Up-regulation of SR-BI promotes progression and serves as a prognostic biomarker in clear cell renal cell carcinoma

Patients and clinical tissue specimens

A total of 113 ccRCC samples and matched normal kidney tissues were collected from patients who underwent radical or partial nephrectomy at the Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). It was the first time for the patients to be diagnosed as ccRCC pathologically and none of them received any adjuvant treatments such as radiotherapy, chemotherapy or immunotherapy prior to surgery. All specimens were freshly snap-frozen in liquid nitrogen and stored in a deep freezer at −80 °C for RNA and protein extraction. Histological and pathological diagnosis of all specimens was confirmed by Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.

Follow up of patients

Patients’ survival condition was monitored after the operation. All the patients were followed up every 3 month in the first year, every 6 month in the second year, and then once every year thereafter. All follow-up related data or document files were acquired by phone contact or clinical examination at outpatient. The follow-up ranged from 5.06 to 90.12 months. PFS of patients was defined as the time interval from the date of first operation to the date of progression, or recurrence, or death. During the follow-up, patients whom we could not contact were recorded as censored.

Cell culture

Human ccRCC cell lines 786-O and Caki-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). HK-2, the human proximal tubule epithelial cell line, was also obtained from ATCC. All cells were cultured in Dulbecco’s modified Eagle medium (DMEM) /High Glucose (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) and antibiotics (100 μg/μl streptomycin and 100 U/μl penicillin, HyClone, Logan, UT, USA) at 37 °C in 5% CO₂.

Oil Red O staining and hematoxylin-eosin staining

Oil Red O (ORO) and hematoxylin-eosin (HE) staining were conducted to compare the lipids accumulation in ccRCC and non-cancerous kidney tissues according to the manufacturer’s instructions. Classical and typical images were captured as our intention.

RNA extraction and real-time quantitative reverse transcription PCR (qRT-PCR) analysis

Total RNA from cells and tissues was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) based on manufactures [39]. The concentration and quality of RNA were measured using Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA). Reverse transcription (RT) of RNA was conducted using Revertaid™ First Strand cDNA synthesis Kit (Fermentas, Vilnius, Lithuania) according to the manufacturer’s protocol. The qRT-PCR was performed with Platinum SYBR Green qPCR supermix UDG (Invitrogen, Carlsbad, CA) using synthesized primers from Genewiz (Suzhou, China). The amplification reaction conditions for SR-BI and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 10 min at 95 °C, 45 cycles at 95 °C for 10s, then 10s at 60 °C and 15 s at 72 °C. The primer set for SR-BI was 5′-CCTATCCCCTTCTATCTCTCCG-3′ (forward), and 5′-GGATGTTGGGCATGACGATGT-3′ (reverse). GAPDH gene served as an endogenous control with primers 5′-GGTGAAGGTCGGAGTCAACGG-3′ (forward) and 5′-GAGGTCAATGAAGGGGTCATTG-3′ (reverse). The relative expression of SR-BI was analyzed by normalizing to internal control GAPDH using the 2^−ΔΔCt method.

Protein extraction and western blot analysis

Total protein of tissues and cells was extracted using Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) containing protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) and phenylmethanesulfonyl fluoride (PMSF) (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The concentration of protein samples was determined using the Bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, USA) according to the manufacturer’s procedures. Equivalent amounts of protein sample (40-60 μg) were separated by 8% dodecyl sulfate, sodium salt (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Roche, Germany). After blocking with 5% (w/v) non-fat skimmed milk at room temperature for 1 h, the membranes were washed by phosphate buffer saline (PBS)-Tween20 (PBST) for 3 times with 10 min once, and then incubated with the indicated specific primary antibody overnight at 4 °C. The primary antibodies used for western blot analysis were polyclonal rabbit anti-SR-BI (Sigma-Aldrich, SAB2700477, USA, dilution at 1:1000), polyclonal mouse anti-β-actin (Beyotime, AF0003, China, dilution at 1:3000). After washing membranes with PBST three times, the membranes were then incubated with goat anti-mouse or goat anti-rabbit secondary antibodies (Promega, Madison, WI, USA) conjugated with horseradish peroxidase (HRP) at a dilution of 1:2500 at room temperature for 2 h. After washing three times again, the stained proteins on bands were visualized and quantified using ChemiDoc-XRS+ (Bio-Rad, USA) by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific Inc., Waltham, MA).

