Introduction

Renal cell carcinoma (RCC) accounts for approximately 2–3% of all malignancies and is the 12th most common cancer worldwide [1,2,3]. Currently, the only potential curative treatment for localized RCC is surgery; however, 20–30% of patients with RCC experience local or distant recurrence within 5 years after radical nephrectomy [4]. This is a major factor limiting patient survival; therefore, identification of new molecular markers to predict patient survival and tumor relapse remains to be a subject of fundamental importance. At present, studies concerning novel molecular targets in RCC were very limited.

Integrins are heterodimeric transmembrane receptors that could mediate interactions of cells with the extracellular matrix (ECM) [5]. Integrins are formed by specific noncovalent associations between different α and β subunits [6], and each subunit contains a cytoplasmic tail, a transmembrane and an extracellular region [7, 8]. The integrin family is classified according to the associated β-subunit, mainly including β1 (CD29) and β2 (CD18) [9, 10]. The β2 integrin family has a common β2 chain paired with homologous α subunits and consists of the following four members: αMβ2 (CD11b/CD18, or Mac-1); αLβ2 (CD11a/CD18, or LFA-1); αDβ2 (CD11d/CD18); and αXβ2 (CD11c/CD18).

Integrins exhibit a very broad ligand-binding specificity with the component of ECM, which allows for its diverse cell functions, such as cell interactions, adhesion and migration [11, 12]. Abnormal expression of integrins often correlates with irregular processes like inflammation or tumor. Integrin αMβ2 is mainly expressed in myeloid, NK and T cells [6], which not only participate in regulating monocyte differentiation and mediating adhesive reactions of leukocytes during the inflammatory response [7, 13, 14], but also take part in the maintenance of tolerance and control of inflammation [15, 16]. However, the specific roles of integrin αMβ2 in the progression of tumor cells remain unclear.

Our previous study inspired us that LEF1 and the integrin αMβ2 may be related to RCC cells, which still needs to be confirmed in further study [17, 18]. LEF1, initially identified as a pre-B and T lymphoid-specific gene belonging to the family of high-mobility group transcription factors [19, 20], contains a strong DNA binding domain near the C terminus and a domain at the N terminus that binds the transcription activator, β-catenin [21]. Chang et al. indicated that β-catenin could regulate integrin α5β1 expression, and LEF1 binding sites in the promoter regions of integrin α5/β1 were also confirmed [22]. Thus, whether LEF1 is involved in the regulation of integrin αMβ2 in RCC should be further investigated.

The TGF-β signaling pathway has been confirmed to modulate numerous physiologic processes, including proliferation, migration and invasion of tumors [23,24,25], and TGF-β1 is involved in promoting the proliferation of RCC cells [26,27,28]. Moreover, Lebdai et al. identified and validated TGF-β1 as a promising prognosis marker of clear cell renal cell carcinoma [29], and Huang et al. found TGF-β1 could induce Fascin1 to promote cell invasion and metastasis of human 786-0 RCC cells [30]. Previous study demonstrated that Smad7 interacted with β-catenin and LEF1/TCF, transcriptional regulators in Wnt signaling, in a TGF-β-dependent manner [31]. Also, integrin signaling was found to potentiate TGF-β1 with important implications for epithelial to mesenchymal transition (EMT) in RCC [32]. However, it is still unknown whether TGF-β1 participated in regulating LEF1/integrin αMβ2 expression in RCC cells. In this study, we tried to figure out whether integrin αMβ2 is associated with RCC and its detail role in the development of RCC. We assume that TGF-β1 may employ LEF1/integrin αMβ2 to further enhance the proliferative and metastatic potential in human renal cell.

