The uremic toxin p-cresyl sulfate induces proliferation and migration of clear cell renal cell carcinoma via microRNA-21/ HIF-1α axis signals

p-Cresyl sulfate (pCS), a uremic toxin, can cause renal damage and dysfunction. Studies suggest that renal dysfunction increases the prevalence of renal cancer. However, the effect of pCS on the proliferation and migration of renal cancer is unclear. Clear cell renal cell carcinoma (ccRCC) expresses mutant von Hippel-Lindau gene and is difficult to treat. Hypoxia-inducible factor-1α and 2-α (HIF-1α and HIF-2α) as well as microRNA-21 (miR-21) can regulate the proliferation and migration of ccRCC cells. However, the association between HIF-α and miR-21 in ccRCC remains unclear. Therefore, the effects of pCS on ccRCC cells were investigated for HIF-α and miR-21 signals. Our results showed that pCS induced overexpression of HIF-1α and promoted the proliferation and regulated epithelial-mesenchymal transition-related proteins, including E-cadherin, fibronectin, twist and vimentin in ccRCC cells. pCS treatment increased miR-21 expression. Specifically, inhibition of miR-21 blocked pCS-induced proliferation and migration. Taken together, the present results demonstrate that pCS directly induced the proliferation and migration of ccRCC cells through mechanisms involving miR-21/HIF-1α signaling pathways.

impaired; in patients with hemodialysis, protein-bound pCS is difficult remove by dialysis. pCS accumulates as renal function deteriorates, with 100-200 μM as the mean serum total levels of pCS observed in uremic patients 15 . Accumulation of pCS causes renal dysfunction and disease progression by inducing oxidative damage and endothelial dysfunction 16,17 . Previous studies found that renal dysfunction is related to the risk of renal cell carcinoma, particularly ccRCC 18,19 . pCS can induce proliferation and migration of rat aortic vascular smooth muscle cells 20 and epithelial-mesenchymal transition (EMT) in kidney fibrosis 21 . Based on these studies, we predicted that pCS influences proliferation, EMT, and migration in ccRCC. Therefore, the aim of this study was to determine the effect of pCS on the proliferation and migration of ccRCC cells and the related mechanisms.
HIF-1α and HIF-2α are transcription factors 22,23 . Most patients with ccRCC have high expression of HIF-1α 24 , and studies have shown that HIF-1α plays an important role in the proliferation of ccRCC 10,24,25 and regulates EMT in ccRCC and tubular epithelial cells 26,27 . Fibronectin, twist, vimentin, and E-cadherin are associated with EMT and are required for cell migration 28,29 . Additionally, inhibition of HIF-1α can reduce the migration of ccRCC cells 30 . These results suggest that HIF-1α plays an important role in the proliferation, EMT, and cell migration of ccRCC. HIF-2α also regulates cell proliferation and migration in ccRCC 7,31 . Therefore, the expression of HIF-α, EMT-related proteins, and migration were investigated in pCS-treated ccRCC cells in this study.
MicroRNAs, which are 19-25 nucleotides in length, can influence gene expression 32 . MicroRNAs can interact with the 3′-untranslated regions of mRNAs to cause mRNA degradation and inhibit translation 33 . MicroRNA-21 (miR-21) is highly expressed in various cancers 34 . Studies have indicated that miR-21 can induce cell proliferation and EMT in ccRCC 35,36 . MiR-21 can activate HIF-1α expression in various cells including prostate cancer, retinal pigment epithelia, cervical cancer, and human stem cells [37][38][39] , but its role in ccRCC remains unclear. Therefore, the miR-21/HIF-1α axis signals were studied in pCS-treated ccRCC cells to examine cell proliferation and migration.

