Renal cancer cells, OS-RC-2 and 786-O, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Corning Inc., Corning, NY, USA) as described in our previous studies (17,18).

All of the following manipulations were performed in full accordance with prior review, consent and approval provided by the Institutional Ethics Committee of the State Key Laboratory of Biotherapy, West China Hospital of Sichuan University. Eighty pairs of clear cell renal cell carcinoma tissues (RCTs) and their corresponding autologous paracancerous kidney tissues (PKTs) were obtained from RCC patients who underwent surgery at West China Hospital, Sichuan University (Chengdu, China) from July 2006 to February 2008. These patients included 44 men and 36 women. The average age was 59 years (range age, 29–82 years) and all patients did not receive radiotherapy, chemotherapy and immunotherapy prior to surgery. All tissues were identified through pathologic biopsy, and the tissues were frozen in liquid nitrogen. The RCC patient demographic and clinical information, including age, gender and histological type of tumor differentiation (26), was collected following provision of informed consent. The demographic and clinical information of the 80 RCC patients was presented in our previous study (18). Follow-up information was obtained from review of the medical records of the patients.

The expression plasmids pFlag-PGRMC1 and pYR-ATP1A1 were constructed for exogenous overexpression of PGRMC1 and ATP1A1 in RCC OS-RC-2 and 786-O cells (17,18). After RCC cells were seeded on a 6-well plate for culture overnight, cells were transiently transfected with 2.5 µg pFlag-PGRMC1 or pYR-ATP1A1 plasmids per well using Invitrogen™ Lipofectamine 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The siRNA for PGRMC1 (siPGRMC1) was synthesized by the RiboBio Co. (RiboBio, Guangzhou, China). The PGRMC1-specific siRNA sequences (siPGRMC1) were designed as 5′-CTGGGAGTCTCAGTTCACT-3′, and negative control oligonucleotides (siNC) were 5′-UUCUCCGAACGUGUCACGU-3′. The OS-RC-2 and 786-O cells were seeded on a 6-well plate for incubation with 50 nM siPGRMC1 or siNC per well using Invitrogen™ Lipofectamine 2000.

To detect the protein expression level, 60–80 µg proteins, extracted from cell pellets or tissues, were separated on a 10% SDS-PAGE gel to test by western blotting. The primary antibodies included PGRMC1 (1:500; cat. no. ab48012; Abcam, Cambridge, UK), ATP1A1 (1:200; cat. no. ab2872; Abcam), GAPDH (1:1,000; cat. no. sc-365062; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), AKT (1:500; cat. no. 4961; Cell Signaling Technology, Inc., Danvers, MA, USA) and phosphorylated AKT (p-AKT, 1:500; cat. no. ab18206; Abcam). The HRP-conjugated secondary antibodies, including goat anti-rabbit (cat. no. ZB-2301; Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China) and goat anti-mouse (cat. no. ZB-2305; Beijing Zhongshan Golden Bridge Biotechnology), were diluted at 1:10,000 for incubation with the PVDF membrane at 37°C for 1 h. Final detection was performed with western blot analysis reagent ECL (Amersham Biosciences; GE Healthcare, Chicago, IL, USA).

Eighty pairs of RCTs and their corresponding PKTs were paraffin-embedded and cut into sections with 5-µm thickness for immunohistochemistry (IHC) analysis according to our previous procedures (27). Tissue sections were incubated with the primary antibody of ATP1A1 (1:400; cat. no. ab2872; Abcam), PGRMC1 (1:1,000; cat. no. ab48012; Abcam) and p-AKT (1:500; cat. no. ab18206; Abcam) at 37°C for 2 h followed by quenching the endogenous peroxidase activity and antigen retrieval. Subsequently, the sections were incubated with the secondary antibody, biotinylated anti-goat IgG (cat. no. ZB-2306; ZSGB-BIO; OriGene Technologies, Inc., Beijing, China) at 37°C for 40 min, following reacting with 3,3′-diaminobenzidine substrate solution (Dako Cytomation GmbH, Shanghai, China) and counterstaining with hematoxylin. Five independent fields at ×200 magnification for positive cells were chosen to evaluate the immunostaining intensity and percentage. The staining intensity was defined as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The staining percentage was defined as follows: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). The IHC scores for each tissue sample, ranging from 0 to 12, were measured as immunostaining intensity multiplied by the percentage of positive cells (28,29). The expression levels of PGRMC1 and ATP1A1 were determined according to the final IHC scores (17,18). Similarly, the p-AKT level in tissues was defined as low (scores <3) and high expression (scores 3–12).

