciferase reporter assay was performed to investigate whether let-7a-5p may directly target HMGA2. As demonstrated in Fig. 5, transfection of let-7a-5p mimics induced significant decreases in the luciferase activities within wild-type HMGA2 3′-UTR (HMGA2-3′-UTR)-transfected 293 cells, while transfection with let-7a-5p mimics exhibited no significant effects on mutant HMGA2 3′-UTR (HMGA2-MUT)-transfected cells (Fig. 5B; P<0.01). These results indicated that HMGA2 is a direct target of let-7a-5p.