All samples (paired tumor and normal tissues) were surgically resected from patients with RCC between 2012 and 2014, at Union Hospital of Huazhong University of Science and Technology (Wuhan, China). Resected tissues were immediately frozen in liquid nitrogen for subsequent experiments. None of the patients had received any antitumor therapy before surgery. Informed written consent was obtained from each of the RCC patients. This study and experiment procedures were approved by the Institutional Review Board of Huazhong University of Science and Technology.

All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA), and 1% penicillin-streptomycin at 37°C in a humidified 5% CO₂ atmosphere. For TGFβ1 treatment, the HK-2 cells were cultured in DMEM containing 10% fetal bovine serum, and 1% penicillin-streptomycin and treated with 2.5 ng/ml of TGFβ1 for 0, 3, 7, 10 day(s), respectively.

The si-RNA duplexes targeting SOX4 (si-SOX4) and si-RNA Negative Control (si-NC) were synthesized and purified by GenePharma (Shanghai, China). All si-RNA sequences were subjected to BLAST analysis to guarantee that there is no homology to any other known coding sequences in the Human Genome Database. The si-SOX4 and si-NC were transfected at a final concentration of 50 nM with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's recommendations. The plasmid vectors expressing SOX4 (SOX4) or Negative Control (NC) were constructed by Vigene Biosciences (Shandong, China). A total 2 µg of SOX4 or NC plasmid were transfected with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's recommendations. At 48 h after transfection, cells were harvested for subsequent experiments. The si-RNA sequences were as follows: si-SOX4 #1: 5′-UUUGCC CAGCCGCUUGGAGAUCUCG-3′, si-SOX4 #2: 5′-UUGUC GCUGUCUUUGAGCAGCUUCC-3′, si-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′.

Immunohistochemistry (IHC) assay was performed as previously described (27). Briefly, RCC tissues and corresponding normal tissues were fixed in 10% formalin, dehydrated, and embedded in paraffin sequentially. The sections were incubated with rabbit polyclonal antibody SOX4 (1:100, ab80261, Abcam, Cambridge, MA, USA) overnight at 4°C. After washing three times with PBS for 10 min each time, the sections were incubated with anti-rabbit secondary antibodies conjugated to horseradish peroxidase-labeled polymers. Finally, the sections were counterstained with hematoxylin.

Appropriate amounts of cells were seeded on glass coverslips in 12-well plates, washed with PBS three times, fixed in 4% paraformaldehyde and permeabilized with 0.3% TritonX-100 for 10 min. Cells were blocked with 3% BSA for 1 h. Grass coverslips were incubated with primary antibodies overnight at 4°C, followed by incubation with CY3-conjugated secondary antibodies for 1 h at room temperature, and then stained with DAPI. After washing with PBS, glass coverslips were photographed under a fluorescence microscope.

In vitro cell migration and invasion assays were performed using 24-well Transwell chambers with 8.0-µm pore polycarbonate membrane inserts (Corning). For the migration assay, 1×10⁴ (786-O, SN12-PM6) or 2.5×10⁴ (HK-2) cells were added to the top chambers. For the invasion assay, 2×10⁴ (786-O, SN12-PM6) or 5×10⁴ (HK-2) cells were added to the top chambers precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). As a chemoattractant, complete medium supplemented with 10% FBS was added to the bottom chambers to stimulate migration or invasion. After cells were incubated for 24 h, cells on the top surface of the membrane were gently scraped with a cotton swab and cells on the lower surface of the membrane were fixed with 100% methanol and then stained with 0.1% crystal violet. The cells migrating or invading through the membrane were counted in 5 random fields. For wound healing assays, cells transfected with si-RNA were seeded in 6-well plates. When cells reached 70–80% confluence, a scratch was made using 10-µl pipette tip, washed, then images were at 0 and 24 h.

