The authors have declared that no competing interests exist.
Conceived and designed the experiments: MSZ GD RD. Performed the experiments: MSZ ST VS GD TC. Analyzed the data: MSZ GD SS SM. Contributed reagents/materials/analysis tools: MSZ SF IC SA HH KU KS YT. Wrote the paper: MSZ.
miR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported.
We measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3′UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the
The current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.
miRNAs have been shown to be involved in various kidney diseases
In this study, we examined the role of miR-23b-3p in renal clear cell carcinoma. We found that expression of miR-23b-3p was increased in A-498 and Caki-2 cell lines and a majority of tissue samples. Increased expression also correlated with lower survival rate for renal cell carcinoma patients. Inhibition of miR-23b-3p expression in A-498 and Caki-2 cells caused an increase in apoptosis and a decrease in invasive properties. PTEN protein expression was found to be increased after knocking down miR-23b-3p in A-498 cells with a concomitant decrease in the expression of PI3-kinase, total Akt and IL-32. The PTEN gene was shown to be a direct target of miR-23b-3p by 3′ luciferase reporter assays. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high.
To understand the clinical relevance of miR-23b in kidney cancer we measured miR-23b-3p levels in 29 pairs of human kidney cancers (all clear cell renal cell carcinoma) and matched normal tissues by real-time PCR. In 15 cases (52%), miR-23b-3p was found to be increased (T/N [Tumor/Normal] was greater than 1.2). In 5 samples (17%) the expression was moderate (T/N 0.8–1.2). Expression of miR-23b-3p was also measured in cultured renal cancer cell lines, A-498, ACHN, Caki-1, Caki-2 and non malignant normal HK-2 cells. miR-23b-3p expression in A-498 cells was approximately 11x that of HK-2 cells while that of ACHN was 4.1x, Caki-1 4.8x, and Caki-2 5.8x (
A) Ratio of T/N expression in tissue samples was found to be high in a majority of samples. B) A-498 and Caki-2 cells had high expression of miR-23b-3p as compared to non malignant normal HK-2 cells (T-Tumor,N-Normal), [p value (A) 0.032; p value (B) 0.018)].
The expression level of miR-23b-3p correlated with 5 year survival of the patients. For those patients with moderate to low miR-23b-3p expression (n = 12) (
To elucidate the functional role of miR-23b-3p in renal cancer we knocked down the expression of miR-23b-3p in A-498 and Caki-2 renal cancer cells with a commercially available miR-23b-3p inhibitor. Anti-miR-Negative control #1 was also used for these experiments. The miR-23b-3p level was reduced by more than 99%, as compared to the negative control in A-498 cells (
Expression of miR-23b-3p was decreased by more than 99% after its inhibition in both A-498 (A) and Caki-2 cells (B).
The effects of miR-23b-3p knock down on the cell cycle and apoptosis were analyzed by flow cytometry. Apoptosis assay showed a significant increase in the number of total apoptotic cells (early apoptotic plus apoptotic) in both A-498 (8.87%) (
A) Apoptosis assay showed a significant increase in the number of total apoptotic cells (8.87%) in the anti- miR-23b-3p inhibitor transfected cells as compared to the negative control (3.37%) in A-498 cells (p value 0.029). B) In Caki-2 cells the total number of apoptotic cells (11.74%) in the anti- miR-23b-3p inhibitor transfected cells as compared to the negative control (3.68%) (p value 0.030).
To determine whether miR-23b-3p affects renal cancer cell migration and invasion a cytoselect 24-well cell migration and invasion kit was used. A-498 and Caki-2 cells were transfected with anti-miR-23b-3p inhibitor and anti-miR-Negative control. Both A-498 and Caki-2 cells showed a 30% decrease in cell invasion in the samples where miR-23b-3p was inhibited as compared to negative controls (
Invasive properties of A-498 (A) and Caki-2 (B) cells were decreased by 30% after miR-23b-3p knock down compared to controls. Y-axis-Absorbance at 560 nm [p value(A) 0.026; p value(B) 0.030].
To see the effect of miR-23b-3p expression on different genes involved in apoptosis and invasion we checked the protein expression of several genes involved in these processes. The pro apoptotic gene PTEN was found to be up regulated after miR-23b-3p knock down in A-498 cells (
A) Expression of PTEN, PI3-kinase, Akt and IL-32 in A-498 cells, compared to negative control. GAPDH was used as a normalizing control. B) Quantitative data for Western blot analysis. PTEN was up regulated, whereas PI3-kinase, Akt and IL-32 expression were down regulated.
Since a significant increase in apoptosis was observed in both A-498 and Caki-2 cells and increased PTEN expression in A-498 cells after miR-23b-3p knock down, we looked to see if miR-23b-3p targets the pro apoptotic gene PTEN. Using TargetScan (targetscan.org) and microrna.org we identified complementary sequences to miR-23b in the 3′UTR of the PTEN gene (
A) Software analysis shows sequences complementary to the seed sequences of miR-23b-3p in the 3′ UTR of PTEN gene. B) Luciferase assay showed a decrease in relative luciferase units in samples co transfected with miR-23b-3p was and PTEN 3′ UTR gene construct as compared to control constructs (Neg Con-negative control; Con-Control plasmid construct lacking PTEN 3′UTR sites for miR-23b-3p; PTEN-PTEN plasmid construct having PTEN gene 3′UTR sites for miR-23b-3p; (p value 0.017).
To further confirm that PTEN is a target of miR-23b-3p we checked for expression of PTEN protein in normal and cancer regions of patient tissue samples which had a high expression of miR-23b-3p, using immunohistochemistry (IHC). Ten samples having high miR-23b-3p expression were selected. A representative example of PTEN immunohistochemistry is shown in
A) Representative pictures of Immunohistochemistry (IHC) analyses. B) Graphical representation of the data showing that PTEN expression was high in normal regions of renal cancer patient samples whereas in cancerous regions having high miR-23b-3p expression PTEN was not observed.
