The lentivirus encoding miR-200a mimics or its negative control was purchased from the GenePharma (Shanghai, China). Recombinant human TGF-β1 was provided by the MedChemExpress (China). miR-200a mimics and the negative control (mimics NC), and miR-200a inhibitor and the negative control (inhibitor NC) were synthetized by the RiBoBio Co. (Guangzhou, China). Masson trichrome staining kit, DAB reagent and hematoxylin were obtained from the Solarbio (Beijing, China). Primary antibodies and secondary antibodies were purchased from Abcam (USA). Lipofectamine 3000, TRIzol reagent, SYBR Premix Ex Taq™ II assay and RIPA lysis buffer were obtained from the Invitrogen (USA). RevertAid™ First Strand cDNA Synthesis kit and the kit for miRNA-specific TaqMan MicroRNA assay were obtained from the Thermo Fisher Scientific (Waltham, MA, USA).

For carrying out animal experiment, healthy Wistar rats (male, weighting at about 180g, and aging at 6–8 weeks) were obtained from the Shanghai Experimental Animal Center (Shanghai, China). All rats were raised in accordance with standard conditions, including but not limit to free food and water, constant temperature at 22°C and 12h cycle of light–dark. At one week after acclimatization, rats were randomly divided into 7 groups (n=6): Sham, UUO-7d, UUO-14d, Sham+mimics NC, Sham+miR-200a mimics, UUO+mimics NC and UUO+miR-200a mimics. UUO surgery was carried out according to previous study.17 In brief, after anaesthetization with pentobarbital sodium (1%), the left ureter was uncovered and subsequent ligated at two points. The rats in Sham group were anaesthetized and exposed the left ureter, but without ligation. The rats in Sham+mimics NC, Sham+miR-200a mimics, UUO+mimics NC, and UUO+miR-200a mimics groups were injected with the lentivirus (1.0×1012vg) encoding miR-200a mimics or mimics negative control through tail vein at one week after UUO surgery. At the 14 days after operation, rats were euthanatized, and then renal tissues were collected for next study. All animal procedures were approved by the Committee of Animal Use and Care of Zhuzhou Central Hospital. All animal experiments were carried out strictly in accordance with the Guideline for Animal Research.

In order to analyze the fibrotic levels in renal tissues, the tissues were embedded with paraffin, and then were cut into 4μm-thick slices. After that, slices were soaked successively with dimethylbenzene I for 10min, dimethylbenzene II for 10min, ethanol absolute I for 1min, ethanol absolute II for 1min, 95% ethanol for 1min, 90% ethanol for 1min, 90% ethanol for 1min, and 85% ethanol for 1min, and were washed with double distilled water. Subsequently, slices were maintained with Masson trichrome staining according to the manufacture's protocol.

The expression of α-SMA in the renal tissues of the rats experienced UUO or without was measured by immunohistochemistry assay. The 4μm-thick slices also were soaked successively with dimethylbenzene and ethanol. After that, the slices were maintained successively with 3% H2O2 for 20min at room temperature, 0.01M trisodium citrate dihydrate for 10min at 95°C, 10% normal goat serum for 20min at room temperature, and anti-α-SMA primary antibody (1:200 dilution). Subsequently, the slices were soaked with the goat anti-rabbit antibody for 30min at 37°C. All slices were maintained with HRP-labeled streptavidin for 20min at 37°C. Finally, the slices were maintained with DAB reagent and hematoxylin. Five fields were randomly selected from each slices at ×200 magnification, and the integrated optical density in positive area was analyzed utilizing the Image J software (NIH, Bethesda, MD).

