Rat renal tubular epithelial cells and HEK 293T cells were obtained from the Shanghai Academy Sciences Cell Bank (Shanghai, China). Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (25 mmol/l) and low glucose (5 mmol/l), respectively (Gibco-BRL, Carlsbad, CA, USA). The low glucose group served as a control, while the high glucose group represented the environment of renal cells in a patient with diabetes. The media were supplemented with 10% fetal bovine serum (FBS; Gibco-BRL). All cells were grown in a humidified incubator in 5% CO₂ at 37°C.

TargetScan 4.1 (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de/) and miRNA.org (http://www.microrna.org/), GenBank (http://www.ncbi.nlm.nih.gov/gene/) online databases were used to predict the targets of miR-21. These three programs used a different algorithm to identify highly complementary sites of miRNA. SMAD7 was consistently identified as a target of miR-21 with all three programs (Fig. 1).

Luciferase reporter gene constructs containing either the wild-type or mutated full-length 3′ untranslated region (UTR) of SMAD7, were constructed using the luciferase reporter vector psi-CHECK2 (Promega Corporation, Madison, WI, USA). The following primers were used for site direct mutagenesis plasmid construction: Forward: 5′-CACACTTTAATGCGGTTCATTTTTCTAACTACAAAGGTTT-3′; and reverse: 5′-AGTTAGAAAAATGAACCGCATTAAAGTGTGAGCATGCTCA-3′ for MutSmad7. ATAAGCT was mutated to GCGGTTC. The cells were transiently cotransfected into 24-well plates using Lipofectamine™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Mutations to the seed region of miR-21 mimics, which can result in high levels of miR-21 expression, and miR-21 negative controls (NC) which have no function were compounded. These were cotransfected into HEKT cells with the WT or mutant 3′UTR SMAD7. Reporter assays were performed at 48 h posttransfection using the Dual-luciferase Reporter Assay system (E1910; Promega Corporation) according to manufacturer’s instructions. All experiments were performed in triplicate and the standard deviation was calculated.

Rat renal tubular epithelial cells were transfected with 1.2 μl lentivirus overexpressing miR-21 or empty virus (Shanghai Jikai Gene Chemical Co., Ltd, Shanghai, China). Flow cytometry (FACSVantage; BD Biosciences, San Jose, CA, USA) was used 36 h after transfection in order to assess transfection efficiencies. Total RNA was then extracted from the cells and the miR-21 levels were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Following transfection, the cells were seeded into 96-well plates at a density of 10,000 cells/well. Four plates were used in total, and were incubated for 0, 24, 48, or 72 h. The cellular proliferation was analyzed at each time point using an MTT Cell Proliferation Assay kit (Invitrogen Life Technologies) over four days.

Cells were collected following transfection for 72 h and total RNA was extracted using TRIzol™ reagent (Invitrogen Life Technologies). cDNA was synthesized from the extracted RNA using a First-strand cDNA Synthesis kit (Promega Corporation) according to the manufacturer’s instructions. The miRNA reverse transcription-PCR primers for miR-21, and endogenous control U6RT were purchased from Applied Biosystems (Applied Biosystems, Invitrogen Life Technologies). The primer sequences used in the experiment were designed as follows: Forward: 5′-ACACTCCAGCTGGGTAGCTTATCAGACTGAT-3′; and reverse: 5′-ACTGGTGTCGTGGAGTCG-3′ for Rno-mir-21. qPCR analysis was performed using an ABI PRISM^® 7500 Sequence Detection system (Applied Biosystems). The conditions for the PCR were as follows: 95°C for 5 min, 95°C for 15 sec and 62°C for 30 sec, for a total of 40 cycles. Each sample was run in triplicate. The comparative threshold cycle (CT) method was used to evaluate the relative gene expression.

