*Contributed equally
A-kinase interacting protein 1 (AKIP1) has previously been demonstrated to be overexpressed in clear cell renal cell carcinoma (ccRCC) tissues and is associated with patient prognosis. The aim of the present study was to explore whether AKIP1 can affect the proliferation, invasion, migration and angiogenesis of ccRCC cells via its interaction with Rac1. Furthermore, the influence of AKIP1 and therefore Rac1 on the expression of the downstream ERK/cellular (c)-Myc signaling pathway was explored. The interaction between AKIP1 and Rac1 was determined using co-immunoprecipitation. The mRNA and protein expression levels of AKIP1 and Rac1 in normal renal epithelial cell lines and ccRCC cell lines were detected using reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively. The transfection efficiency of small interfering RNA-AKIP1 and the Rac1 overexpression vector were also confirmed using RT-qPCR and western blotting. The viability, proliferation, invasion and migration of ccRCC cells following transfection were analyzed using the Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Transwell and wound healing assays, respectively. The tube formation ability of HUVECs was assessed using the tube formation assay. The protein expression levels of proliferation, invasion, migration and tube-formation-associated proteins as well as proteins associated with the ERK/c-Myc signaling pathway, were detected via western blotting. The results demonstrated that AKIP1 expression levels were increased in ccRCC cell lines. AKIP1 knockdown inhibited the proliferation, invasion and migration of ccRCC cells and HUVEC tube-formation. In addition, AKIP1 was demonstrated to bind to Rac1 in ccRCC cells and AKIP1 downregulation inhibited Rac1 expression. Furthermore, Rac1 overexpression reversed the effects of AKIP1 knockdown on ccRCC cells. AKIP1 knockdown also suppressed the ERK/c-Myc signaling pathway, which was reversed by Rac1 overexpression. In conclusion, AKIP1 knockdown potentially suppressed the proliferation, invasion, migration and angiogenesis of ccRCC cells and inhibited the ERK/c-Myc signaling pathway by binding to Rac1.
Clear cell renal cell carcinoma (ccRCC) is one of the commonest tumors of the urinary system. Each year 202,000 cases of ccRCC are diagnosed and there are 102,000 ccRCC-related deaths worldwide (
A-kinase interaction protein 1 (AKIP1) was initially reported as novel human breast cancer-associated gene 3, which encodes a selectively splicing proline-rich protein (
A previous study reported that AKIP1 binds to Rac1(
The aim of the present study was to investigate whether AKIP1 can affect the proliferation, invasion, migration and angiogenesis of ccRCC cells via its interaction with Rac1. Furthermore, the influence of AKIP1 and Rac1 on the expression of the downstream ERK/c-Myc signaling pathway was explored.
Normal renal epithelial cell lines (HKC-5 and HK-2), human umbilical vein endothelial cells (HUVECs) and ccRCC cell lines (NRCC, Caki-1 and A498) were purchased from BioVector NTCC, Inc. HKC-5 and HK-2 cells were cultured in minimum essential medium (MEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin. ccRCC cells were cultured in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS and 1% penicillin-streptomycin. HUVECs were cultured in endothelial cell basal medium (EBM; Cambrex Bio Science Rockland Ltd.) containing endothelial cell growth medium supplements (EGM; Cambrex Bio Science Rockland Ltd.), 10% FBS, 100 IU/ml penicillin, and 100 mg/ml streptomycin. All cells were cultured at 37˚C in a humidified atmosphere of 95% air and 5% CO2. The approval of Ethics Committee of Beijing Ditan Hospital was received for the use of human primary cell lines.
