miR-543 regulates high glucose-induced fibrosis and autophagy in diabetic nephropathy by targeting TSPAN8

Eight-week-old male db/db mice (n=12) on C57BL/KsJ background and control C57BL/KsJ mice (n=12) were obtained from NBRI (Nanjing, China). Mice were maintained under a temperature of 21 ± 2 °C and humidity of 55 ± 2%, with a cycle of 12-h light/dark. Mice were allowed to diet and water freely. The db/db mice were commonly used as DN models and previous studies demonstrated that db/db mice could be considered as the early stage of DN at 8 weeks [18‐20]. Animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals at The First Affiliated Hospital of Bengbu Medical College and approved by the ethics committee of The First Affiliated Hospital of Bengbu Medical College. Euthanasia of mice was performed using pentobarbital solution (150mg/kg, i.p.).

Human HK2 cell line purchased from the American Type Culture Collection, ATCC) was cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) with 15% fetal bovine serum (FBS; Gibco, Grand Island, NY). The medium contained additional 100U/ml penicillin (Gibco, Grand Island, NY) and 100μg/ml streptomycin (Gibco, Grand Island, NY). Cells were cultured in an incubator with 5% CO₂ at 37°C. For normal growth condition, cells were cultured in medium containing D-glucose at 5.5 mmol/L glucose plus 19.5 mmol/L mannitol. For high glucose (HG) treatment, cells were maintained in medium with 25 mmol/L glucose.

Trizol reagent (Thermo Fisher Scientific) was used to extract RNA from tissues and cells according to the instructions. The extracted total RNA was dissolved in DEPC water and 5 μg of total RNA was used for reverse-transcription into cDNA using RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). The resulted cDNA was diluted to 50 ng/μL and analyzed in a 7500 Real Time PCR System (Applied Biosystems) using SYBR premix EX TAQ II kit (RR820A, Takara). The PCR cycling condition used: 95^oC 2 mins, 40 cycles of 95^oC 30 sec, 60^oC 30 sec and 72^oC 60 sec. Finally, the 2–∆∆Ct method was used to analyze the relative expression level and GAPDH/U6 was used as the internal reference gene. All primer sequences were synthesized and purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China):

miR-543-forward, 5’- CAGTGCTAAAACATTCGCGG-3’; miR-543- reverse,5’-TATGGTTGTTCACGACTCCTTCAC-3’; TSPAN8-forward, 5′- GAGUUUAAAUGCUGCGGUU -3′; TSPAN8- reverse: 5′- AACCGCAGCAUUUAAACUC-3′; GAPDH-forward, 5’-AGAGGCAGGGATGATGTTCTG-3’; GAPDH- reverse, 5’-GACTCATGACCACAGTCCATGC-3’; U6-forward, 5’-ACAGTAGTCTGCACATTGGTTA-3’.

For TSPAN8 overexpression, the cDNA sequence of TSPAN8 was cloned into pcDNA3.1 expression vector. miR-543 mimic and miR-543-inhibitor were purchased from Shanghai GenePharma. For dual luciferase assay, the wide-type (WT) or mutant 3′-untranslated regions (3′-UTRs) of TSPAN8 was cloned into the pmirGLO Dual-Luciferase Vector (Promega, Madison, WI).

Cell transfection was performed using Lipofectamine® 3000 reagent (Thermo Fisher Scientific, L3000001). In 6 well plate, 60% confluent cells were transfected with 100 nM of microRNA mimic or inhibitor or 6 ug of pcDNA3.1- TSPAN8 plasmid according to manufacturer’s instruction. Transfected cells were subjected to subsequent analysis 48 hours post-transfection.

48 hours after transfection, cells were seeded in to a 96 -well plate at a density of 1500 cell/well and cultured in a humidified cell culture incubator for 0, 24, 48, and 72 hours, respectively. Subsequently, 10 μL CCK8 reaction solution (Dojindo Laboratory, Japan) was added to the cell culture for 3-hour incubation in a humidified cell culture incubator. The light absorption value (OD value) in each condition was captured at 450nm wavelength on a Synergy H1 microplate reader.

