Contributed equally
Tumor necrosis factor (TNF)-α has been reported to be important in glomerulonephritis, which is closely associated with podocyte dysfunction and apoptosis. However, the precise mechanisms by which TNF-α expression are regulated remain unclear. The purpose of the present study was to investigate the role of microRNA (miR)-130a-3p/301a-3p in the post-transcriptional control of TNF-α expression and high glucose (HG)-induced podocyte dysfunction. Mice MPC5 podocytes were incubated with HG and transfected with miR-130a-3p/301a-3p mimics or inhibitors, reactive oxygen species (ROS) levels were measured by flow cytometry assay, and the mRNA and protein levels were assayed by using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The targeted genes were predicted by a bioinformatics algorithm and verified using a dual luciferase reporter assay. It was observed that miR-130a-3p/301a-3p was a novel regulator of TNF-α in mouse podocytes. miR-130a-3p/301a-3p mimics inhibited TNF-α 3′-untranslated region luciferase reporter activity, in addition to endogenous TNF-α protein expression. Furthermore, forced expression of miR-130a-3p or miR-301a-3p resulted in the downregulation of ROS and malondialdehyde (MDA) and the upregulation of superoxide dismutase (SOD) 1 in the presence of HG. Inhibition of TNF-α level prevented a remarkable reduction in SOD activity and a marked increase in ROS and MDA levels in HG-treated podocytes. Furthermore, TNF-α loss-of-function significantly reversed HG-induced podocyte apoptosis. These data demonstrated a novel up-stream role for miR-130a-3p/301a-3p in TNF-α-mediated podocyte dysfunction and apoptosis in the presence of HG.
Glomerular podocytes are highly differentiated cells that play a key role in maintaining the integrity of the glomerular filtration barrier (
MicroRNAs (miRs) are endogenous, noncoding, short, single-stranded RNAs (18–25 nucleotides) that are evolutionarily highly conserved, believed to regulate the translation of target messenger RNAs (mRNAs) by binding to its 3′-untranslated regions (3′-UTRs) and verified to play a role in controlling a variety of biological processes (
miR-130a-3p and miR-301a-3p belong to miR-130 and miR-301 family individually, which are mainly identified in mouse (
Conditionally immortalized mouse podocytes (MPC5) were obtained from Dr Peter MUNDEL (Mount Sinai School of Medicine, New York, USA) and maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) at 37°C in a humidified incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 5% CO2, 95% air atmosphere. All of the experiments were performed after 48 h incubation with RPMI-1640 medium containing high glucose (HG, 30 mM D-glucose) or normal glucose (NG, 5 mM D-glucose). Human embryonic kidney (HEK) 293 T cells (American Type Culture Collection, ATCC, Manassas, USA) were incubated in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) and supplemented with 10% FBS, 100 µg/ml streptomycin and 100 IU/ml penicillin (all of them from Sigma-Aldrich, St. Louis, MO, USA). All of the experiments were repeated with at least three different cell preparations in triplicate.
Total superoxide dismutase (SOD; cat. no: S0101) and malondialdehyde (MDA; cat. no: S0131) were determined using assay kits (Beyotime Institute of Biotechnology, Haimen, China), following the manufacturer's protocol.
The generation of ROS in the podocytes was evaluated using a fluorometry assay via the intracellular oxidation of dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich). The cells (4×105) were incubated in a 6-well plate for 48 h, following DCFH-DA (50 µg/ml) incubation for 30 min at 37, the cells were harvested and washed with PBS 2 times and finally added into 1 ml PBS, which were detected and analyzed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The fluorescent product 2′,7′-dichlorofluorescein (DCF) was detected at an emission wavelength of 530 nm and excitation wavelength of 485 nm, and the result was analyzed using the flow cytometry analysis software BD CellQuest (v.5.1; Becton Dickinson, San Jose, CA, USA).
Podocytes (1×106) were lysated by NP-40 buffer, and the supernatant was collected for assay. In brief, 20 µl of cell lysate incubated with anti-caspase-3 antibody (cat. no: sc-7272; dilution, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37°C for 1 h. The immunocomplexes were then incubated with peptide substrate (2 µl of 10 mM acetyl-Asp-Glu-Val-Asp-p-nitroanilide) in assay buffer (100 mM Hepes, pH 7.5, 20% v/v glycerol, 5 mM dithiothreitol, and 0.5 mM EDTA) for 2 h at 37°C. The release of p-nitroaniline was measured at 405 nm using an ELISA reader (MD SpectraMax M5; Molecular Devices, LLC, Sunnyvale, CA, USA).