Material and method

Cell culture and agents

Four commercially available human RCC cell lines (ACHN, Caki-1, NC 65 and A498) were obtained from the ATCC (Manassas, VA, USA). All cells were incubated with complete medium, consisting of 10% heat-inactivated fetal bovine serum and supplemented with RPMI-1640, 2 mM l-glutamine, 1% nonessential amino acids, 25 mM HEPES and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA). RCC cell lines were cultured as a monolayer in an incubator at 37 °C with a humidified atmosphere of 5% CO2. TGF-β1 was purchased from Sigma-Aldrich, St Louis, MO, USA, and RCC cells were treated with TGF-β1 (10 ng/ml) in the following experiments.

Reverse transcription-PCR

Total RNA of RCC cells was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was performed using a cDNA synthesis kit (Applied Biosystems, Carlsbad, CA, USA). The cDNA was amplified by PCR using TaqMan gene expression assays, and the PCR conditions were set according to the manufacturer’s instructions (Applied Biosystems). The PCR product was separated by 3% agarose gel electrophoresis, and GAPDH was used as the internal control. All primer sequences in this study are shown in Table 1.

Table 1 Primer sequences and siRNA oligonucleotides used in this study
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Western blot and immunoprecipitation

Total protein was extracted with protease inhibitor (Roche, Basel, Switzerland) and cell lysis buffer (Cell Signaling, Cambridge, UK). Protein (60 mg/well) was separated by SDS–polyacrylamide gel electrophoresis; then, proteins were transferred to 0.2 μm nitrocellulose (Life Technologies, Carlsbad, CA, USA) and incubated with a blocking solution for 2 h at room temperature. The membranes were incubated with primary antibodies overnight at 4 °C. Integrin αM/CD11B (D6X1N) rabbit mAb, integrin β2 (D4N5Z) rabbit mAb, LEF1 (C12A5) rabbit mAb, β-catenin (D10A8) XP® rabbit mAb, mouse (G3A1) mAb IgG isotype control, matrix metalloproteinases (MMPs)-2 (D8N9Y) rabbit mAb and MMP-9 (D6O3H) XP® rabbit mAb were purchased from Cell Signaling Technology. β-actin monoclonal antibody (ab6276) and calpain 2 antibody (ab39165) were purchased from Abcam (Cambridge, UK). The immune complexes were detected with an ECL system (Amersham, Aylesbury, UK) according to the manufacturer’s instructions. Integrin function-blocking monoclonal antibodies (CD18 (CBL158) and CD11b (CBL145)) were obtained from Chemicon (Temecula, CA, USA).

RNA interference and transfection

The siRNA oligonucleotide or scrambled siRNA (negative control) was designed using siDirect software. The oligonucleotides used in this study are shown in Table 1. RCC cells were seeded at the density of 1 × 105 cells per well into a 6-well culture dish and incubated until confluence reached 50–60%; then, cells were transfected with siRNA oligonucleotides by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The cDNA coding sequence of LEF1 was cloned, as previously described [33]. RCC cell lines were also transfected with LEF1 vector containing full-length cDNA for LEF1 or with a blank vector without inserting the LEF1 by Lipofectamine 2000. RCC monoclonal cell lines were selected by G418, and the expression of LEF1 was detected using Western blot analysis.

Cell viability assay

The proliferative ability of RCC cells was analyzed using WST-1 assay. Briefly, RCC cells were seeded into 96-well plates at a density of 0.5 × 104 cells per well. After 48 h of continuous incubation, 20 μL of WST-1 reagent (Roche, Penzberg, Germany) was added to each well. Following incubation for 2 h at 37 °C, the viable cells were detected by measuring absorbance at 450 nm using an absorbance reader (Immunoreader NJ-2000; Japan Intermed, Tokyo, Japan).

Cell migration and invasion assays

For the migration assays, chemotaxis was detected using a Transwell system (Poretics Corp., Livermore, CA, USA) containing 8-μm pore polycarbonate membrane filters. The invasion assay was analyzed using a Transwell system incorporating a polycarbonate filter membrane (Corning, NY, USA). Briefly, 1 × 105 RCC cells were selected in 100 μL of serum-free medium and seeded into the upper chamber. After continuous incubation at 37 °C for 48 h, the invading cells on the bottom of each well and the migrating cells in the lower chamber were fixed with methyl alcohol, and the number of RCC cells was counted by a CX23 microscope (Olympus Corporation, Tokyo, Japan) in five randomly microscopic fields.