For carrying out cell experiments, human renal proximal tubular epithelial cells HK-2 were obtained from the China Center for Type Culture Collection. Cell culture was carried out in accordance with the previous study.3 Recombinant human TGF-β1 at a dosage of 5ng/ml was utilized to induce HK-2 cells for 12, 24, or 48h. In addition, to overexpress miR-200a, miR-200a mimics and the negative control (mimics NC) was synthesized. To downregulate miR-200a, a specific inhibitor of miR-200a and the negative control (inhibitor NC) was synthesized. To overexpress GAB1, the recombined pcDNA 3.1 vector expressing GAB1 was established, and the empty vector served as control. According to the different experiment aims, above mimics, inhibitor, GAB1 overexpression vector or the corresponding control were transfected into TGF-β1-induced HK-2 cells utilizing Lipofectamine 3000. 48h after TGF-β1 induction or transfection, cells were collected for next step.

qRT-PCR was carried out to indicate the relative levels of miR-200a, Collagen I, α-SMA, N-cadherin, E-cadherin and GAB1 genes. Total RNA was isolated from renal tissues or HK-2 cells in accordance with the protocol of TRIzol reagent. Then, cDNA was synthesized using the RevertAid™ First Strand cDNA Synthesis kit. After that, real-time PCR reactions for mRNA or miR-200a were accomplished utilizing the SYBR Premix Ex Taq™ II assay or a miRNA-specific TaqMan MicroRNA assay, respectively. The relative expression of mRNAs was normalized to GAPDH, and the relative expression of miR-200a was normalized to U6. Relative expression of mRNA and miR-200a was analyzed utilizing the 2−ΔΔCt method. The primers sequences as follows: α-SMA: 5′-AAAAGACAGCTACGTGGGTGA-3′ (F) and 5′-GCCATGTTC TATCGGGTACTTC-3′ (R); E-cadherin: 5′-CGAGAGCTACACGTTCACGG-3′ (F) and 5′-GGGTGTCGAGGGAAAAATAGG-3′ (R); U6: 5′-CTCGCTTCGGCAGCAC A-3′ (F) and 5′-AACGCTTCACGAATTTGCGT-3′ (R); GAPDH: 5′-CACCCACTCC TCCACCTTTG-3′ (F) and 5′-CCACCACCCTGTTGCTGTAG-3′ (R); Collagen I: 5′-G AGGGCCAAGACGAAGACATC-3′ (F) and 5′-CAGATCACGTCATCGCACAAC-3′ (R); N-cadherin: 5′-GCTTATCCTTGTGCTGATGTTT-3′ (F) and 5′-GTCTTCTTCTC CTCCACCTTCT-3′ (R); GAB1: 5′-GAAGTTGAAGCGTTATGCGTG-3′ (F) and 5′-T CCAGGACATCCGGGTCTC-3′ (R).

Western blot was carried out to determine the relative expression of Collagen I, α-SMA, N-cadherin, E-cadherin, GAB1, p-Ser9GSK3β, GSK3β, p-Ser675β-catenin and β-catenin. Total protein was extracted from renal tissues and HK-2 cells utilizing RIPA lysis buffer. An equal quality (25μg) proteins from each group were separated by 12% SDS-PAGE. Following transferring all proteins into PVDF membranes, the membranes were blocked with 5% non-fat milk, and subsequent were maintained with the primary antibodies (1:2000 dilution) for overnight at 4°C. Then, membranes were maintained the horseradish peroxidase-labeled secondary antibodies, and were then exposed to the enhanced chemiluminescence reagent for protein bands visualization. The relative levels of proteins were analyzed utilizing the Image J software.