Cell viability was measured by CCK-8 assay as previously described (30). The OS-RC-2 and 786-O cells were seeded on a 6-well plate and incubated with 50 nM siPGRMC1 or siNC per well. On the first day after siPGRMC1 treatment for RCC cells, cells were transfected with 2.5 µg pYR-ATP1A1 plasmids to further observe cell growth (31). After 24 h, 5×10³ cells/well were seeded in a 96-well plate to culture for 24, 48, 72 and 96 h. Then, 10% CCK-8 reagent (ZP328-3; Zoman Biotechnology, Beijing, China) was added to incubate for another 2 h at 37°C to assess optical density (OD) values at 450 nm. Three independent experiments were performed.

Cell migration was detected within a 24-well Transwell chamber system (PIEP12R48; EMD Millipore, Billerica, MA, USA) (28–30). The OS-RC-2 and 786-O cells were seeded on a 6-well plate for incubation with 50 nM siPGRMC1 or siNC per well. After siPGRMC1 treatment for 24 h, cells were transfected with 2.5 µg pYR-ATP1A1 plasmids to culture for 48 h. Then, 8×10³ cells in 500 µl serum-free RPMI-1640 medium were seeded in the upper chamber of the Τranswell apparatus, and 500 µl RPMI-1640 medium with 10% FBS was supplemented in the bottom chamber. Migratory cells were fixed by methanol and stained with crystal violet. The migration cells were counted in five visual fields randomly selected from each membrane under an Olympus inverted microscope (Olympus Corp., Lake Success, NY, USA). Three independent experiments were performed. The data of experimental group and control group were input for statistical analysis.

RCC patients were divided into four groups based on PGRMC1 and ATP1A1 expression levels, including a high PGRMC1/negative ATP1A1 group (n=15), low PGRMC1/positive ATP1A1 group (n=16), low PGRMC1/negative ATP1A1 group (n=18) and high PGRMC1/positive ATP1A1 group (n=31). OS outcomes were evaluated by the Kaplan-Meier survival analysis method, with the log-rank test to compare groups. The Student's t test and post hoc test with ANOVA were used to compare the factors across groups. P<0.05 was considered to indicate a statistically significant difference.

The protein expression levels of PGRMC1 and ATP1A1 are shown in three randomly selected RCTs and their counterparts (Fig. 1A). PGRMC1 was overexpressed in RCTs when compared with their corresponding PKTs, whereas ATP1A1 was largely decreased. We further detected expression levels of these two proteins in 80 pairs of RCTs and PKTs by IHC analysis (Table I). The results demonstrated that PGRMC1 had an upregulated expression in RCTs compared with PKTs, but ATP1A1 protein was significantly downregulated (Fig. 1B). The average immunoreactivity score of PGRMC1 was 5.56±2.94 in 80 RCTs, which was higher than the average staining score 3.70±1.83 in 80 corresponding PKTs (P<0.001). On the other hand, the IHC score of ATP1A1 was 1.27±1.85 in 80 RCTs, which was significantly lower than the average staining score 8.35±2.96 in 80 RCTs (P<0.001). The IHC scores and clinical information for the RCC cases are provided in detail in Table II.

PGRMC1 and ATP1A1 are confirmed to be two proteins associated with RCC prognosis (17,18). We further validated whether the combination of two biomarkers can have a more favorable prognostic performance than each biomarker alone.