Immunohistochemical staining

The expression pattern and localization of SR-BI protein were assessed by immunohistochemical (IHC) analysis in paraffin-embedded sections of ccRCC tissues and normal kidney tissues based on the operating steps. The primary polyclonal rabbit antibody against SR-BI (Sigma-Aldrich, SAB2700477, USA) was used at a dilution of 1:250. Representative images under the microscope were captured and recorded.

RNA interference and transfection

Small interfering RNA (siRNA) targeting SR-BI mRNA (si-SR-BI) and scrambled negative control (si-NC) was designed and synthesized by Genepharma (Shanghai, China). The sequences of si-SR-BI were as follows: 5′-CAAGUUCGGAUUAUUUGCUTT-3′ (sense), and 5′-AGCAAAUAAUCCGAACUUGTT-3′ (antisense). Twenty-four hours before transfection, cells were seeded into a 6-well plate (Corning Costar, New York, NY, USA) at a density of (3-5) × 10⁵/ml, making sure that the confluence of cells was at about 80% the next day. Transfection of si-SR-BI or si-NC was then performed by using lipofectime2000 (Invitrogen) according to the manufacturer’s protocol, with a final concentration of 100 nmol/L. Neither antibiotics nor serum was involved during the process of transfection and the medium was replaced by new complete medium 6 h later.

Wound closure assay

According to procedures [40], cells were seeded into a 6-well plate and cultured in complete growth medium until 80% confluences. After 48 h of siRNA transfection, a 10 μL pipette tip was used to make a vertical wound against the surface of well bottom through the cell monolayer carefully and quickly. The medium and cell debris were aspirated away carefully and cells were washed 3 times with warm PBS. Appropriate fresh culture medium was then added to the well against the wall slowly. Initial pictures of the wound at time 0 were taken under light microscope. The plate was then incubated at 37 °C in 5% CO₂ and typical images of the wound were captured at indicated time points.

Cell proliferation assay

The ability of cell proliferation was measured by using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Sigma, USA). Briefly, 200 μL complete DMEM/High Glucose containing 3 × 10³ cells was added into each well of a 96-well plate 48 h after transfection. Then, 20 μL MTT solution per well was added and the plate was incubated at 37 °C for another 4 h. At the indicated time points, the medium was removed and 150 μL dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals with shaking gently. Subsequently, absorbance values at 490 nm wavelength were measured and quantified using a microplate reader (BioTek, USA). All experiments at each time point were performed in triplicate with six replicate wells.

Plate colony formation assay

Cells transfected with si-NC or si-SR-BI for 48 h were plated into 6-well plates at a density of 1.0 × 10³ cells per well and maintained in an incubator for 2 weeks with the medium replaced every 3 days. Then, cells were washed with PBS 3 times, fixed with methanol for 20 min and stained by 0.1% crystal violet for 15 min. After washing with PBS 3 times again, the images of colonies were captured and the numbers of colonies containing no less than 50 cells were counted.