Statistical analysis

Statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). All results in this study were shown as the mean ± standard deviation (SD). Comparisons between two groups were made by unpaired or paired Student’s t tests. All statistical tests were 2-tailed, and p value < 0.05 was regarded as significant different.

Results

RCC cells possess higher expression of LEF1 and integrin αMβ via TGF-β1-dependent mechanism

We employed RT-PCR and Western blot to detect the expression of LEF1, integrin αMβ2 and TGF-β1 in four pairs of RCC and corresponding normal kidney tissue. Our results demonstrated that the expression of LEF1, integrin αMβ2 and TGF-β1 was up-regulated in RCC compared to normal kidney tissue (Fig. 1), which suggested their involvement in RCC development. Of the four RCC cell lines, the result was consistent, and ACHN and Caki-1 were selected for following experiments (Fig. 2a, b). To determine whether TGF-β1 exerts its affection on LEF1 and integrin αMβ2, TGF-β1 (10 ng/ml for 48 h) was added to ACHN and Caki-1 for further detection of protein expression. The results showed that TGF-β1 could significantly up-regulate the expression of LEF1 and integrin αMβ2 in RCC cells (Fig. 2c). These findings suggested that TGF-β1-dependent mechanism in RCC cells may contribute greatly to the up-regulation of LEF1 and integrin αMβ2.

Fig. 1
figure 1

The expression of LEF1, integrin αMβ2 and TGF-β1 was detected by RT-PCR (a) and Western blot (b) in RCC and normal kidney with four pairs of samples shown

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Fig. 2
figure 2

The expression of LEF1 and integrin αMβ2 was detected by RT-PCR (a) and Western blot (b), and TGF-β1 increased the expression of LEF1 and integrin αMβ2 in RCC cells (C)

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LEF1 may interact with β-catenin and regulate the expression of integrin αMβ2

We investigated the relationship among LEF1, integrin αMβ2 and TGF-β1 to see how the three proteins affect each other. Firstly, overexpression and knockdown of LEF1 in RCC cells were achieved by different vectors, and Western blot was used to detect the change of three protein expression. As demonstrated, the protein level of integrin αMβ2 was significantly increased in RCC cells with high expression of LEF1, whereas markedly decreased in LEF1 knockdown RCC cells. Meanwhile, the expression of TGF-β1 was not affected by LEF1 transfections in RCC cells. In addition, RCC cell lines with varying expression of LEF1 were subjected to immunoprecipitation test to evaluate the interaction of LEF1 and β-catenin. Although LEF1 did not affect β-catenin expression, higher amount of LEF1/β-catenin complex was detected in RCC cells with high expression of LEF1 (Fig. 3). These findings suggested that LEF1 may interact with β-catenin and regulate the expression of integrin αMβ2 in human RCC cells.

Fig. 3
figure 3

LEF1 regulated the expression of integrin αMβ2 in RCC cells, and the expression of LEF1 was detected by Western blot and immunoprecipitation after transfection with LEF1 siRNA oligonucleotide and a vector containing full-length cDNA for LEF1

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LEF1 enhances the proliferation and metastasis of RCC cells

The proliferation and metastasis are considered as important steps in the development of RCC, so the proliferative and metastatic potential of RCC cells were evaluated concerning the LEF1 status. As shown in Fig. 4a, the effect of LEF1 on the proliferation of RCC cells was analyzed by the WST-1 assay. RCC cell lines with overexpressed LEF1 showed increased proliferative ability compared to the control cells, but significantly reduced proliferative ability upon knockdown of LEF1. In addition, RCC cells with lower expression of LEF1 had less capacity for migration and invasion than those cell lines with higher expression of LEF1 (Fig. 4b, c). These results suggested that LEF1 was involved in the proliferation and metastasis of RCC cells.