We compared the combined clinical value of ATP1A1 and PGRMC1 with the single molecule evaluation in RCC prognosis. The Kaplan-Meier estimates showed significantly higher OS rates for the RCC patients with low PGRMC1/positive ATP1A1 than the other three groups. The individuals with low PGRMC1/positive ATP1A1 also had the longest average OS. Conversely, the patients with high PGRMC1/negative ATP1A1 had the worst average OS time (73.1±8.87 months, P=0.04, Fig. 2). The 7-year OS rate of the low PGRMC1/positive ATP1A1 group was the highest of all groups (92.3%). It was significantly higher than those patients with high PGRMC1/negative ATP1A1 (46.7%, Table III).

PGRMC1 promotes activation of the PI3K/AKT signaling pathway (4,32). On the other hand, inhibitors of Na⁺/K⁺-ATPase can activate PI3K/AKT signaling pathways (33). We confirmed that the ATP1A1-mediated Raf/MEK/ERK signaling pathway is suppressed in RCC cells (18). Therefore, we hypothesized that PGRMC1 and ATP1A1 could play a combined role in RCC development via AKT phosphorylation.

In OS-RC-2 and 786-O cells, when PGRMC1 expression was upregulated due to a transient transfection of pFlag-PGRMC1 plasmids for 48 h, the relative level of p-AKT was also increased (Fig. 3A). In contrast, when PGRMC1 expression was suppressed by siRNA treatment for 48 h, the p-AKT level was correspondingly decreased (Fig. 3A). However, the expression level of ATP1A1 was not significantly changed in response to PGRMC1 overexpression or knockdown. On the other hand, ATP1A1 upregulation by transient pYR-ATP1A1 transfections inhibited the p-AKT level in OS-RC-2 and 786-O cells (Fig. 3B), and there was no significant change in PGRMC1 expression when ATP1A1 was upregulated. In general, the expression levels of PGRMC1 and ATP1A1 are independent of each other, but elevated PGRMC1 and downregulated ATP1A1 both enhanced the p-AKT level in the RCC cells.

To further explore the association of the interplaying signaling molecule p-AKT by PGRMC1 and ATP1A1 in RCC development, we analyzed p-AKT expression in RCTs. The p-AKT levels were increased in RCTs compared with their corresponding PKTs by western blot analysis (Fig. 4A). In addition, we evaluated p-AKT expression in 80 pairs of RCTs and PKTs by IHC. The average immunoreactivity score of p-AKT was 2.61±0.32 in 80 RCTs, which was significantly higher than the average staining score 0.69±0.11 in PKTs (Fig. 4B). The negative, weak, moderate and strong staining patterns of p-AKT are respectively shown in RCTs and PKTs (Fig. 4C).

Furthermore, we investigated the association of p-AKT with RCC patient survival outcomes. The Kaplan-Meier method indicated RCC patients with a low p-AKT expression (n=50) had a significant higher survival rate than those patients with a high p-AKT expression (n=30) (Fig. 5, P=0.011).

We desired to know whether a therapy targeting both PGRMC1 and ATP1A1 would have a better effect in vitro. Compared with the control group, co-transfection of pYR-ATP1A1 and siPGRMC1 exerted more enhanced inhibitory effects on cell proliferation (Fig. 6A and B) in 786-O and OS-RC-2 cells.

Meanwhile, cell migration by acting on PGRMC1/ATP1A1 proteins was significantly decreased by approximately 50% compared with targeting a single protein in the RCC cell lines (Fig. 6C) (P<0.05). We measured cell migration under the condition of PGRMC1 downregulation combined with ATP1A1 upregulation. The number of migrating cells was 34 following a combined regulation of PGRMC1 downregulation and ATP1A1 upregulation in the OS-RC-2 cells (Fig. 6C, the right bar marked with siPGRMC1+ATP1A1). On the other hand, the mean migrating cell number was 73 in OS-RC-2 cells following inhibition of PGRMC1 expression by siRNA, and the cell number was 62 in OS-RC-2 cells by only increasing the ATP1A1 level. A similar result was obtained in the 786-O cells. These results confirmed that the synergistic regulation of PGRMC1 downregulation along with ATP1A1 upregulation improved tumor inhibition potentials in RCC cells, which could contribute to the longer survival of RCC patients with low PGRMC1/positive ATP1A1 levels (Fig. 2).