Transwell cell migration and invasion assay

The abilities of cell migration and invasion in vitro were evaluated by performing transwell assays. As described before [41], A 24-well transwell chamber with 8 μm pore size (Corning Costar, New York, NY, USA) was used for this experiment. Briefly, cells were seeded into 6-well plates and transfected with si-NC or si-SR-BI. After transfection for 24 h, cells were harvested, centrifuged and resuspended with serum-free culture medium. Cells were then counted under microscope to get the concentration. Subsequently, cells contained in 100 μL culture medium with no serum were loaded into upper chamber at a pre-determined density (3.0 × 10⁴/well for 786-O and 5.0 × 10⁴/well for Caki-1). While in the lower insert, 500 μL complete medium with 10% FBS was added as a kind of chemo-attractant. It should be noted that the upper chamber used for invasion assay was pre-coated appropriate amount matrigel (BD Biosciences) at a dilution of 1:8 and dried in a cell incubator for 5 h prior to cell seeding. Twenty-four hours post-incubation at 37 °C in 5% CO₂, cells remained on the upper surface of the membrane were removed gently, whereas those migrated the filter membrane and attached to the lower side of it were fixed by methanol and stained with 0.1% crystal violet after washing with PBS. Photographs were taken and cells number was counted microscopically.

Determination of HDL-cholesterol content

To explore whether HDL-cholesterol content was changed after down-regulation of SR-BI, we tested and quantified the content in ccRCC cells and extracellular media after transfection with siRNA in 786-O and Caki-1 cells. HDL-cholesterol content was determined by using the HDL-cholesterol kit (Nanjing Jiancheng bioengineering Institute, A112-1, Nanjing, China) following manufacturer’s instructions. Transfected cells were also stained with ORO to assess the changes.

Statistical analysis

All statistical analyses were performed by SPSS16.0 software (SPSS, Inc., Chicago, IL, USA) or GraphPad Prism version5.01 (GraphPad Software, Inc., CA, USA). All continuous results were expressed as means ± standard deviation (SD). A P-value <0.05 was considered statistically significant.

Enhanced SR-BI expression in ccRCC tissues and cell lines

It has been reported that SR-BI played vital roles in several human cancers. However, we know little about SR-BI in ccRCC. Hence, to explore the role of SR-BI in ccRCC, we examined SR-BI expression in ccRCC tissues and cell lines by using qRT-PCR and western blot. As shown in Fig. 1a-c, the expression of SR-BI was significantly increased in ccRCC tissues compared with normal matched tissues. Furthermore, a similar result was observed in tumor cell lines. As shown in Fig. 1b-d, the expression of SR-BI was significantly increased in 786-O and Caki-1 cells compared with HK-2 cells. IHC staining also showed that protein level of SR-BI was much higher in cancerous tissues (Fig. 1e).

Increased accumulation of lipid droplets in ccRCC tissues

To verify that there are many lipid droplets in ccRCC tissues, ORO and HE staining was performed. By using the light microscope, we found that lipid droplets were widely stained red in ccRCC tissues, whereas no obvious difference was observed in paired normal kidney samples by ORO staining. HE staining also showed much higher content of lipid droplets in cancerous tissues (Fig. 1e).

Associations between SR-BI mRNA expression and clinicalpathological features of ccRCC

Prognostic value of SR-BI mRNA expression in ccRCC

To further clarify the clinical value of SR-BI mRNA expression in ccRCC patients, Kaplan-Meier analysis and log-rank test was conducted to determine whether the PFS was associated with the expression of SR-BI in tumors. As shown in Fig. 2a, the patients with tumors that expressed high levels of SR-BI had a shorter PFS survival (P = 0.0062). In addition, we also detected the mRNA expression of SR-BI in ccRCC cancerous tissues with distant metastasis. As shown in Fig. 2b, the expression of SR-BI in patients with distant metastasis was significantly higher than those without metastasis (P = 0.0019). To further confirm whether SR-BI mRNA level was an independent prognostic factor of clinical outcome, Cox proportional hazards regression models were performed. By using univariate analysis (Table 2), the gender (P = 0.029), tumor size (P = 0.004), lymph node metastasis (P < 0.001), distant metastasis (P = 0.001), TNM stage (P < 0.001) and SR-BI mRNA level (P = 0.009) were significantly related to the PFS of ccRCC patients, suggesting SR-BI serves as a prognostic factor. However, multivariate analysis (Table 2) indicated that only increased SR-BI mRNA expression (P = 0.021), gender (P < 0.001) and tumor size (P = 0.002) were independent prognostic factors.