To demonstrate the functional interaction between miR-543 and TSPAN8 mRNA, the sequence containing the wild type binding site and the sequence with mutated binding site in the 3’UTR of TSPAN8 mRNA were cloned into the PmirGLO vector (Promega). The reporter plasmid and Renilla luciferase (hRlucneo) control plasmid were co-transfected into cells with either miR-543 mimic or miR-NC using Lipofectamine 3000 reagent according to the manufacturer’s instructions. 48 h post transfection, the relative luciferase activities were measured using Dual-Luciferase Reporter Assay Kit (Promega, E1910) on a luminescence microplate reader (Infinite 200 PRO; Tecan). The relative firefly luciferase activity in the reporter plasmid was normalized to that of Renilla luciferase.

Western blotting was conducted as in a previous study [21]. Total protein was extracted from cells using RIPA lysis buffer containing protease inhibitor cocktail (Thermo Fisher Scientific 78429). Cells suspended in RIPA buffer were lysed on ice for 10 mins and lysed cells were centrifuged at 14000 rpm for 10 mins. The supernatant containing total protein lysate was quantified by a BCA Protein assay kit (Beyotime Biotechnology P0009; Shanghai, China). 20 ug of protein was used for SDS-PAGE electrophoresis. Separated protein in SDS_PAGE gel was transferred onto the PVDF membrane (BioRad 1620177, Irvine, CA, USA). After blocking with 5% skimmed milk for 1 hour, the membrane was then incubated with primary antibodies: anti-actin (1:2000, ab8227); fibronectin (1:1000, ab2413), collagen IV (1:1000, ab6586), TSPAN8(1:1000, ab 70007), LC3A/B (1:1000, ab128025), Beclin1 (1:1000, ab62557) and p62 (1:1000, ab91526) overnight. The membrane was washed 3 times with TBST for 5 minutes each. After wash, the membrane was further incubated with HRP-linked secondary antibody (1:3000; Cell signaling #7074, MA, USA) at room temperature for 1 hour. Then the membrane was washed 4 times with TBST and the protein bands were visualized using an enhanced chemiluminescence kit (ECL, Solarbio, Beijing, China) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States). The densitometry analysis was performed with Image J software (Bethesda, MD, USA).

RNAscope kit (Invitrogen, CA, United States) was used to perform RNA FISH according to manufacturer’s instructions. Briefly, the kidney tissues were fixed with 4% paraformaldehyde and embedded in paraffin. 5 μm sections were obtained by a microtome. After deparaffinization and hydration, the tissue sections were hybridized with miR-543 probe with Cy3 fluorescent dye (RiboBio Co. Ltd., Guangzhou, China) at 50°C in hybridization buffer for 3 hours, and the section was mounted onto a slide using the mounting media containing DAPI (Vector Lab, Inc., Burlingame, CA, United States). Fluorescent images were captured by Leica AM6000 microscope.

Starbase (http://​starbase.​sysu.​edu.​cn/​) online resources was used for bioinformatic prediction of the binding site between miR-543 and TSPAN8 mRNA analysis.

Statistical analyses were performed with SPSS 20.0 software (IBM SPSS, Armonk, NY, USA). The relationship between the expression of miR-543 and TSPAN8 was analyzed by the spearman correlation coefficient test. The statistical difference between two groups was compared using unpaired student’s t tests. Comparisons among multiple groups were analyzed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test for pairwise comparison. Comparisons of data at multiple time points were examined using two-way ANOVA. Data were reported as the mean ± standard deviation (SD). P < 0.05, P < 0.01 or P < 0.001 was considered as statistically significant.

We first examined the expression level of miR-543 and TSPAN8 in kidney tissue of DN mice and control mice by qRT-PCR. The results showed that miR-543 was significantly downregulated in DN group compared with the control group (Fig. 1A). We also performed RNA FISH to examine the localization of miR-543 in kidney, and miR-543 seemed to be expressed by different cells in both the cortex and medulla (Supplementary Fig.1) Meanwhile, the expression of TSPAN8 was significantly increased in kidney tissue of DN mice (Fig.1B). Spearman correlation coefficient test demonstrated that miR-543 expression level was negatively correlated with TSPAN8 expression in kidney tissues of DN mice (Fig.1C). These data suggest that both miR-543 and TSPAN8 are dysregulated in DN mouse model.

