miR-34a regulates mesangial cell proliferation via the PDGFR-β/Ras-MAPK signaling pathway

The main pathological characteristic of glomerulonephritis is diffuse mesangial cell proliferation. MiR-34a is associated with the proliferation of various organs and cancer cells. However, the role of miR-34a in renal proliferation diseases is not clear. Therefore, this study aimed to elucidate the mechanism of miR-34a in the regulation of renal mesangial cell proliferation. The miR-34a expression level at different time points in an anti-Thy1 mesangial proliferative nephritis rat model was determined by qRT-PCR. The cell proliferation rate and cell cycle changes were measured in the in vitro cultured rat mesangial cells (RMCs). Our results suggested that miR-34a expression was negatively correlated with the degree of cell proliferation in the anti-Thy1 nephritis model. MiR-34a could extend the G0/G1 phase and block cell proliferation in RMCs. Dual-luciferase assay results showed that there were binding sites of miR-34a at 3′-UTR of platelet-derived growth factor receptor-β (PDGFR-β). MiR-34a can inhibit PDGFR-β protein expression at a post-transcriptional level, suppress Ras/MAPK signaling pathways, and down-regulate expression of cell cycle proteins at the G0/G1 phase, such as cyclin D1, CDK4/CDK6. In addition, miR-34a may also inhibit RMC proliferation by directly targeting cyclin E and CDK2. MiR-34a inhibits exogenous stimuli-induced proliferation of mesangial cells. Expression levels of phospho-PDGFR-β and phospho-MEK1 (an important downstream molecule in PDGFR-β-induced signaling pathway) were significantly increased in the anti-Thy-1 nephritis rat model. These results suggest that miR-34a may regulate RMC proliferation by directly inhibiting expressions of PDGFR-β, MEK1, and cell cycle proteins, cyclin E and CDK2.


Introduction
Mesangial proliferative glomerulonephritis (MsPGN) can be divided into IgA nephropathy and non-IgA nephropathy, which show diffuse hyperplasia of glomerular mesangial cells (GMCs) and different degrees of extracellular matrix (ECM) accumulation [1,2]. Inhibition of mesangial cell proliferation is an important treatment strategy in proliferating glomerular diseases [3].
Anti-Thy1 nephritis is a rat model of human mesangial proliferative glomerulonephritis, involving a combination Abstract The main pathological characteristic of glomerulonephritis is diffuse mesangial cell proliferation. MiR-34a is associated with the proliferation of various organs and cancer cells. However, the role of miR-34a in renal proliferation diseases is not clear. Therefore, this study aimed to elucidate the mechanism of miR-34a in the regulation of renal mesangial cell proliferation. The miR-34a expression level at different time points in an anti-Thy1 mesangial proliferative nephritis rat model was determined by qRT-PCR. The cell proliferation rate and cell cycle changes were measured in the in vitro cultured rat mesangial cells (RMCs). Our results suggested that miR-34a expression was negatively correlated with the degree of cell proliferation in the anti-Thy1 nephritis model. MiR-34a 1 3 of Thy1 antibody and Thy1 antigen in mesangial cells. Macromolecular immune complexes are formed in situ and deposited in the mesangial area, leading to mesangial cell proliferation and inflammation [4,5]. These inflammatory mediators in turn stimulate activation, shrinkage, and proliferation of mesangial cells; release a variety of inflammatory mediators and ECM components [6,7]; and aggravate inflammation, all of which result in glomerular damages, including damage to the molecular barrier and charge barrier of the filtration membrane, and proteinuria [8].
MicroRNAs (miRNAs) are 21-to 25-nucleotide (nt)long, single-stranded RNA molecules that serve as posttranscriptional regulators of gene expression in eukaryotes and that do not code for any protein [9][10][11]. miRNAs can regulate the expression of thousands of proteins by degrading target mRNA or inhibiting translation as a result of complementary matching between miRNAs and specific sites in target genes [12]. miRNAs are widely involved in a series of biological functions and are closely associated with human diseases, including kidney diseases [13].
miR-34 was first found in the worm Caenorhabditis elegans in 2001 [14]. In recent years, it has been shown that miR-34a is involved in tumor proliferation of neuroblastoma [15,16], colon cancer [17], uveal melanoma [18], brain tumors [19], and cervical cancer [20] through regulation of different target genes. However, the role of miR-34a in mesangial proliferative glomerulonephritis is unclear. We thus aimed to investigate the role of miR-34a in renal proliferation diseases.