Results
Effect of pCS on cell proliferation in dose-and time-dependent manner. Cells from two cell lines, ccRCC 786-O and A498, were treated with 20, 50, 100, 200, and 500 μM of pCS for 48 h. Cell proliferation was measured by the WST-1 assay and the optical density of the cell culture was determined. As shown in Fig. 1, cell proliferation by pCS treatment of both cells was increased significantly at 12 h for 786-O cells and 24 h for A498 cells. pCS-induced cell proliferation was time-dependent in both 786-O and A498 cells (Fig. 1a,b). Cells treated with control, 100 or 200 μM pCS for 48 h are shown in Fig. 1c. www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of pCS on HIF-1α, HIF-2α and VHL levels. HIF-1α and HIF-2α transcription factors can regulate the cell proliferation of renal cancer 5 . We investigated whether pCS-induced proliferation was related to HIF-1α and HIF-2α. 786-O and A498 cells were treated with 100 or 200 μM pCS for 5 days. HIF-1α, HIF-2α, and VHL levels and the ratios of their expressions are shown in Fig. 2. pCS-induced cell proliferation of 786-O cells was related to HIF-1α signals and decreased levels of HIF-2α ( Fig. 2a-d). Similarly, HIF-1α was increased and HIF-2α was decreased in pCS-treated A498 cells (Fig. 2b,f,g). Interestingly, VHL levels were also increased in both pCS-treated 786-O (Fig. 2a,e) and A498 cells (Fig. 2b,h). This result suggests that HIF-1α signals play an important role in pCS-induced proliferation of ccRCC cells.

Effect of pCS on EMT and cell migration.
Because HIF-1α regulates EMT-related proteins 29 , we studied the effect of pCS on EMT in ccRCC cells. EMT-related proteins (including fibronectin, twist, vimentin, and E-cadherin) were determined by Western blotting in pCS-treated 786-O and A498 cells (Fig. 3a,b). The results showed that fibronectin, twist, and vimentin expression was increased, while E-cadherin was decreased in pCS-treated 786-O and A498 cells (Fig. 3) and indicated that pCS induced EMT in ccRCC cells. The effect of pCS on cell migration was further investigated in ccRCC cells. As shown in Fig. 3k, pCS promoted the migration of 786-O cells as compared to the control. The relative density of migration is shown in Fig. 3k. Similarly, pCS promoted the migration of A498 cells (data not shown). These results indicate that pCS induces EMT and migration of ccRCC cells.

miR-21 mediates pCS-induced EMT and proliferation of ccRCC.
Previous studies demonstrated that miR-21 promotes the proliferation and EMT of ccRCC cells 38,39 . We examined the role of miR-21 in pCS-induced proliferation and EMT-mediated protein expression. The results showed that the miR21 inhibitor reduced the expression of HIF-1α mRNA at days 1 and 3 (Fig. 6a). Additionally, overexpression of HIF-1α and VHL proteins was inhibited and expression of HIF-2α was promoted in pCS-treated cells with miR-21 inhibitor (Fig. 6b). The inhibitor of miR-21 also significantly inhibited the expression of fibronectin and vimentin in pCS-treated cells (Fig. 6g,j). Overall, our results suggest that pCS increases miR-21 to cause cell proliferation and EMT in ccRCC cells.

Discussion
Previous reports showed that patients with end-stage renal disease (ESRD) generally have high levels of uremic toxins containing IS and pCS 40,41 . Clinical cases showed that patients with ccRCC are closely associated with ESRD 42,43 . However, direct evidence linking pCS and ccRCC progression is lacking. Our results demonstrated that pCS induced the proliferation and migration of ccRCC cells and suggested that the progression of ccRCC is related to pCS in the kidneys of patients. Therefore, removing pCS may prevent ccRCC progression in ESRD patients. AST-120, an approved clinical drug, decreases pCS levels 44,45 and may be useful for preventing ccRCC progression, but further studies are needed to confirm this aspect. Other methods are also available for decreasing pCS such as renal replacement therapy of hemodiafiltration, dietary intervention, laxative, and pro-, pre-, and syn-biotics 13,14 .
Accumulation of pCS increases oxidative stress in endothelial cells, reactive oxygen species in cardiomyocytes, and expression of DNA methyltransferase in HK2 cells 13 . A study showed that pCS activated the renin angiotensin aldosterone system and induced EMT 21 . Some studies showed that accumulation of reactive oxygen species leads to stabilization of HIF-1α 46,47 . Another study showed that the angiotensin system increased HIF-1α signals 48 .