Total RNA of tissues and cells was extracted by using TRIzol reagent (Invitrogen), and 1 µg RNA samples were reverse transcribed to cDNA by using reverse transcriptase M-MLV (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using the SYBR-Green PCR master mix (Invitrogen) on a Roche LightCycler 480 system (Roche Diagnostics, Mannheim, Germany). Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. Relative expression of genes was calculated using the power formula: 2^−ΔCt (ΔCt = Cttarget - Ctcontrol), as previously described (28). The gene primer sequences were as follows: SOX4: forward, 5′-GGCCTCGAGCTGGGAATCGC-3′, reverse, 5′-GCCCACTCGGGGTCTTGCAC-3′; E-cadherin: forward, 5′-GACAACAAGCCCGAATT-3′, reverse, 5′-GGAAACTCTCTCGGTCCA-3′; N-cadherin: forward, 5′-CGGGTAATCCTCCCAAATCA-3′, reverse, 5′-CTTTATCCCGGCGTTTCATC-3′; Vimentin: forward, 5′-GAGAACTTTGCCGTTGAAGC-3′, reverse, 5′-GCTTCCTGTAGGTGGCAATC-3′; Twist: forward, 5′-CTGCCCTCGGACAAGCTGAG-3′, reverse, 5′-CTAGTGGGACGCGGACATGG-3′; ZEB1: forward, 5′-TGCTCCCTGTGCAGTTACACCTT-3′, reverse, 5′-CCAGACTGCGTCACATGTCTTTGA-3′; GAPDH: forward, 5′-GAGTCAACGGATTTGGTCGT-3′, reverse, 5′-GACAAGCTTCCCGTTCTCAG-3′.

Tissues or cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS1% and sodium deoxycholate, pH 7.4) containing a protease inhibitor cocktail tablet (Roche Diagnostics) and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysate protein concentration was measured by using BCA kit (Beyotime Institute of Biotechnology) according to manufacturer's instructions. Total protein (40 µg) were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After being blocked in PBS containing 5% nonfat milk for 1 h, the membranes were incubated with antibody against SOX4, N-cadherin, Vimentin, MMP-9, p-AKT, AKT (all from Abcam), E-cadherin (Cell Signaling Technology, Beverly, MA, USA), Twist, GAPDH (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The membranes were then incubated with secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature. After washing with PBS-Tween-20, the proteins were visualized and quantified using ChemiDoc-XRS+ (Bio-Rad, Laboratories, Inc., Hercules, CA, USA).

Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons of different groups were performed using Student's two-tailed t-test. Data of each group were expressed as mean ± SEM and P<0.05 was considered to indicate a statistically significant difference.

Our previous findings demonstrated low miR-129-3p levels were associated with short disease-free and overall survival of RCC patients. miR-129-3p impaired RCC cell migratory and invasive properties by decreased multiple metastasis-related genes, including SOX4 (29). Based on these observations, we first aimed to examine the levels of SOX4 expression in RCC cell lines and tissues by qRT-PCR and western blot analysis. Of note, the levels of SOX4 mRNA and protein expression were dramatically upregulated in RCC cell lines (786-O, SN12-PM6, ACHN and A498) compared with the normal renal epithelial cell line HK-2 (Fig. 1A and B). Unexpectedly, no remarkable difference of SOX4 mRNA was observed between ccRCC and normal tissues. However, RCC tissues exhibited significantly higher SOX4 protein expression in 17 of 20 pairs RCC compared with corresponding normal tissues by western blotting and IHC (Fig. 1C and D). Moreover, immunofluorescence revealed that SOX4 was overexpressed in RCC cells compared with the normal renal cells (Fig. 1E). Collectively, these results indicate SOX4 expression is up regulated in RCC.

To explore the roles of SOX4 upregulation in RCC cell lines, we then performed a loss-of-function assay by using small interfering RNA specifically targeting SOX4 (Fig. 2A and D). The effects of SOX4 knockdown on migration and invasion of these two cell lines were examined using wound healing and Transwell chamber assays. The results revealed that SOX4 knockdown markedly undermined the migration and invasion of these two RCC cell lines (Fig. 2B and C, and E and F), however, it did not influence cell proliferation and apoptosis (data not shown). To further confirm the involvement of SOX4 in migration and invasion, we then carried out a gain-of-function assay in HK-2 cell line by constructing plasmid vector expressing SOX4 (Fig. 2G). The results indicated that SOX4 overexpression prominently promoted the migration and invasion of HK2 cell line (Fig. 2H), but it did not affect cell proliferation and apoptosis (data not shown). In addition, downregulation or upregulation of SOX4 did not cause significant changes in cell morphology. Therefore, our loss-of-function and gain-of-function assays demonstrated that SOX4 might promote migration and invasion in RCC cell lines in vitro.