The small interfering (si)RNA-negative control (NC), siRNA-AKIP1-1/2, empty vector (Ov-NC) and Ov-Rac1 constructs were purchased from Guangzhou Ribo Biotechnology Co., Ltd. (
Total RNA was extracted from ccRCC cells using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and complementary DNA was synthesized from RNA using the PrimeScript RT reagent kit (Takara Bio, Inc.). The following temperature protocol was used for reverse transcription: 37˚C for 50 min followed by 70˚C for 15 min. Subsequently, qPCR was performed using the Kapa SYBR® FAST qPCR Master Mix (Takara Bio, Inc.) and was analyzed using the MyGo PCR detection system (IT-IS Life Science Ltd.). The thermocycling conditions were: 10 min initial denaturation at 94˚C, 15 sec denaturation at 94˚C and 30 sec of annealing at 55˚C (40 cycles) and final extension for 1 min at 72˚C. The mRNA expression levels of AKIP1, VEGFA, VEGFR2 and Rac1 were quantified using the 2-∆∆Cq method (
Total protein in ccRCC cells was extracted using RIPA buffer (Beyotime Institute of Biotechnology) and the protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). Total protein (40 µg total protein/lane) was separated using 10% SDS-PAGE. Separated protein was transferred to a PVDF membrane (MilliporeSigma). Subsequently membranes were blocked with 5% non-fat milk for 1 h at room temperature and were then incubated with primary antibodies overnight at 4˚C. The primary antibodies used were as follows: Anti-AKIP1 (1:1,000; cat. no. ab135996; Abcam), anti-Ki67 (1:5,000; cat. no. ab92742; Abcam), anti-proliferating cell nuclear antigen (PCNA; 1:5,000; ab92552; Abcam), anti-MMP2 (1:5,000; ab92536; Abcam), anti-MMP9 (1:5,000; ab76003; Abcam), anti-VEGFA (1:1,000; ab214424; Abcam), anti-VEGFR2 (1:5,000; ab134191; Abcam), anti-Rac1 (1:1,000; ab155938; Abcam), anti-p-ERK (1:1,000; ab201015; Abcam), anti-ERK (1:10,000; ab184699; Abcam), anti-c-Myc (1:1,000; ab32072; Abcam) and anti-GAPDH (1:2,500; ab9485; Abcam). Membranes were then washed in TBS with 0.1% Tween-20 three times. Following the primary incubation membranes were incubated with the HRP-conjugated secondary antibody (1:2,000; cat. no. ab6721; Abcam) at room temperature for 1 h. The blots were visualized using ECL reagent (Pierce; Thermo Fisher Scientific, Inc.) and the band intensity was semi-quantified using ImageJ 1.51 software (National Institutes of Health).
Following transfection, ccRCC cells were seeded into 96-well plates at a density of 2x103 cells/well and were incubated for 24, 48 and 72 h at 37˚C. A total of 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added to each well for 2 h at 37˚C. The optical density at 450 nm was assessed using a microplate reader.
Following transfection, ccRCC cells were seeded into a culture flask and incubated with diluted EdU solvent for 24 h at room temperature. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 min and stained with Alexa Fluor 594 (cat. no. ab150084; 1:200; Abcam) overnight at room temperature. Cell nuclei were counterstained with DAPI for 15 min at room temperature. Cell proliferation was observed using a fluorescence microscope (Eclipse 80i; Nikon Corporation).
Following transfection, ccRCC cells were seeded at a density of 4x104 cells into the upper Transwell chamber that was pre-coated with Matrigel (BD Biosciences) for 1 h at 37˚C. In the lower chamber 1 ml DMEM/F12 medium was added. Subsequently the cells were incubated at 37˚C for 24 h. After removing the cells on the upper surface of the chamber the remaining cells were fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were then stained with 0.1% crystal violet (Sangon Biotech Co., Ltd.) for 1 h at room temperature. The number of cells was quantified using a light microscope (Olympus Corporation) and invasive ability was assessed using ImageJ 1.51 software.
Following transfection, ccRCC cells were seeded into 6-well plates and incubated until >90% confluency. A sterile 200 µl pipette tip was used to create a 0.5-1 cm horizontal line. After washing with PBS the scratched cells were cultured in serum-free medium for 24 h. The width of the scratch was imaged at 0 and 24 h using a light microscope (Olympus Corporation) and the migratory ability of the cells was quantified using ImageJ 1.51 software (National Institutes of Health).
The transfected ccRCC cells were cultured in MEM until they reached 70% confluency. Following washing with PBS, ccRCC cells were cultured in serum-free MEM for 48 h at 37˚C. The culture media (CM) was collected following centrifugation at 500 x g at room temperature for 10 min. Early passage (<Phase 4) HUVECs were seeded at 3x104 cells/well on Matrigel-coated 48-well plates. HUVECs were then incubated with the CM for 6 h at 37˚C. HUVEC tube formation was observed and imaged using a phase-contrast inverted microscope.
Following transfection, ccRCC cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing 1% PMSF and 1% protease inhibitor, centrifuged at 24,148.8 x g for 10 min at 4˚C. A part of the cell lysate was isolated as input and 250 µl of lysates were incubated with 1 µg anti-AKIP1 (cat. no. LS-C309384; LSBio) or anti-Rac1 (cat. no. sc-514583; Santa Cruz Biotechnology, Inc.) and homologous IgG antibodies (cat. no. ab172730; Abcam and cat. no. sc-2025; Santa Cruz Biotechnology, Inc.) at 4˚C overnight. Subsequently, 25 µl protein A/G beads (Cell Signaling Technology, Inc.) were added to the lysis solution and the solution was incubated for 1 h at 4˚C. Then, the supernatant was centrifuged for 3 min at 1,509.3 x g at 4˚C. The beads were extracted and were incubated with protein loading buffer at 100˚C for 15 min to isolate the proteins, which were subsequently detected via western blotting.