The target genes of miR-200a were predicted through three databases, including TargetScan Human, starBase and miRDB. Three binding site sequence between miR-200a and GAB1 3′-UTR were predicted by starBase and verified utilizing luciferase reporter assay. Three wide type (WT-1, WT-2 and WT3) and three mutant (Mut-1, Mut-2 and Mut-3) human GAB1 3′-UTR sequences which containing the predicted binding sites for miR-200a were sub-cloned into pMIR vector. The GAB1 3′-UTR-WT-1, 3′-UTR-WT-2, or 3′-UTR-WT-1 and GAB1 3′-UTR-Mut-1, 3′-UTR-Mut-2 or 3′-UTR-Mut-3 were co-transfected with miR-200a mimics or mimics NC into 293T cells for 48h. Then, the luciferase activity of each group cells was examined utilizing a luciferase reporter system (Promega, WI, USA)

All data were showed as mean±standard deviation, and were analyzed using the GraphPad Prism software. Student's t-test or one-way ANOVA followed by the Bonferroni's multiple comparison test were utilized to the comparison between two groups or among multiple groups, respectively. P<0.05 was recognized as statistically significant.

It was uncovered that miR-200a is downregulated in the renal tissues of UUO rats, and may be participate in ureteral obstruction-induced renal fibrosis.12 Consistently, we also found decreased miR-200a in the renal tissues of UUO rats after 7 days or 14 days of surgery, and the decreasing following with the longer time after operation (Fig. 1A). Then, to dig the functions of miR-200a in the ureteral obstruction-induced renal fibrosis, we overexpressed it in normal and UUO rats utilizing the lentivirus which expressing miR-200a mimics. Masson staining results exhibited fibrotic renal tissues in UUO rats. Overexpression of miR-200a had no influence on the renal tissues in normal rats, but obviously diminished renal fibrosis in the rats experienced UUO (Fig. 1B). The importantly component of extracellular matrix, Collagen I and α-SMA, and E-cadherin are three major indicators for tissues fibrosis.18 Here, the data revealed that Collagen I and α-SMA mRNA levels were enlarged in UUO rats when compared to normal rats. Overexpression of miR-200a declined Collagen I and α-SMA mRNA levels in UUO rats, but had no effect on these gene expression in normal rats (Fig. 1C and D). Oppositely, E-cadherin mRNA level was decreased in the rats went through UUO. Overexpression of miR-200a obviously facilitated E-cadherin mRNA level in UUO rats, but not normal rats (Fig. 1E). Western blot data showed the consistent results with qRT-PCR assay. Collagen I and α-SMA were increased, but E-cadherin was decreased in the renal tissues of UUO rats. miR-200a overexpression dropped the expression of Collagen I and α-SMA, while facilitated E-cadherin expression in only UUO rats (Fig. 1F). The inhibition of miR-200a to upregulated α-SMA protein level in UUO rats also was proved by immunohistochemistry assay (Fig. 1G). Overall, miR-200a overexpression significantly alleviated the renal tissues in rats after UUO surgery.

Fig. 1.

The influence of miR-200a overexpression on the renal fibrosis of the rats experienced UUO. (A) The expression of miR-200a in renal tissues was analyzed utilizing qRT-PCR after the 7 days and 14 days of UUO surgery. The normal and UUO rats were injected with the lentivirus expressing miR-200a mimics or mimics negative control at the 7 days post-UUO. Next, (B) the fibrotic level of renal tissues was analyzed through Masson staining after the 14 days of UUO rats. (C–E) The relative expression levels of Collagen I, α-SMA, and E-cadherin mRNA in the renal tissues of rats were analyzed using qRT-PCR assay. (F) The relative expression levels of Collagen I, α-SMA, and E-cadherin proteins were determined through Western blot. (G) The expression level of α-SMA in renal tissues of rats was also evaluated through immunohistochemistry assay.

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Through the miRDB, TargetScan Human, and starBase databases, we predicted that GAB1 was a downstream target of miR-200a (Fig. 4E). We then inspected the mRNA and protein levels of GAB1 in the renal tissues of the rats experienced UUO. GAB1 gene and protein expression was enhanced in the renal tissues of the rats experienced UUO, and the expression of them positively associated with the time after UUO surgery (Fig. 2A and B). Crucially, miR-200a overexpression had no influence on the expression of GAB1 gene and protein in normal rats, but noticeably suppressed GAB1 gene and protein expression in UUO rats (Fig. 2C and D). Meantime, we determined the activation of Wnt/β-catenin signaling that has been proved to be activated in the renal tissues of rats following UUO surgery.19 Our results revealed that the phosphorylation level of GSK3β and β-catenin was increased in UUO rats. Overexpression of miR-200a does not change the level of p-GSK3β and p-β-catenin in normal rats, but markedly declined GSK3β and β-catenin phosphorylation in UUO rats (Fig. 2D). In summary, miR-200a overexpression limited GAB1 expression and Wnt/β-catenin signaling activation.