The sequences of the miR-130-3p mimics (5′-CAGUGCAAUGUUAAAAGGGCAU-3′), mutant miR-130-3p (5′-CA
The potential binding site between miR-130a-3p/301a-3p and TNF-α was obtained using online predict software Targetscan (
si-TNF-α (AGATTGAGGTGAAATCTTC) was designed using siDesigner (
Total RNA in podocyte was extracted by TRIzol (Invitrogen) according to the manufacturer's protocol. The cDNA was synthesized by reverse transcription reactions with 2 µg of total RNA using moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. PCR reaction mixtures were contained 12.5 µl SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 1 µl cDNA, 300 nM of each primer, and DEPC H2O to a final volume of 25 µl, and then RT-qPCR was performed using the Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.). The Cq (quantification cycle fluorescence value) was calculated using SDS software, version 2.1 (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the relative expression levels of miRs and mRNA were calculated using the 2−ΔΔCq method (
Proteins were extracted with radio immunoprecipitation assay (RIPA) buffer (cat. no: P0013B; Beyotime Institute of Biotechnology) with protease inhibitors, and the concentrations were determined using the Bicinchoninic Acid Kit for Protein Determination (cat. no: BCA1-1KT; Sigma-Aldrich; Merck KGaA). 50 µg of protein for each sample was separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% non-fat dry milk at room temperature for 2 h, the membrances were incubated with the primary antibody of TNF-α (cat. no: sc-52746; dilution: 1:1,000), Bcl-2 (cat. no: sc-509; dilution: 1:1,000), BAX (cat. no: sc-70405; dilution: 1:1,000) and β-actin (cat. no: sc-130301; dilution: 1:2,000; all of them from Santa Cruz Biotechnology) at room temperature for 2 h. β-actin signals were used to correct for unequal loading. Following three washes with TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (cat. no: sc-516102; dilution: 1:10,000; Santa Cruz Biotechnology) at room temperature for 2 h and visualized by chemiluminescence (Thermo Fisher Scientific, Inc.). Signals were analyzed with Quantity One® software version 4.5 (Bio Rad Laboratories, Inc.).
Data were presented as the mean ± standard deviation (SD) for each group. All statistical analyses were performed using PRISM v5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Inter-group differences were analyzed by one-way analysis of variance, followed by a post hoc Tukey test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
Our preliminary analysis indicated that miR-130a-3p/301a-3p and TNF-α were closely related to HG-induced cell dysfunction. Thus, for the first steps we focused our experiments on the expression of miR-130a-3p/301a-3p and TNF-α in podocytes exposure to HG. The findings demonstrated that both miR-130a-3p and miR-301a-3p were significantly down-regulated in HG-incubated podocytes compared with control group (
To investigate whether TNF-α was a direct target of miR-130a-3p/301a-3p, the online predict software Targetscan and miRanda were used for prediction. The results demonstrated that TNF-α was a direct target gene for both miR-130a-3p and miR-301a-3p that share the same 3′-UTR of target gene (
To confirm this, either the WT sequence of TNF-α or its mutant-type (MUT) sequence was transfected into the luciferase-reporter plasmids, and then the reporters were co-transfected with mimics, mutant-mimics, antisense or mutant-antisense of miR-130a-3p and miR-301a-3p into 293 T cells, and the levels of luciferase enzyme activity were measured. As shown in
To further determine whether TNF-α protein expression was regulated by miR-130a-3p or miR-301a-3p, the MPC5 podocytes were transfected with mimics, mutant-mimics, antisense or mutant-antisense of miR-130a-3p and miR-301a-3p, respectively, and the protein quantification assay of TNF-α was performed by western blotting. Our results showed that miR-130a-3p or miR-301a-3p mimics significantly reduced the protein expression of TNF-α. On the contrary, miR-130a-3p or miR-301a-3p antisense markedly increased TNF-α protein expression compared with corresponding control group (
Oxidative stress occurs when ROS affect the balance between oxidative pressure and antioxidant defense (
We have shown that downregulation of miR-130a-3p/−301a-3p is accompanied by upregulation of TNF-α in HG-treated MPC5 podocytes. TNF-α is a cognate target for miR-130a-3p and miR-301a-3p. It is conceivable that TNF-α mediates HG-induced MPC5 podocytes dysfunction. TNF-α si-RNA was designed to confirm the hypothesis. We had confirmed that podocytes transfected with si-TNF-α or treated with TNF-α inhibitor could significantly reverse HG-induced the up-regulation of ROS (
This study demonstrated that miR-130a-3p/301a-3p was a novel regulator of TNF-α in mouse podocytes. Results of our bioinformatic analysis and luciferase reporter assay showed that miR-130a-3p/301a-3p directly interacted with the 3′-UTR of TNF-α. In MPC5 podocytes, the miR-130a-3p or miR-301a-3p mimic decreased the TNF-α protein expression, which was rescued by the miR-130a-3p or miR-301a-3p inhibitor. These results provide strong evidence that TNF-α was a direct target gene of miR-130a-3p/301a-3p. Here we showed forced expression of miR-130a-3p or miR-301a-3p via transfection of its mimic in cultured podocytes resulted in the down-regulation of ROS and MDA and the up-regulation of SOD in the present of HG. Moreover, inhibition of TNF-α level also prevented a remarkable reduction in SOD activity and a dramatic increase in ROS and MDA levels in HG-treated podocytes. The combined results above strongly suggested that overexpressed miR-130a-3p/301a-3p could significantly ameliorate HG-induced MPC5 podocytes dysfunction, and the underlying mechanism was mediated, at least partially, through the suppression of TNF-α expression.