Data are presented as the mean ± SD. All statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). One-way ANOVA followed by Tukey's post hoc test was used to perform statistical analysis among more than two groups. P<0.05 was considered to indicate a statistically significant difference.
The results demonstrated that AKIP1 mRNA and protein expression levels were increased in ccRCC cells compared with normal renal epithelial cells. Furthermore, AKIP1 mRNA and protein expression levels in Caki-1 cells were the highest among all ccRCC cell lines tested (
ccRCC cells were transfected with siRNA-NC or siRNA-AKIP1-1/2 and AKIP1 mRNA, and protein expression levels were determined. The results demonstrated that AKIP1 mRNA and protein expression levels were markedly decreased in the siRNA-AKIP1-1/2 groups compared with siRNA-NC group. The transfection efficiency in the siRNA-AKIP1-1 group was greater than that of the siRNA-AKIP1-2 group and therefore the siRNA-AKIP1-1 group was selected for use in the subsequent experiments (
The results demonstrated that the invasion and migration of Caki-1 cells were inhibited in the siRNA-AKIP1 group compared with the control group, whereas no change was observed between the siRNA-NC and control groups (
The CM of siRNA-NC-transfected Caki-1 cells had no effect on HUVEC tube-formation, whereas tube formation was suppressed by AKIP1 knockdown (
The results demonstrated that Rac1 mRNA and protein expression levels were increased in ccRCC cells and that the highest expression levels of Rac1 were demonstrated in Caki-1 cells (
The results demonstrated that Ov-Rac1-transfected Caki-1 cells exhibited upregulated Rac1 mRNA and protein expression levels (
The results demonstrated that Rac1 overexpression increased the invasion and migration of Caki-1 cells transfected with siRNA-AKIP1 (
The results demonstrated that HUVEC tube-formation was increased in the CM of Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1, compared with the CM of Caki-1 cells transfected with siRNA-AKIP1 alone (
The results demonstrated that AKIP1 knockdown inhibited the expression of phosphorylated (p)/total (t)-ERK and c-Myc in Caki-1 cells. Furthermore, Rac1 overexpression increased the expression levels of p/t-ERK and c-Myc in Caki-1 cells transfected with siRNA-AKIP1 (
AKIP1 was first identified in breast and prostate cancer cell lines and serves an important role in normal physiological processes (
A previous study reported that AKIP1 can bind to Rac1(
Rac1 activates the ERK signaling pathway in hepatocellular carcinoma and colon cancer to regulate the proliferation, invasion and migration of cancer cells (
In conclusion, AKIP1 knockdown potentially suppressed the cell proliferation, invasion, migration and angiogenesis of ccRCC cells and may have suppressed the ERK/c-Myc signaling pathway by binding to Rac1. However, this phenomenon was potentially reversed by Rac1 overexpression. Therefore, the results of the present study provided a theoretical basis for the treatment of ccRCC and showed an interaction between AKIP1 and Rac1, while the functional relationship between these two proteins will be explored in the future.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YZ, HZ, ZH and QL contributed to the study design. XW, XL, PY and SJ performed the experiments and analyzed the data. YZ and HZ contributed to the experiments and writing and performed the secondary data analyses and revising the manuscript for intellectual and scientific content. ZH and XW contributed to the conception of the study. YZ and HZ confirm the authenticity of all the raw data. All authors read and approved the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
AKIP1 expression increases in ccRCC cell lines. (A) mRNA and (B) protein expression of AKIP1 in ccRCC and normal renal epithelial cell lines were detected by reverse transcription-quantitative PCR and western blotting. ***P<0.001 vs. HKC-5 group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1.
AKIP1 knockdown inhibits the proliferation of ccRCC cells. (A) mRNA (B) and protein expression of AKIP1 in Caki-1 cells transfected with siRNA-AKIP1-1 and siRNA-AKIP1-2 were detected by reverse transcription-quantitative PCR and western blotting. (C) The viability of Caki-1 cells transfected with siRNA-AKIP1 was analyzed by Cell Counting kit-8 assay. (D) The expression of proliferation-related proteins in Caki-1 cells transfected with siRNA-AKIP1 was detected by western blotting. (E) The proliferation of Caki-1 cells transfected with siRNA-AKIP1 was analyzed by 5-Ethynyl-2'-deoxyuridine staining (magnification, x200). **P<0.01 and ***P<0.001 vs. control group; ##P<0.01 and ###P<0.001 vs. siRNA-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control.