HIF-1α and HIF-2α have similar structures containing transcription and oxygen-dependent degradation domains, but have different functions 49 . Additionally, both HIF-1α and HIF-2α can regulate cell proliferation www.nature.com/scientificreports www.nature.com/scientificreports/ and migration 7,24,25,28,31 . Some studies indicated that HIF1α acts as a renal tumor suppressor and HIF2α as a renal tumor inducer 50,51 . However, another study showed that HIF-1α inhibits and HIF-2α induces apoptosis 52 . Nevertheless, both HIF-1α and HIF-2α induce the proliferation of ccRCC cells 7,10,25,53 . Our results showed that pCS induced HIF-1α overexpression and promoted the proliferation of ccRCC cells. This suggests that HIF-1α plays an important role in promoting the proliferation of ccRCC cells. Our results on pCS-induced HIF-1α and reduced HIF-2α expression agreed with those of previous studies 54,55 .
VHL can interact with HIF-α to promote the degradation of HIF-α via the ubiquitin system, leading to blockage of HIF-α signals 56,57 . However, mutant VHL genes are found in most ccRCC cells, including in 786-O and A498 cells 58 . A study indicated that mutant VHL does not regulate HIF-α signals 52 . Our results showed that pCS induced HIF-1α and VHL and suppressed HIF-2α expression. We found that overexpression of mutant VHL in pCS-treated 786-O and A498 cells did not inhibit HIF-1α-induced proliferation. This may be explained by the data showing that overexpression of mutant VHL in ccRCC prevented UV-induced apoptosis 59 . Although the mechanism of pCS-induced overexpression of VHL remains unclear, pCS-induced overexpression of VHL may decrease apoptosis and lead to the progression of ccRCC. miR-21 in various cancers can activate HIF-1α signals to promote cell proliferation 37,38 . Both miR-21 and HIF-1α induced proliferation and EMT of ccRCC cells 25,28,35,36 , but it was unclear whether miR-21 upregulates HIF-1α in ccRCC. Our results showed that inhibition of miR-21 decreased HIF-1α expression in pCS-treated ccRCC cells. Therefore, miR-21 functions upstream of HIF-1α to regulate the proliferation and EMT of ccRCC cells. A hypothetical mechanism is shown in Fig. 7 and summarizes our study results. Briefly, pCS induced overexpression of miR-21, resulting in increased expression of HIF-1α and VHL and decreased HIF-2α. HIF-1α further promote the proliferation and migration of ccRCC cells. VHL is commonly mutated in ccRCC, and there are no reports regarding the relationship between miR-21 and VHL in ccRCC. However, a previous study indicated miR-21 can target VHL to regulate cell growth and that miR-21 inhibition induces VHL expression and inhibits the proliferation of glioblastomas 60 . In contrast, our results showed that inhibition of miR-21 decreased the expression of VHL and inhibited pCS-induced proliferation in ccRCC cells. Because wild-type VHL in glioblastoma inhibits cell growth while mutant VHL in ccRCC inhibits apoptosis, miR-21 has distinct effects on VHL expression and regulates cell growth in different cells.
In summary, we demonstrated that pCS-induced proliferation and EMT of ccRCC was mainly mediated by miR-21/HIF-1α signals.   inhibitor-treated group (miR21-5P), pCS-treated-1 and 3 day groups (1D and 3DPCS200), as well as pCS plus miR-21 inhibitor-treated-1 and 3 day groups (1DPCS200 miR21-5PI and 3DPCS200 miR21-5PI). Ratios of HIF-1α (c), HIF-2α (d), and VHL (e) to α-tubulin were compared. miR-21 inhibition regulated pCS-induced expression of EMT-related proteins (f). Ratios of fibronectin (g), E-cadherin (h), twist (i), and vimentin (j) to α-tubulin were also compared. The inhibitor of miR-21 significantly inhibited the expression of HIF-1α, fibronectin and vimentin in pCS-treated cells, suggesting that pCS increases miR-21 to cause overexpression of HIF-1α and EMT in 786-O cells. Values are expressed as the mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, compared to the pCS-treated group. www.nature.com/scientificreports www.nature.com/scientificreports/ SDS-PAGE and western blotting. The cells were collected and washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ), and then lysed with NETN buffer (20 mM Tris at pH 8.0, 150 mM NaCl, 1 mM EDTA at pH 8.0, 0.5% Nonidet P-40) plus protease and phosphatase inhibitors (25 mM NaF, 2 mM Na 3 VO 4 , 0.1 mM PMSF, 20 μg/mL aprotinin) and sonicated with a sonicator (ChromTech UP-800). After centrifugation at 15,000 × g for 15 min at 4 °C, the soluble extraction containing proteins was collected from the supernatant. The protein concentration was measured with a protein assay kit (Thermo Fischer Scientific, Waltham, MA, USA). Equal quantities (approximately 40 μg) of samples were loaded onto 6%, 10%, and 15% SDS-PAGE gel and separated at a voltage of 100V. Proteins in the SDS-PAGE gel were then transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was treated with 5% skim milk in TBST buffer (TBS containing 0.1% Tween-20) for 1 h at 26.5 °C and then hybridized with primary antibody at 4 °C with gentle agitation overnight. After washing with TBST three times, the membrane was incubated with secondary antibody for 1 h at 26.5 °C. The protein was detected by using the enhanced chemiluminescence detection reagent (GE Healthcare, Little Chalfont, UK) and observed with a Luminescence Image Analysis system (LAS-4000, GE Healthcare). The protein levels were quantified by using Image J software (NIH, Bethesda, MD, USA) and protein percentage was indicated as target protein level/tubulin protein level × 100%.