Fig. 4
figure 4

LEF1 played an important role in the proliferation (a), migration (b) and invasion (c) of RCC cells. All experiments were performed in triplicate, and the error bar represents the SD. *p < 0.05 vs control

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Integrin αMβ2 is necessary for TGF-β1-induced proliferation and metastasis of human RCC

TGF-β1 plays an important role in the carcinogenesis of RCC; however, whether its function depends on integrin αMβ2 is unclear, so blocking antibody against integrin αM or β2 was used to detect the effect that integrin αMβ2 exerts on TGF-β1 function. As shown in Fig. 5a–c, RCC cells treated with TGF-β1 (10 ng/ml) for 48 h significantly improved cell growth and enhanced cell migration and invasion capacity. But administration of blocking antibodies significantly decreased proliferative ability of untreated cells and TGF-β1 treated cells (Fig. 5a), and similar results were found in regard as cell migration and invasion capacity.

Fig. 5
figure 5

TGF-β1 induced the proliferation (a), migration (b) and invasion (c) depended on the activity of integrin αMβ2 in RCC. All experiments were performed in triplicate, and the error bar represents the SD. *p < 0.05 vs 1. Control vs 2. TGF-β1

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These findings indicated that integrin αMβ2 was necessary for TGF-β1-induced proliferation and metastasis of human RCC.

Integrin αMβ2 is crucial for LEF1 up-regulating the expression of MMPs and calpain-2

The effect of LEF1 and integrin αMβ2 on regulation of MMP2, MMP9 and calpain-2 expression was evaluated in this study, which aimed to further investigate the molecular mechanism of LEF1/integrin αMβ2 involved in the metastasis of RCC cells. Our results found that the amount of LEF1 was positively associated with the expression of MMPs and calpain-2, mainly embodied by overexpression of LEF1 with increased MMPs and calpain-2 expression, and knockdown of LEF1 with decreased MMPs and calpain-2 expression (Fig. 6a). However, MMPs and calpain-2 expression could be suppressed after treatment with blocking antibody against integrin αM or β2 (1 µg/ml) for 48 h in LEF1 highly expressed RCC cells (Fig. 6b). These findings suggested that LEF1 enhanced the metastatic potential depending on the regulation of MMPs and calpain-2 secretion by integrin αMβ2 in human RCC.

Fig. 6
figure 6

LEF1 enhanced the metastatic potential by up-regulating MMP-2, MMP-9 and calpain-2 secretion depending on the activity of integrin αMβ2 in RCC

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Discussion

Our study gained a new finding that integrin αMβ2 positively promoted RCC development, based on its up-regulation in RCC cells and facilitating proliferative and metastatic potential of RCC cells. This helped to amplify function of integrin αMβ2 besides its regulation on inflammation and shed light on understanding and control of RCC.

The positive correlation between integrin αMβ2 expression and LEF1 suggested that LEF1 may directly act as the transcription factor for αMβ2 to govern αMβ2 expression, and this should be more definite if the evidence that LEF1 has binding sites in the promoter regions of integrin αMβ2 is added. As known, LEF1 asks for other transcription activators to exert its function, and LEF1/β-catenin complex is confirmed to act downstream of the Wnt/β-catenin signaling [34, 35], which is regarded as great contribution for tumor cells progression [36]. Here in our study, accordingly, LEF1 formed complex with β-catenin to up-regulate integrin αMβ2 in RCC cells. Consequently, as the key component of Wnt/β-catenin pathway, LEF1 could enhance the proliferation, migration and invasion of RCC cells, largely owning to its up-regulation of genes related to these processes, of course, including integrin αMβ2.