To investigate the potential role of miR-543 in DN, we applied miR-543 mimic in high glucose (HG)-induced HK2 cells as a cellular model of DN. HG-treated cells displayed a more elongated and fibroblast-like cell shape, which is reminiscent of fibrotic phenotype (Supplementary Fig.2). The efficiency of miR-543-mimic transfection was confirmed by qRT-PCR (Fig.2A). Compared to miR-NC transfection, miR-543 was significantly increased after miR-543 mimic transfection in HG-induced cells (Fig. 2A). CCK8 cell proliferation assays demonstrated that the HG treatment suppressed cell proliferation, which was further exacerbated by the overexpression of miR-543 (Fig.2B). Moreover, high-glucose treatment increased the expression of collagen IV (Col. IV) and fibronectin (FN), and miR-543 mimic further enhanced their expression (Fig. 2C). We also investigated the change of autophagy-related proteins, which showed that the ratio of LC3-II/LC3-I, Beclin-1 level decreased, but the level of P62 increased upon HG treatment (2D). The overexpression of miR-543 further augmented these changes. These results indicate that high glucose impairs cell proliferation, but promotes fibrosis and autophagy in HK2 cells. miR-543 acts as a promoting factor to further suppress HK-2 cell proliferation and autophagy, but promotes fibrosis in HG-induced HK2 cells.

To identify the downstream target gene, the potential binding site of miR-543 was predicted by starbase (http://​starbase.​sysu.​edu.​cn/​) and there is a putative binding site of miR-543 in the 3’UTR of TSPAN8 (Fig.3A). To confirm their functional interaction, we performed dual luciferase reporter assay with WT and mutated binding site, with the empty vector (luci-NC) as the negative control. We found that the presence of miR-543 mimic significantly suppressed the luciferase activity of WT reporter, which was not observed in the mutated reporter or the negative control vector (Fig.3B). Besides, the mRNA and protein levels of TSPAN8 were reduced after miR-543 mimic transfection, while miR-543 inhibitor increased TSPAN8 expression. (Fig. 3C-D). Together, these data suggest miR-543 negatively regulates TSPAN8 in HG-induced cells.

To explore the functional role of TSPAN8 in HG-induced cell model, we transfected HK2 cells with TSPAN8 overexpression vector. As shown in Fig.4A, the expression level of TSPAN8 was upregulated in HG-induced HK2 cells after transfecting with TSPAN8 vector. CCK8 assay demonstrated that high glucose could lead to suppression of cell proliferation and TSPAN8 overexpression partially rescued this effect (Fig.4B). In addition, the relative protein levels of LC3-II/LC3-I, Beclin-1 decreased, while fibronectin (FN), collagen type IV (Col. IV), P62 increased in HG-induced HK2 cells upon high glucose induction. TSPAN8 overexpression could partially reverse these effects (Fig.4C-D). These results indicate that TSPAN8 alleviates cell damages induced by HG through suppressing fibrosis and promoting autophagy.

Given the observation that miR-543 negatively regulates TSPAN8, we further attempted to investigate whether miR-543 mediates high glucose-induced cell damage by targeting TSPAN8. In HG-induced HK2 cells, the cell proliferation was greatly reduced by miR-543-mimic as compared with miR-543-NC and the effect of miR-543 mimic was abrogated by TSPAN8 overexpression (Fig. 5A). The proteins in fibrosis and autophagy were examined in HG-induced HK2 cells with miR-543-mimic and TSPAN8 overexpression. The results showed that miR-543 promoted the decrease of relative ratio of LC3-II/LC3-I and Beclin-1, and increased the levels of FN, Col. IV, and P62, suggesting that miR-543 promotes fibrosis and inhibits autophagy. However, TSPAN8 overexpression could reverse the effects of miR-543 in HG-induced HK2 cells (Fig.5B-C). Together, these data imply that miR-543 mediates high glucose-induced cell damage by targeting TSPAN8.