AKIP1 knockdown inhibits the invasion and migration of ccRCC cells. (A) The invasion (magnification, x100) and (B) migration of Caki-1 cells transfected with siRNA-AKIP1 were determined by Transwell and wound healing assays (magnification, x200). (C) The expression of metastasis-related proteins in Caki-1 cells transfected with siRNA-AKIP1 was detected by western blotting. ***P<0.001 vs. control group; ###P<0.001 vs. siRNA-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control.
AKIP1 knockdown inhibits the angiogenesis in ccRCC. (A) The angiogenesis in CM from Caki-1 cells transfected with siRNA-AKIP1 was detected by HUVEC tube-formation assay. The (B) mRNA and (C) protein expression of angiogenesis-related proteins in CM from Caki-1 cells transfected with siRNA-AKIP1 were detected by reverse transcription-quantitative PCR and western blotting. **P<0.01 and ***P<0.001 vs. control (CM)+HUVEC group; ##P<0.01 and ###P<0.001 vs. siRNA-NC (CM)+HUVEC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control; CM, culture media.
AKIP1 binds to Rac1 in ccRCC cells and its downregulation inhibits the Rac1 expression. The (A) mRNA and (B) protein expression of Rac1 in ccRCC cell lines and normal renal epithelial cell lines were detected by reverse transcription-quantitative PCR and western blotting. ***P<0.001 vs. HKC-5 group. The interaction of (C) AKIP1 and (D) Rac1 was confirmed by co-immunoprecipitation assay. ***P<0.001 vs. input group; ###P<0.001 vs. IgG group. (E) The expression of Rac1 in Caki-1 cells transfected with siRNA-AKIP1 was detected by western blotting. (F) The expression of AKIP1 in Caki-1 cells transfected with siRNA-AKIP1 was detected by western blotting. ***P<0.001 vs. control group; ###P<0.001 vs. siRNA-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control.
AKIP1 participates in the proliferation of ccRCC cells by binding to Rac1. The (A) mRNA and (B) protein expression of Rac1 in Caki-1 cells transfected with Ov-Rac1 were detected by reverse transcription-quantitative PCR and western blotting. ***P<0.001 vs. Control group; ###P<0.001 vs. Ov-NC group. (C) The viability of Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 was analyzed by Cell Counting Kit-8 assay. (D) The expression of proliferation-related proteins in Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 was detected by western blotting. (E) The proliferation of Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 was analyzed by 5'-Ethynyl-2'-deoxyuridine staining. **P<0.01 and ***P<0.001 vs. control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. siRNA-AKIP1 group; ++P<0.01 and +++P<0.001 vs. siRNA-AKIP1+Ov-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control; Ov, overexpression.
AKIP1 participates in the invasion and migration of ccRCC cells by binding to Rac1. (A) The invasion and (B) migration of Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 were determined by Transwell and wound healing assays. (C) The expression of metastasis-related proteins in Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 was detected by western blotting. ***P<0.001 vs. control group; ###P<0.001 vs. siRNA-AKIP1 group; +++P<0.001 vs. siRNA-AKIP1+Ov-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control; Ov, overexpression.
AKIP1 participates in the angiogenesis in ccRCC by binding to Rac1. (A) The angiogenesis in CM from Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 was detected by HUVEC tube-formation assay. The (B) mRNA and (C) protein expression of angiogenesis-related proteins in CM from Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 were detected by reverse transcription-quantitative PCR and western blotting. ***P<0.001 vs. control (CM)+ HUVEC group; ##P<0.01 and ###P<0.001 vs. siRNA-AKIP1 (CM)+HUVEC group; ++P<0.01 and +++P<0.001 vs. siRNA-AKIP1+Ov-NC (CM)+HUVEC group. CM, culture media; ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control; Ov, overexpression.
AKIP1 regulates the expression of ERK/c-Myc signaling pathway in ccRCC cells by binding to Rac1. The expression of p-ERK, ERK and c-Myc in Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 was detected by western blotting. ***P<0.001 vs. control group; ###P<0.001 vs. siRNA-AKIP1 group; +++P<0.001 vs. siRNA-AKIP1+Ov-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control; Ov, overexpression; c-Myc, cellular-Myc.