Cell migration assay. Cell migration was determined by using Millicell cell culture chambers (24-well, 8-μm chambers, Millipore) according to the manufacturer's instructions. Briefly, the Matrigel was re-hydrated with RPMI 1640 media (1:4) immediately for 1 h before the migration assay. Cells (5 × 10 4 ) were suspended in 200 μL serum-free medium then added to the upper chamber of Matrigel-coated filter inserts. After treatment with surfactin, 700 μL RPMI 1640 (containing 10% fetal bovine serum) was added to the bottom well as a chemoattractant. Next, the chambers were incubated for 24 h. Migrated cells attached to the lower surface of the filter. The cells were fixed and stained with 2% ethanol containing 0.2% crystal violet. Migrated cells were counted under a light microscope (40x) (OLYMPUS, IX-71, Tokyo, Japan) and absorbance was measured at 470 nm. The migration percentage was indicated as A470 experimental group/A470 control group × 100%.
Inhibition of miR-21. Cells were cultured to 50-60% confluence and transfected with a miR-21-5P inhibitor and negative control miRNA inhibitor (Integrated DNA Technologies) by using siLenFect TM lipid reagent (Bio-Rad, Hercules, CA, USA) in serum-free Opti-MEM medium according to the manufacturer's instructions. The final concentration of the oligomers was 25 nM. After transfection for 24 h, the medium was replaced with fresh RPMI medium containing 10% fetal bovine serum. The levels of miR-21 were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).
Determination of RNA expression levels. The RNA expression levels of miR-21, HIF1α and were determined by qRT-PCR. The optimized PCR assay of 20 μL PCR volume contained 10 µL of iTaq Universal Probes Supermix, 2 μL of TaqMan Gene Expression Assay, and water to a volume of 20 μL. All reagents were mixed and distributed into a 96-well PCR plate before adding 2 µL of cDNA (1-100 ng). The PCR program was as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 1 s and 60 °C for 60 s, during which fluorescence data were collected. Total RNA was extracted using the Purezol kit (Bio-Rad) according to the manufacturer's protocol. Next, 1 μg of total RNA was used to synthesize cDNA with a cDNA Synthesis kit (Bio-Rad). The expression levels of B2M and HIF1α were quantified by qRT-PCR using the iTaq Universal probe Supermix kit (Bio-Rad) and StepOne plus Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Primers used in this experiment were as follows: HIF1α: 5′-CAACCCAGACA-TATCCACCTC-3′ (forward (F)), 5′-CTCTGATCATCTGACCAAAACTCTA-3′ (reverse (R)). The relative expression level of each gene was calculated by using the 2 −ΔΔCt method). All data were obtained from three independent experiments. Statistical analysis. Data are presented as the mean ± SE from at least three independent experiments.
One-way analysis of variance was used to compare the experimental data. Two-way analysis of variance was used to compare data obtained from different treatment concentrations and incubation times. The data were analyzed with SPSS Statistics v18.0 (SPSS, Inc., Chicago, IL, USA). A P value < 0.05 was considered statistically significant.