Fig. 4.

The regulation of miR-200a to GAB1 expression. (A, B) TGF-β1 (5ng/ml) was used to induce HK-2 cells for 0, 12, 24, and 48h, and then qRT-PCR was accomplished to analyze GAB1 gene expression and Western blot was accomplished to measure GAB1 protein expression. (C, D) The TGF-β1 (5ng/ml)-induced HK-2 cells were transfected with miR-200a mimics and mimics negative control for 48h. Next, qRT-PCR and Western blot were carried out to analyze the relative expression levels of GAB1 mRNA and proteins in the cells. (E) Three different binding sites sequences of miR-200a and GAB1 mRNA 3′-UTR was predicted utilizing starBase database. (F–H) Luciferase activity reporter assay was carried out to verify which binding sites truly effect the regulation of miR-200a to GAB1 expression.

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Fig. 2.

The regulation of miR-200a to GAB1 expression and activation of Wnt/β-catenin signaling. (A, B) The relative levels of GAB1 mRNA and protein in renal tissues was analyzed utilizing qRT-PCR and Western blot after the 7 days and 14 days of UUO surgery, respectively. After that, (C) the relative expression level of GAB1 mRNA in renal tissues of the rats, which injected with miR-200a mimics- or mimics negative control-expressed lentivirus, was determined at the 14 days post-UUO. (D) After the 14 days of UUO surgery, the relative expression levels of GAB1, GSK3β, and β-catenin proteins, and the phosphorylation of GSK3β and β-catenin in renal tissues were analyzed through Western blot.

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According to above data, we guessed that overexpression of miR-200a contributes to the improvement of ureteral obstruction-induced renal fibrosis. We further examined the influence of miR-200a overexpression on the TGF-β1-indued human renal proximal tubular epithelial (HK-2) cells EMT considered as a pivotal change in the development of fibrosis. Our results showed that the level of miR-200a was declined in the TGF-β1-induced HK-2 cells in a time-dependent manner (Fig. 3A). However, TGF-β1-induced downregulating in miR-200a expression was markedly recused by miR-200a mimics transfection (Fig. 3B). In addition, our results corroborated that the gene expression of ECM-related proteins, Collagen I and α-SMA, was facilitated in TGF-β1-indued HK-2 cells, but it was reversed by miR-200a overexpression (Fig. 3C and D). The gene expression of epithelial marker E-cadherin was suppressed, but the gene expression of mesenchymal marker N-cadherin was facilitated in the HK-2 cells treated with TGF-β1, however, there were partly recused by miR-200a expression (Fig. 3E and F). The western blotting assay also confirmed that TGF-β1 treatment resulted in an increasing in Collagen I, α-SMA, and N-cadherin protein expression, and decreasing in E-cadherin protein expression. Whereas, overexpression of miR-200a notably suppressed Collagen I, α-SMA, and N-cadherin protein expression, and enhanced the protein expression of E-cadherin in the HK-2 cells treated with TGF-β1 (Fig. 3G). All in all, miR-200a overexpression effectively alleviated TGF-β1-induced EMT and ECM deposition in HK-2 cells.

Fig. 3.