In our study, we found that miR-130a-3p and miR-301a-3p shared the identical seed site of TNF-α. However, there has been no relevant report about the functional similarity between miR-130a-3p and miR-301a-3p in mammals. Our findings indicated that miR-130a-3p and miR-301a-3p had played a similar role in HG-induced MPC5 podocytes dysfunction. A recent study shows that miR-130a-3p is aberrantly reduced in livers of a mouse model with nonalcoholic fibrosing steatohepatitis, and the overexpression of miR-130a-3p inhibits hepatic stellate cells (HSC) activation and proliferation and promotes HSC apoptosis (
Inflammatory reaction is tightly regulated by altering TNF-α (
Numerous studies have shown that TNF-α is a potential therapeutic target for chronic kidney disease (CKD) and glomerulonephritis (
Taken together, the data obtained in our study support the conclusion that miR-130a-3p/301a-3p targeting to TNF-α inhibits HG-induced oxidative stress and apoposis in podocytes, and suggest that a novel function for miR-130a-3p/301a-3p in podocytes injury-related renal disease. Our results are also important in providing new perspective for the understanding of the underlying molecular mechanisms in podocytes dysfunction and potential therapeutic target for glomerulonephritis.
This work was supported by the United Fund of the Department of Science and Technology in Guizhou Province of China (Grant no: LH-2016-7395).
Reciprocal changes of miR-130a-3p/301a-3p and TNF-α expression in HG-incubated MPC5 podocytes. (A) The levels of miR-130a-3p and miR-301a-3p were performed by qRT-PCR after 48 h incubation with high glucose (HG, 30 mM D-glucose) or normal glucose (NG, 5 mM D-glucose). The mRNA (B) and protein (C) expression of TNF-α were measured by qRT-PCR and western blotting, respectively, after treatment with HG or NG for 48 h. n=3 in each group, *P<0.05 vs. NG group.
(A) Bioinformatics prediction of TNF-α as a candidate target gene for miR-130a-3p/301a-3p. Schematic representation of the putative miR-130a-3p/301a-3p binding site in the 3′UTR of TNF-α was predicted by the Targetscan and miRanda, and miR-130a-3p and miR-301a-3p shared the identical seed site of TNF-α. (B) The conserved sequence of binding sites between miR-130a-3p/301a-3p and TNF-α was shown across various species.
(A, B) TNF-α was a direct target gene of miR-130a-3p/301a-3p. The 293 T cells were co-transfected with the WT and MUT 3′-UTR of TNF-α and miR-130a-3p or miR-301a-3p mimics, antisense or corresponding mutation sequence, and the luciferase activity assay was performed after 48 h transfection. (C, D) The podocytes were transfected with miR-130a-3p or miR-301a-3p mimics, antisense or corresponding mutation sequence, the protein expression of TNF-α was measured by western blotting after 48 h transfection. n=3 in each group, *P<0.05 vs. corresponding control group.
Overexpressed of miR-130a-3p and miR-301a-3p inhibited HG-induced ROS and oxidative stress in podocytes. The levels of (A) ROS, (B) SOD and (C) MDA were analyzed in podocytes with or without HG treatment, as well as transfected miR-130a-3p or miR-301a-3p mimics, antisense or corresponding mutation sequence. n = 3 in each group, *P<0.05 vs. corresponding control group; #P < 0.05 vs. HG group.
TNF-α loss-of-function alleviated HG-induced MPC5 podocytes dysfunction and apoptosis. The levels of (A) ROS, (B) SOD, (C) MDA and (D) caspase-3 were analyzed in podocytes with si-TNF-α transfection or TNF-α inhibitor treatment in the present or absence of HG. (E, F) The protein expression of Bcl-2 and BAX were measured by western blotting after 48 h treatment. n=3 in each group, *P<0.05 vs. NC group; #P<0.05 vs. HG group.