Variables	Univariate analysis			Multivariate analysis
	HR	95%CI	P value	HR	95%CI	P value
Gender	0.384	0.163–0.908	0.029	0.131	0.046–0.374	0.000
Age	1.144	0.526–2.489	0.735
Tumor size	8.742	2.036–37.546	0.004	11.696	2.433–56.216	0.002
Furman grade	1.704	0.800–3.628	0.167
T stage	1.610	0.700–3.703	0.263
N metastasis	5.593	2.409–12.984	0.000	2.786	0.904–8.580	0.074
M metastasis	5.730	2.107–15.583	0.001	2.221	0.714–6.909	0.168
TNM stage	4.363	2.014–9.451	0.000	1.895	0.654–5.494	0.239
Venous invasion	1.605	0.483–5.338	0.440
Location side	1.022	0.487–2.147	0.954
SR-BI expression	3.014	1.317–6.898	0.009	2.926	1.175–7.285	0.021

HR hazard ratio, CI confidence interval

Knockdown of SR-BI suppresses ccRCC cells proliferation and plate colony formation

To exploit the biological function of SR-BI in ccRCC carcinogenesis and progression, we used siRNA to knockdown endogenous SR-BI expression in vitro. As shown in Fig. 3a-b, SR-BI expression was effectively inhibited by siRNA in ccRCC cell lines. MTT assay was then conducted to assess the impact of SR-BI on cell proliferation. The results showed that knockdown of SR-BI could significantly decrease the proliferative ability of ccRCC cells (Fig. 3c). In consistence with the results, ccRCC cells transfected with si-SR-BI formed fewer colonies than those transfected with si-NC (Fig. 3d-e).

Knockdown of SR-BI impairs ccRCC cells invasion and migration in vitro

The capacity of cell invasion and migration means much in cancer development, progression and metastasis. Next, we explored whether depletion of endogenous SR-BI could attenuate the invasion and migration of ccRCC cells in vitro. Transwell invasion and wound healing assays were performed to verify the hypothesis. As shown in Fig. 4a-b, ccRcc cells treated with si-SR-BI reduced the potential of migration and invasion dramatically compared with the cells transfected with si-NC. Meanwhile, the number of migrated cells treated with si-SR-BI was much fewer. Accompanied by the results, wound healing assay proved that SR-BI knockdown closed the gap much slower (Fig. 4c-f).

Knockdown of SR-BI inhibited the up-take of HDL-cholesterol

As shown in Fig. 5a, ccRCC cells transfected with siRNA were stained by ORO, and we found that knockdown of SR-BI inhibited the proliferation of cancer cells. More importantly, cells transfected with si-NC showed much fresher red color than cells treated with si-SR-BI, which meant that there was much lower HDL-cholesterol content in cells treated with si-SR-BI, when HDL receptor was knocked down. The quantification test also showed that there was much less HDL-cholesterol in cells (P = 0.0147 for 786-O, P = 0.0015 for Caki-1) while much more in extracellular media (P = 0.0050 for 786-O, P = 0.0007 for Caki-1) with down-regulation of SR-BI (Fig. 5b-c).

Knockdown of SR-BI induced the reduction of AKT, p-AKT and MMP-2/9

It has been widely recognized that PI3K/AKT pathway is tightly correlated with cell metabolisms [42, 43]. More persuasively, the concept was verified in prostate cancer [16]. Recently, It is well known that matrix metalloproteinases (MMP) especially MMP-2/9 played vital roles in cancer progression [44, 45]. Hence, we confirmed whether knockdown of SR-BI would influence the expression of AKT, p-AKT and MMP-2/9. As shown in Fig. 6, the protein expression of AKT, p-AKT and MMP-2/9 were all dramatically decreased when ccRCC cells were transfected by si-SR-BI (Fig. 6).