The regulation of miR-200a to EMT and ECM deposition in HK-2 cells caused by TGF-β1. (A) 5ng/ml of TGF-β1 was utilized to induce HK-2 cells for 0, 12, 24, and 48h. Then, the relative level of miR-200a in the cells was analyzed by qRT-PCR. After that, TGF-β1-induced HK-2 cells were transfected with miR-200a mimics or mimics negative control for 48h. (B) The relative level of miR-200a in the cells was examined by qRT-PCR. (C, D) The relative mRNA levels of ECM-related proteins, Collagen I and α-SMA, in the cells were analyzed by qRT-PCR. (E, F) The relative gene expression levels of EMT-related markers, N-cadherin and E-cadherin, in the cells were examined by qRT-PCR. (G) Collagen I, α-SMA, N-cadherin, and E-cadherin proteins expression in the cells was measured by Western blot.

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To probe whether miR-200a regulates TGF-β1-induced EMT and ECM deposition via targeting GAB1, we analyzed the relationship between miR-200a and GAB1. Our data proved that GAB1 mRNA and protein levels was upregulated in the TGF-β1-induced HK-2 cells in a time-dependent manner (Fig. 4A and B). Nevertheless, the promotion of TGF-β1 to GAB1 mRNA and protein levels in HK-2 cells was limited by miR-200a overexpression through transfection of miR-200a mimics (Fig. 4C and D). Through the miRDB, TargetScan Human, and starBase databases, we conjectured GAB1 maybe a downstream objective of miR-200a. Based on the prediction, the three different binding sites between miR-200a and GAB1 mRNA 3′-UTR was shown in Fig. 4E. Subsequently, we verified the combination of miR-200a and GAB1 mRNA 3′-UTR through luciferase reporter assay. The results confirmed that miR-200a bound with GAB1 mRNA 3′-UTR through the first binding site (Fig. 4F–H). In conclusion, miR-200a inhibited GAB1 expression via binding GAB1 mRNA 3′-UTR region.

Furthermore, to probe whether miR-200a regulates TGF-β1-induced EMT and ECM deposition via targeting GAB1, we increased GAB1 in the HK-2 cells treated with TGF-β1 following miR-200a overexpression. The results revealed that miR-200a notably suppressed the gene and protein expression of GAB1 in the TGF-β1-induced HK-2 cell, which was recused by GAB1 overexpression (Fig. 5A and B). Importantly, the inhibition of miR-200a overexpression to Collagen I, N-cadherin, and α-SMA gene expression, and the promotion to E-cadherin gene expression were partly reversed by GAB1 increasing (Fig. 5C–F). The western blotting assay results also indicated that the suppression of miR-200a overexpression to Collagen I, α-SMA, and N-cadherin protein expression, and the promotion to E-cadherin protein expression were recused by GAB1 increasing in the HK-2 cells treated with TGF-β1 (Fig. 5G). Meantime, miR-200a overexpression obviously limited the phosphorylation of GSK3β and β-catenin in TGF-β1-induced HK-2 cells, which was reversed following GAB1 increasing (Fig. 5H). Overall, these data indicated that the inhibition of miR-200a to TGF-β1-induced EMT, ECM accumulation and activation of Wnt/β-catenin signaling in HK-2 cells was reversed by GAB1 increasing.

Fig. 5.

The regulation mechanism of miR-200a to TGF-β1-induced EMT and ECM deposition in HK-2 cells. TGF-β1-induced HK-2 cells were co-transfected miR-200a mimics with or without GAB1 overexpression vector. At 48h after transfection, (A) GAB1 gene expression was analyzed utilizing qRT-PCR. (B) Western blot was accomplished to ensure the expression level of GAB1 protein. (C–F) qRT-PCR was implemented to ensure the expression levels of Collagen I, N-cadherin, α-SMA, and E-cadherin genes in the cells. (G) The ECM-related proteins, Collagen and α-SMA, and the EMT-related markers, N-cadherin and E-cadherin, expression were confirmed utilizing Western blot. (H) The activation of Wnt/β-catenin signaling pathway was analyzed through examining the phosphorylation levels of GSK3β and β-catenin by Western blot.

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