The targets of miR-499a-5p were predicted by starBase [21] under the parameters of medium stringency in Degradome data, strict stringency in CLIP data, 5 programs in program number.

Human renal proximal tubule (HK-2) cells and renal cell adenocarcinoma cells (786-O) were commercially provided by the American Type Culture Collection (ATCC, Rockville, IN, United States). The HK-2 cells were cultured under a 5% CO₂ atmosphere at 37°C in Dulbecco’s modified Eagle medium (DMEM) containing% fetal bovine serum (FBS), streptomycin (100 mg/ml) and penicillin (100 units/mL, 1 ml). Upon reaching 60% confluence rate, cells were conducted to serum-starvation for 12 h. For HG treatment, HK-2 cells were cultured with 45 mmol/L of glucose for 24 h, while for control treatment, cells were cultured with 5.5 mmol/L of glucose for 24 h. Hyperoside at concentrations of 0, 10, 50, and 100 μmol/L were applied to treat HK-2 cells at room temperature for 6 h.

The short hairpin RNA targeting nuclear receptor-interacting protein 1 (NRIP1), termed sh-NRIP1, miR-499a-5p mimics and NC mimics were provided by Biomics Biotechnologies (Jiangsu, China). Before transfection, treated HK-2 cells (5 × 10⁵ cells/well) were inoculated into 6-well plates. Next, cells were transiently transfected with these oligonucleotides using Lipofectamine 3000 (Invitrogen, CA, United States). Forty-eight hours after transfection, cells were harvested.

Total RNA was extracted from treated HK-2 cells using the TRIzol reagent (Invitrogen). MiR-499a-5p was reverse transcribed into cDNA using a miRNA reverse transcription kit (Thermo Fisher Scientific). For NRIP1 reverse transcription, a PrimeScript reverse transcription reagent kit (Takara, Dalian, China) was applied. qPCR reactions were conducted on an ABI Prism 7500 RT PCR instrument (Applied Biosystems) using SYBR Premix Ex Taq (Takara). The thermocycling conditions were as follows: 50°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. 2^−ΔΔC_t method [22] was utilized to analyze relative expression of miR-499a-5p or NRIP1 normalized to U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences for RT-qPCR are shown in Table 1.

A CCK-8 assay was used to measure cell viability. HK-2 cells (5 × 10³ cell/well) in different groups: control, HG, HG + 1 μmol/L of hyperoside, HG + 10 μmol/L of hyperoside, HG + 50 μmol/L of hyperoside, and HG + 100 μmol/L of hyperoside were inoculated into 96-well plate. After incubation, 10 μL of CCK-8 solution (Dojindo, Kyushu, Japan) was added to culture plate and cells were incubated for another 1 h at 37°C. Optical density of each well was evaluated by a Microplate Reader at wavelength of 450 nm.

Cell apoptosis was investigated by a TUNEL assay kit. Treated HK-2 cells (4 × 10³ cell/well) were seeded on chamber slides. After 48 h of incubation, HK-2 cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and cultured with TUNEL reagents (Millipore; Merck KGaA, Darmstadt, Germany). TUNEL positive stained cells were observed by an optical microscope (Olympus, Tokyo, Japan).

An apoptosis kit (Solarbio, Beijing, China) was used to measure apoptosis. HK-2 cells by different treatments were transferred to for. Cells were washed and resuspended in 100 μL of 1 × Binding Buffer after 5 min of centrifugation in a centrifuge tube. Next, 5 μL of Annexin V-FITC solution was added to the cells and cells were incubated at 37°C for 15 min in the dark. Afterward, cells were washed again and re-suspended in 200 μL of 1 × Binding Buffer, and 5 μL of PI solution was added. Cell apoptosis rate was analyzed by flow cytometry within 1–2 h.

Western blotting was conducted according to a previous study [23]. The primary antibodies used in the study are listed as follows: anti-Bax (ab32503, 1/1000), anti-Bcl-2 (ab32124, 1/1000), anti-IL-10 (ab133575, 1/2000), anti-IL-6 (ab233706, 1/1000), anti-IL-1β (ab234437, 1/1000), and anti-GAPDH (ab9485, 1/2500).

HK-2 cells by different treatments were centrifuged at 1,000 × g for 20 min. The levels of IL-10, IL-6 and IL-1β in supernatants were examined by corresponding ELISA kits (ab185986, ab178013, ab214025, Abcam). The absorbance values at 450 nm were detected using a microplate reader (BioTek Instruments).

Binding sequences between miR-499a-5p and NRIP1 3′ untranslated region (UTR) were predicted from starBase. The HK-2 cells were plated into 24 well plates and reached 80% confluency. Next, wide-type (Wt) and mutant-type (Mut) NRIP1 3′ UTR were inserted into pmirGLO vectors (Invitrogen) to generate pmirGLO-NRIP1-Wt and pmirGLO-NRIP1-Mut plasmids. Afterward, these vectors were cotransfected with miR-499a-5p inhibitor or NC inhibitor into HK-2 cells. After 48 h, the luciferase activities were measured using a Lucifer Reporter Assay System (Promega, Madison, WI, United States) and were normalized to Renilla luciferase activity.