Western blot analysis was performed according to standard protocols. Briefly, the cells were solubilized in lysis buffer (containing posphatase inhibitors, protease inhibitors and PMSF) and the protein was extracted using a Protein Extraction reagent (Sigma, St. Louis, MO, USA). Protein concentrations were determined using a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA). The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Then the membranes were blocked and incubated overnight at 4°C with a rabbit anti-SMAD7 antibody (Abcam, Cambridge, MA, USA). The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Abcam) at 37°C for two hours. Proteins were visualized by an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). When required, the membranes were stripped and reprobed with β-actin, as an internal loading control.

Statistical analyses were performed using a one-way analysis of variance or a Kruskal-Wallis non-parametric test, according to the Gaussian or non-Gaussian distribution of the data. P<0.05 was considered to indicate a statistically significant difference in miRNA expression profiling. All data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS software 17.00 (SPSS Inc., Chicago, IL, USA).

In order to determine whether SMAD7 was a predicted target of miR-21, the miRNA GenBank was used. Using the TargetScan software, it was predicted that SMAD7 was a target of miR-21. One binding site for miR-21 was identified in the 3′UTR of the SMAD7 gene, complementary to the 3′ region of miR-21. The predicted binding site was located at 1192–1198 and contained seven conservative target sites (Fig. 1).

To confirm whether SMAD7 could be regulated by miR-21 in vitro, wild-type and mutant plasmids expressing the 3′UTR region predicted to bind miR-21, were constructed. Then miR-21-mimics with a mutated seed region and miR-21-negative control (NC) were synthetized (Shanghai Jima Company, Shanghai, China). Subsequently, the two luciferase reporter vectors with miR-21 response were cotransfected into HEK 293T cells using a Dual-Luciferase Reporter Assay system. As shown in Fig. 2A, following transfection in the HEK 293T cells, a significant difference was observed in luciferase activity between the miR-21 and blank groups (P<0.05) and between the miR-21 and NC groups (P<0.01). These data showed that miR-21 could interact with the 3′UTR of SMAD7, which was not observed in the NC and blank groups (P>0.05). As shown in Fig. 2B, in HEK 293T cells cotransfected with the mutated 3′UTR rat SMAD7 gene, there was no statistical significance among the miR-21, blank and NC groups (P>0.05). These data indicate that the SMAD7 3′UTR could not interact with miR-21 when mutated. Therefore, these results confirm that the SMAD7 3′UTR may be regulated by miR-21.

To further confirm whether miR-21 directly targets the 3′UTR of SMAD7, lentiviral vectors overexpressing miR-21 and empty lentiviral vectors were constructed. The plasmids were transfected into rat renal tubular epithelial cells respectively (Fig. 3) and the transfection efficiency was analyzed using flow cytometry (Fig. 3E). The RNA and protein was extracted from renal tubular epithelial cells, 72 h post-transfection. The results of RT-qPCR demonstrated that the expression of miR-21 was higher in the lentivirus-transfected rat renal tubular epithelial cells (Fig. 4b) cultured in high glucose compared with untransfected cells cultured in high glucose (Fig. 4c). There was no difference between the transfected and untransfected cells in the low glucose groups (data not shown). By western blot analysis, it was shown that the protein expression of SMAD7 was increased in the low glucose groups. In the high glucose groups, the SMAD7 expression was lower compared with that in the low glucose group. When lentivirus expressing miR-21 and empty lentivirus were transfected the protein expression of SMAD7 was lower in the miR-expressing groups in high and low glucose conditions (Fig. 5) when compared with the cells transfected with empty lentivirus.

To investigate the effects of miR-21 on renal tubular epithelial cells, miR-21 overexpressing- and empty lentiviruses were transfected into the rat renal tubular epithelial cells and were cultured under high and low glucose conditions. Following transfection for 24, 48 and 72 h, it was detected that the cellular proliferation in the miR-21-transfected lentivirus group was significantly decreased compared with the empty lentivirus and untransfected groups (P<0.01). These data indicated that overexpression of miR-21 may inhibit rat renal tubular epithelial cellular proliferation (Fig. 6).