Materials and methods
Anti-Thy1 nephritis animal model Male Wistar rats (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) weighing between 200 and 220 g were randomly allocated to the control and anti-Thy1.1 groups. Anti-Thy1.1 nephritis was induced by a single intravenous injection of a monoclonal anti-Thy1 antibody (2.5 mg/kg) produced by OX-7 cells. Controls were injected with an identical volume of normal saline. Anti-Thy1-treated animals were killed on days 3, 5, 7, 10, and 14 post-injection (n = 3 per time point), and the control animals were killed on day 0 (n = 3). All rats were provided a diet of standard laboratory chow and free access to water. The renal tissues were examined by routine periodic acid-Schiff staining.

Immunohistochemical analysis and evaluation
The renal tissues were fixed in formalin and embedded with paraffin. The histologic paraffin sections were cut to 3-to 4-μm thickness and mounted on poly-l-lysine-coated slides. Endogenous peroxidase was blocked with 3 % hydrogen peroxide. The sections were heated in a microwave oven for 10 min in sodium citrate buffer (pH 6.0), incubated with 1.5 % normal goat serum for 20 min, and incubated overnight with 1:100 diluted primary antibody (Ki-67). For a negative control, the sections were incubated with PBS. After removal of unbound primary antibody, the sections were incubated with a biotinylated secondary antibody for 60 min at room temperature. The sections were rinsed and incubated with avidin-biotinylated horseradish peroxidase (Vectastain Elite ABC Kit; Vector Laboratories, USA) for 60 min. Incubation with 3,3-diaminobenzidine tetrahydrochloride was performed for 10 min as a substrate chromogen solution to produce a brown color. Finally, the sections were counterstained with hematoxylin. Twenty glomeruli per section at each time point were evaluated under high-power light microscopy (×400) in a blinded fashion. The Ki67 labeling index was calculated as the number of Ki-67-positive cells to total glomerular cells.
Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) TaqMan miRNA assays were performed to quantify mature miR-34 levels. Briefly, cDNA was synthesized using Taqman RT reagents (Applied Biosystems, USA), followed by TaqMan-based quantitative PCR using specific mirVana

Dual-luciferase reporter assay
For reporter assays, RMCs were cultured to approximately 80 % confluence in a 24-well plate and then co-transfected with a dual-luciferase reporter plasmid (psiCHECK-PDGFR-β, or psiCHECK-cyclin E, or psiCHECK-CDK2) and miR-34a mimics for 48 h. The activities of firefly and Renilla luciferases were determined using the Dual-Luciferase Reporter Assay System (Promega cat. no. E1910), and firefly luciferase activity was normalized to that of Renilla luciferase.

Statistical analyses
Measurement data are expressed as mean ± SEM, unless otherwise stated. Statistical analyses were performed using the SPSS 15.0 software package (SPSS, Inc., USA). Comparison among groups was conducted with ANOVA. Statistical differences were evaluated with analysis of variance. A p value <0.05 denoted a statistically significant difference.