Biotin-labeled miR-499a-5p-Wt and miR-499a-5p-Mut were synthesized by RiboBio. The cells were treated with 50 nM of biotin-labeled NC, miR-499a-5p-Wt and miR-499a-5p-Mut for 48 h. Next, cells were rinsed by PBS, and cultured with RIPA lysis buffer for 10 min. Subsequently, cells were cultured at 4°C for 3 h. The beads were washed with precooled lysis buffer twice, low salt buffer 3 times and high salt buffer once. TRIzol was used to purify the binding RNA. Finally, RT-qPCR revealed the relative enrichment of NRIP1.

The data obtained from ≥3 independent experiments are shown as mean ± the standard deviation. Statistical significance of difference was analyzed using Student's t-test (for comparisons between 2 groups) and analysis of variance followed by Tukey’s post hoc test (for comparisons more than 2 groups). p-values less than 0.05 were statistically significant. Statistical analyses were conducted using SPSS 22.0 (IBM, Armonk, NY, United States).

First, viability of HK-2 cells under control condition, HG condition, and HG combined hyperoside condition was measured. As revealed in Figure 1A, cell viability was reduced by HG treatment. The inhibitory effects of HG on cell viability were rescued by hyperoside in a dose dependent way. We used 50 nm of hyperoside in the following assays. Data from TUNEL assays ( Figure 1B) and flow cytometry analysis (Figure 1C ) indicated that cell apoptosis was induced by HG treatment and was reduced by hyperoside. Western blot analysis revealed that increased Bax expression and decreased Bcl-2 expression induced by HG were rescued by hyperoside (Figure 1D). Furthermore, protein levels of IL-10, IL-6 and IL-1β in HG-treated HK-2 cells were measured. HG-induced upregulation of IL-6 and IL-1β protein levels and downregulation of IL-10 protein levels, and hyperoside rescued the influence of HG treatment on IL-10, IL-6 and IL-1β (Figure 1E). Finally, concentrations of these proinflammatory cytokines were detected by ELISA kits. The results indicated that hyperoside reversed the stimulating influence of HG on inflammatory response of HK-2 cells (Figure 1F).

Next, we identified that miR-499a-5p was downregulated by HG and was further dose-dependently upregulated by hyperoside in HK-2 cells (Figure 2A). RT-qPCR was applied to detect the knockdown efficacy of miR-499a-5p in HG treated HK-2 cells. Data in Figure 2B suggested that miR-499a-5p expression was effectively knocked down by miR-499a-5p inhibitor. MiR-499a-5p inhibitor rescued the antiapoptotic effects of hyperoside on HG-treated HK-2 cells (Figures 2C–E). Results from Western blot analysis (Figure 2F) and ELISA (Figure 2G) showed that miR-499a-5p inhibition rescued the increase of IL-10 levels, and the decrease of IL-6 and IL-1β levels induced by hyperoside in HG treated HK-2 cells.

NRIP1 was identified as a downstream target of miR-499a-5p based on starbase prediction. HG resulted in upregulation of NRIP1, and hyperoside treatment dose-dependently reduced its expression in HK-2 cells ( Figure 3A). MiR-499a-5p inhibitor promoted NRIP1 mRNA and protein levels in HK-2 cells regardless of HG treatment (Figures 3B,C). The binding sequences of miR-499a-5p and NRIP1 were predicted from starBase and were shown in Figure 3D. Binding site of NRIP1 was mutated for the next luciferase reporter assay. As indicated in Figure 3E, transfection of miR-499a-5p inhibitor enhanced the luciferase activity of pmirGLO-NRIP1-Wt plasmids and had no significance influence on that of pmirGLO-NRIP1-Mut plasmids in HK-2 cells regardless of HG treatment. Moreover, data of RNA pulldown assay showed that NRIP1 was abundantly enriched in products pulled down by bio-miR-499a-5p Wt, compared with that pulled down by bio-miR-499a-5p Mut, indicating the interaction of miR-499a-5p and NRIP1 ( Figure 3F).

Finally, the rescue assays were conducted. Figures 4A–C revealed that NRIP1 inhibition rescued the proapoptotic influences of miR-499a-5p on HK-2 cells. The stimulating effects of silenced miR-499a-5p on protein levels of IL-10, IL-6 and IL-1β were rescued by downregulation of NRIP1 (Figures 4D,E). Hyperoside induced apoptosis of 786–0 cells, while neither miR-499a-5p nor NRIP1 had rescue effects on hyperoside induced apoptosis of 786–0 cells (Supplementary Figures S1A,B).