Interestingly, we also found that TGF-β1 stimulation could up-regulate the expression of LEF1 and integrin αMβ2 in RCC cells, which may point out the cross talk between TGF-β1 and Wnt/β-catenin pathway just as reported [37]. As known, TGF-β mainly exerts its functions through classical SMAD-dependent mechanism. Reports showed that SMAD3 jointed the complex of LEF1/β-catenin upon TGF-β1 stimulation, and triggered up-regulation of β1 integrin gene expression [33, 38]. Similarly, the complex of LEF1/β-catenin/SMAD7 leads to the up-regulation of LEF1 itself [39]. Here, in RCC cells, it could be deduced that TGF-β1 signaling promotes Wnt/β-catenin pathway, leading to up-regulation of transcription factor LEF1, which served as a positive feedback, and enlarges targeted genes related to tumor progression, including LEF1 itself and integrin αMβ2.

Integrin αMβ2, as a transmembrane receptor itself, has cross talk with TGF-β1 signal. To be noted, integrins could extracellularly activate TGF-β1 which is secreted in a latent form failing to trigger receptor mediated TGF-β signaling [40]. I interacts with TGF-β receptor (TβR) type II, and the interaction enhances TGF-β stimulation of MAPKs and Smad2/3-mediated gene transcription, thereby significantly promoting TGF-β induced EMT in tumor cell [41]. Additionally, cross talk between TGF-β and integrin signaling can also occur on downstream receptors, mainly through affecting the common signal molecules related to the two pathways [42, 43]. All the above information may inspire that TGF-β signaling up-regulates the expression of integrin αMβ2, and then αMβ2 served as a positive feedback, aiming to facilitate and enhance TGF-β stimulated signaling. Finally, the fact in our study that blocking antibody against integrin αMβ2 suppressed TGF-β1’s effects on RCC cells, may be clearly due to the blocker attenuated αMβ2 facilitating TGF-β1 induced EMT, a key index featuring migration and invasion of tumor cells.

Apart from assisting TGF-β signal, integrins govern pathways mediated by its own to transduce the extracellular survival and invasion signal [43]. Though lack of kinase activity, when activated by ligand in ECM, integrin could recruit diverse kinases, including focal adhesion kinase, integrin linked kinase and the SRC kinase family, to induce the cascade signal transduction involving Raf-ERK/MAPK and PI3K/AKT pathway. MMP2 and MMP9 are included in gelatinases belonging to MMPs. Previous studies indicated that MMP2 could mediate migration of vascular smooth muscle cell [44] and enhance pericellular proteolysis and invasion [45]. Downregulation of MMP2 and MMP9 was involved in the inhibition of migration and invasion in RCC cells [46]. Also, calpain-2 has been reported to mediate the invasion of glioma cells and possibly regulate MMP2 [47]. MMPs could just be the targeted prey of integrins through the mentioned pathways [48,49,50], who belong to proteinase family with biological functions in tumor migration and invasion [45, 51,52,53]. This is in accordance with our finding that expression of MMP2 and MMP9 is quite dependent on the status of integrin αMβ2 despite of the overexpression of LEF1. Taken the fact that TGF-β meditating classical SMAD and nonclassical pathways largely contributes to the EMT related gene expression including MMPs [54], it could be deduced that TGF-β together with integrins should be the determinant factors toward EMT and invasion of tumor cells.

In conclusion, our study suggested that integrin αMβ2 up-regulation in RCC cells was dependent on combined effect of TGF-β1 and Wnt/β-catenin pathway leading to high amount of LEF1. Also, we found that integrin αMβ2 played an essential and crucial role in the proliferation, migration and invasion of RCC cells, mainly through assisting TGF-β1 stimulated signal and by its own. All supported the conclusion that TGF-β1 strengthens proliferative and metastatic potential by means of up-regulating LEF1/integrin αMβ2 in human renal cell. These results also suggested that blocking integrin αMβ2 activity could be a new therapeutic option for patients with advanced RCC. Of course, the molecular mechanisms and the expression of integrin αMβ2 in RCC need further investigation.