Results
Pathological changes in rat model of anti-Thy1 mesangial proliferative glomerulonephritis We injected Thy1 antibody into Wistar rats to create an anti-Thy1 mesangial proliferative glomerulonephritis rat model. Following injection of anti-Thy1 antibodies, partial complement-dependent mesangiolysis appeared on day 3; mesangial cell proliferation and ECM accumulation occurred on day 5 and peaked on day 7. On day 10, recovery from glomerular injury began to decrease, and ECM accumulation attenuated on day 14 (Fig. 1a). We also detected the expression changes of cell proliferation marker Ki-67 by immunohistochemistry (Fig. 1b, c). We found higher levels of Ki-67 at every time point during anti-Thy1 nephritis compared with the control, which suggests that the cell cycle remained active from days 3 to 10. Ki-67 increased on days 3 and 5, peaked on day 7, and decreased from days 10 to 14. This suggests that cell cycle activity increased from days 3 to 7 and subsequently gradually decreased from days 10 to 14 (p < 0.05) (Fig. 1b, c).
MiR-34a expression in the kidney tissues of anti-Thy1 nephritis rat model MiR-34a expression was detected in the kidney tissues of the anti-Thy1 nephritis rat model at various time points (days 3, 5, 7, 10, and 14) by real-time qPCR. Compared with the normal group, miR-34a expression gradually decreased as pathological changes progressed from day 3 (p < 0.05). Expression levels and pathologic outcomes became normal on day 14 (p < 0.05) (Fig. 2).
Effect of miR-34a on mesangial cell proliferation rate and cell cycle RMCs were transfected with miR-34a mimics (mimics group) or a miRNA-negative control (Con group), and the proliferation rate of miR-34a mimic-transfected cells was determined using the Cell Counting Kit-8 (Fig. 3a). Proliferation of RMCs was reduced at 24 h (p > 0.05), significantly reduced at 48 h (p < 0.05), and more obvious at 72 h (p < 0.01) in miR-34a mimics-transfected group, compared with the normal group and the miRNA-negative control group, indicating that miR-34a mimics influence RMC proliferation. Cellular DNA content was further assessed by flow cytometry. The results showed that miR-34a mimics markedly extended the G0/G1 phase and reduced the G2/M phase ( Fig. 3b; Table 4), compared with the normal group and the miRNA-negative control group (p < 0.05).
To further confirm the direct action of miR-34a, levels of PDGFR-β mRNA and protein were determined in the miR-34a mimics-transfected cells. There was no significant difference between miR-34a mimic-transfected cells and control cells in terms of PDGFR-β mRNA expression level (p > 0.05) (Fig. 4b). Western blot showed that protein expressions of PDGFR-β and phospho-PDGFR-β were significantly decreased in the miR-34a mimic-transfected cells compared with the control cells (p < 0.05) (Fig. 4c). These results indicate that PDGFR-β may be a target gene of miR-34a. Moreover, miR-34a likely inhibits PDGFR-β primarily through post-transcriptional regulation. At the same time, we detected protein expressions of PDGFR-β and phospho-PDGFR-β (activated PDGFR-β) in the anti-Thy-1 nephritis rat model. Compared with day 0, on day 7 (the most obvious pathological change), phospho-PDGFR-β was significantly increased (p < 0.05) (Fig. 4d), indicating that PDGFR-β plays a key regulative role in proliferation development in the anti-Thy-1 nephritis.
Influence of miR-34a on the molecules in the Ras/MAPK signaling pathway RMCs were transfected with miR-34a mimics or a miRnegative control, and expression levels of two key targets in the Ras/MAPK signaling pathways were determined. The total MEK1 protein level was significantly decreased (p < 0.05), but those of Raf1 and ERK1/2 were unchanged (p > 0.05) (Fig. 6a), indicating that MEK1 is the target gene of miR-34a, which has also been demonstrated by others [23]. Moreover, protein levels of K-ras, p-Raf1, p-MEK1, and p-ERK1/2 were significantly down-regulated (p < 0.05) (Fig. 6a). We also detected MEK1, which is the most important molecule in the RAS/MAPK signal pathway in the anti-Thy1 mesangial proliferative glomerulonephritis rat model. Compared with 0 days, phospho-MEK1 at 7 days was significantly increased (p < 0.05) (Fig. 6b).
The above results suggested that Ras/MAPK signaling pathway plays a key role in the development of rat mesangial proliferative glomerulonephritis.