Accumulating evidence demonstrates that EMT has been implicated in cancer metastasis (2,30,31). Therefore, in order to investigate the potential metastatic mechanisms in RCC, we examined the EMT marker expression in RCC cell lines and tissues by qRT-PCR and western blotting. As shown in Fig. 3A and B, the RCC cell lines exhibited a significant downregulation of E-cadherin compared with the HK-2 cell line; the mesenchymal cell markers N-cadherin, Twist, and Vimentin were markedly upregulated. Moreover, RCC tissues exhibited a significant downregulation of E-cadherin compared with the corresponding normal tissues; whereas, the mesenchymal markers Vimentin, ZEB1, MMP-9, and Twist were prominently upregulated (Fig. 3C and D). Data above indicated that EMT process might have occurred in the formation and progression of RCC.

Recent studies have identified SOX4 as a master regulator of EMT (32). To explore the potential correlation between SOX4 and EMT in RCC, we successfully knocked down the expression of SOX4 in 786-O and SN12-PM6 cell lines that overexpressed endogenous SOX4, by using small interfering RNA, as confirmed by qRT-PCR and western blotting (Fig. 4A and B). We then examined by western blotting the expression of several EMT markers in the 786-O and SN12-PM6 cells that were subjected to SOX4 knockdown. The results indicated that 786-O and SN12-PM6 cell lines that were knocked down by SOX4 had a significant higher E-cadherin expression compared with corresponding control cells; whereas, the mesenchymal cell markers N-cadherin, Vimentin, and Twist expression were prominently downregulated (Fig. 4A and B). To further corroborate the correlation between SOX4 and EMT, we then overexpressed SOX4 in HK-2 by using plasmid vector expressing SOX4, as confirmed by qRT-PCR and western blotting (Fig. 4C). Next, we examined the EMT markers expression in HK-2 cells that overexpressed the SOX4 by western blotting. As shown in Fig. 4C, the HK-2 of SOX4 overexpression exhibited a significant lower E-cadherin expression compared with the corresponding control cells; whereas, N-cadherin, Vimentin, and Twist expression were significantly upregulated. These results suggested that SOX4 could induce EMT in RCC cell lines, and SOX4 knockdown could partially reverse EMT.

To further substantiate the findings, we next performed SOX4 knockdown and rescue tests in the 786-O and SN12-PM6 and HK-2 cell lines, respectively. As shown, in all the three cell lines tested, downregulation of SOX4 upregulated epithelial marker and downregulated mesenchymal markers in 786-O and SN12-PM6 and HK-2 cell lines; while overexpression of SOX4 yielded opposite effects (Fig. 5A–C). These results further solidified our findings that SOX4 could induce the EMT program.

Multiple lines of evidence have confirmed the involvement of TGFβ signaling in EMT program. To further investigate whether SOX4 was involved in the process of TGFβ signaling-induced EMT in HK-2 cells, we stimulated HK-2 cells by using 2.5 ng/ml concentration TGFβ1 for 0, 3, 7, 10 day(s). Compared with the parental HK-2 cells, the TGFβ1-treated HK-2 cells represented morphologic changes from cobblestone-like to fibroblast-like appearances (Fig. 6A). SOX4 expression was upregulated in HK-2 cells at mRNA and protein level, in a time-dependent manner following the addition of TGFβ1 into the cell culture medium (Fig. 6B and C). Moreover, TGFβ1 modulated the expression of EMT markers and p-AKT at protein level (Fig. 6C). These data indicated SOX4 might be an important component involving TGFβ-induced EMT.

AKT signaling cascade has been reported involved in metastasis in a variety of cancers (33–35); Previous studies also showed that AKT pathway could induce EMT (36–39). To further explore the molecular mechanisms how SOX4 induces EMT and metastasis in RCC, we expanded our study to the metastasis-related protein p-AKT, which may be the potential downstream target of SOX4. As shown in Fig. 7A, SOX4 knockdown in 786-O and SN12-PM6 cell lines downregulated the expression of p-AKT, while overexpression of SOX4 in HK-2 cell line upregulated the expression of p-AKT (Fig. 7B). Furthermore, downregulation of p-AKT was accompanied by the upregulation of E-cadherin, while upregulation of p-AKT reversed this result. Based on these results, we preliminarily established a pathway diagram that SOX4 modulated EMT (Fig. 7C). Collectively, these data indicated that SOX4 might contribute to RCC metastasis via modulating the AKT/EMT signaling cascade.