Discussion
MsPGN is divided into IgA and non-IgA nephropathy, and the main pathological changes associated with this condition are diffuse proliferation of GMCs and different degrees of ECM accumulation [1,2,24]. IgA nephropathy Fig. 5 Expression of G0/G1 phase cell cycle-related proteins in RMCs after transfection with miR-34a mimics. a MiRNA expression levels of cyclin D1, cyclin E, CDK2, CDK4, CDk6, p27 kip1 were detected with qRT-PCR. *p < 0.05 vs. normal (normal group) and Con (miRNA-negative control group). b Proteins expression levels of cyclin D1, cyclin E, CDK2, CDK4, CDk6, p27 kip1 were detected with Western blot. Data are means of three separate experiments ± SD. a,b,c,d,e,f p < 0.05 vs. normal group and Con group, n = 3. Mimics represented miR-34a mimics. c Effect of miR-34a on expression of cyclin E. Data are means of three separate experiments ± SD. *p < 0.01 vs. Mock (psiCHECK-vector) and Con (psiCHECK-cyclin E + negative control). Mimics: psiCHECK-cyclin E + miR-34a mimics. d Effect of miR-34a on expression of CDK2. Data are means of three separate experiments ± SD. # p < 0.01 vs. Mock (psiCHECK-vector) and Con (psiCHECK-CDK2 + negative control). Mimics: psiCHECK-CDK2 + miR-34a mimics is the most common type of glomerulonephritis globally [25][26][27], and occurs mainly among Asians, especially the Japanese and Chinese [28][29][30][31][32][33][34][35][36]. A study of 13,519 Chinese renal biopsies found that primary MsPGN accounted for 70.88 % of all cases of glomerular diseases. IgA nephropathy comprised 45.26 % of the primary glomerular diseases, while non-IgA mesangial proliferative lesions comprised 25.62 % [37]. The risk factors associated with inflammatory reactions have not been determined. Mesangial cells are not only target cells of immune injury, but also proliferate under the influence of various stimulatory molecules, release inflammatory factors, and ECM components, and further promote kidney damage, ultimately leading to glomerular sclerosis and interstitial fibrosis. miR-34 was first discovered in C. elegans in 2001 [14] and is associated with a variety of organ and tumor hyperplasias [15][16][17][18][19][20]. Therefore, this study aimed to investigate the role of miR-34a in proliferative glomerulonephritis. We first established an anti-Thy1 MsPGN rat model. In the anti-Thy1 glomerulonephritis rat model, we detected miR-34a expression in kidney tissues at various time points and found that miR-34a level gradually decreased as proliferation increased, then returned to normal levels when mesangial proliferation normalized. This indicates that miR-34a likely plays a suppressive role in RMC proliferation.
MiRanda, TargetScanS, and PicTar were integrated into MiRGen online biology prediction software (http://www. diana.pcbi.upenn.edu/miRGen.html), which is the bioinformatics software used most commonly to predict miRNA target genes. MiRGen prediction results suggested that PDGFR-β may be a direct target gene of miR-34a.
PDGFR is a receptor protein of PDGF. PDGF is classified into four subtypes, of which PDGF-B and PDGF-D are involved in the regulation of cell proliferation through PDGFR-β [46]. PDGF and PDGFR-β binding activates the Ras/MAPK signaling pathway as well as many downstream target genes, such as cyclins D1 and E [1], which control cell proliferation. The selective PDGFR tyrosine blocker STI157 can block mesangial cell proliferation [47].
We found that the PDGFR-β protein level was markedly lower in the miR-34a-transfected RMCs, while the mRNA level did not change. The results of the dual-luciferase assay indicated that PDGFR-β is a direct target gene of miR-34a. We assumed that cyclin E and CDK2 were target genes of miR-34a. The results of the dual-luciferase assay confirmed this hypothesis. MiR-34a not only regulates PDGFR-β expression but also acts directly in conjunction with cyclin E/CDK2 to regulate the cell cycle and influence proliferation. Other studies have confirmed that cyclin D1 [21], CDK4 [9], and CDK6 [21,22] are target genes of miR-34a. In our study, there were no changes in the mRNA levels of cyclin D1, CDK2, or CDK6 while the proteins expression was significantly reduced after miR-34a transfection; however, the mRNA level of cyclin E was decreased in the miR-34a-transfected RMCs. Moreover, both p27 kip1 protein and mRNA levels were increased in the miR-34a-transfected group, which is consistent with the effect on mesangial cell proliferation [44,48].
To demonstrate that miR-34a modulates the cell cycle by directly inhibiting PDGFR-β, we used siPDGFR-β to inhibit the expression of PDGFR-β. The results showed that the changes in both cell proliferation and the cell cycle were identical to those in the miR-34a-transfected group after siPDGFR-β transfection. The cell cycle proteins also showed the same trend as in the miR-34a group. SiPDGFR-β and miR-34a exert similar influences on   Fig. 9 Proposed mechanisms by which miR-34a regulates mesangial cell proliferation