IHC staining results for tissues were graded according to quick score (percent cells stained × intensity of stain). Quick score obtained from the tumor samples was normalized with the quick score of their normal samples (T/N) and Chi-square test was performed with the miR-23b-3p expression levels in the same patient samples (
We also examined the effect of the chemoprevention agent, genistein (a soy product), on the expression of miR-23b-3p in A-498 cells. Treatment with genistein (25 µM) for 96 hours decreased the expression of miR-23b-3p by 50%, whereas a higher concentration of genistein (50 µM) for the same period of time decreased expression by 45% (
miR-23b-3p expression was reduced by 50% in 25 µM genistein treated A-498 cells as compared to untreated cells, 50 µM genistein decreased miR-23b-3p expression by 45% (p value 0.030).
The present study shows that miR-23b-3p functions as an oncogenic microRNA in renal cell carcinoma. Its expression is increased in renal cell carcinoma cell lines and tissue samples (clear cell carcinoma) as compared to normal renal cells and matched normal tissues, respectively. In addition we found that high levels of miR-23b-3p lead to lower survival for renal cancer patients. To study the functional role of miR-23b-3p its expression was knocked down in A-498 and Caki-2 renal cancer cell lines. This led to an increase in the total number of cells undergoing apoptosis in both cell lines compared to controls. However, there was no significant change in the cell cycle. In addition A-498 and Caki-2 cells showed decreased cell invasion in miR-23b-3p knocked down cells but no significant change in migration. Western analysis of miR-23b-3p knock down showed increased PTEN expression and concomitant decreases in the expression of PI3-kinase, Akt and IL-32. PTEN was found to be a direct target of miR-23b-3p through reporter gene assays.
The significant change in the number of apoptotic cells after knock down of miR-23b-3p in both A-498 and Caki-2 cells lead us to search for genes and pathways involved in apoptosis. Previous studies have shown that PTEN can induce apoptosis through a PI3-kinase dependent pathway
Genistein (40,5,7-trihydroxyflavone), one of the principal soy isoflavones, has a wide array of chemopreventive actions. The anticancer effects of genistein have been ascribed to several signaling pathways and mechanisms that affect cell cycle arrest, apoptosis, invasion, metastasis and angiogenesis, attributes that could potentially prevent tumor initiation and progression
In summary, miR-23b-3p is an oncogene in RCC and directly inhibits the PTEN tumor suppressor gene. Thus miR-23b-3p may be useful as a renal cancer diagnostic marker and as a therapeutic target for the treatment of renal cell carcinoma.
Formalin-fixed, paraffin-embedded (FFPE) renal cancer samples were obtained from the San Francisco Veterans Affairs (VA) Medical Center. Written informed consent was obtained from all patients and the study was approved by the UCSF Committee on Human Research (Approval number: H9058-35751-01).
Human renal cancer cell lines A-498, ACHN, Caki-1, Caki-2, and a normal renal cell line (HK-2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Integrity of the cell lines was confirmed by the ATCC using DNA (STR) profiling. Normal renal HK-2 cells were cultured as a monolayer in Keratinocyte Serum Free Medium (K-SFM) supplemented with 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml human recombinant epidermal growth factor (EGF) (Life Technologies/Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA), 50 mg/ml penicillin and 50 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). The renal cancer cell lines A-498 and ACHN were cultured as a monolayer in Eagle’s Minimum Essential Medium, (UCSF Cell Culture Facility, San Francisco, CA, USA). Caki-1 and Caki-2 were cultured the same way in McCoy’s5A media (UCSF Cell Culture Facility, San Francisco, CA, USA). All cell lines were maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C.
A-498 cells (60–70% confluent) were treated with genistein (25 µM and 50 µM ). Genistein (Sigma-Aldrich Corp., St Louis, MO, USA) was dissolved in DMSO, and cells treated with vehicle (DMSO) served as control. Fresh genistein was administered everyday with a change of medium, and the cells incubated for 4 days.
For real-time polymerase chain reaction (PCR), complementary DNA was amplified with Inventoried Gene Assay Products containing two gene-specific primers and one TaqMan MGB probe (6-FAM dye-labeled) using a TaqMan Universal Fast PCR Master Mix in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Thermal cycling conditions included 95°C for 20 seconds(s), 40 cycles of 95°C for 3s and 60°C for 30s according to the TaqMan Fast Universal PCR protocol. Total microRNA was extracted using a miRNeasy kit from Qiagen (Valencia, CA, USA). For miRNA real-time experiments the cDNA strand was synthesized using an Applied Biosystems Taqman MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), with 200 ng of total extracted miRNA. RNU48 was used as an endogenous control. It was also used as an endogenous control for real-time PCR experiments, where miRNAs were isolated from formalin-fixed paraffin-embedded (FFPE) renal samples. Total miRNA was extracted from FFPE samples using a miRNA FFPE kit from Qiagen.
Adjacent normal and cancerous renal tissues were obtained from 29 representative FFPE tissue blocks of radical nephrectomy specimens from the Pathology Department of the Veterans Affairs (VA) Medical Center of San Francisco. The blocks were from kidney cancer patients who were operated on at the VA Medical Center between 1980–2009. Sections (4 µm) of the blocks were prepared, H&E stained, and slides were reviewed by a board certified pathologist to mark the normal and cancer areas. Subsequently, 12 µm slides were made from the blocks and micro dissection was performed using the marked H&E stained slides as a template. microRNA extraction was done using a Qiagen FFPE miRNA extraction kit. The levels of miR-23b-3p were assessed by the Taqman miR assay as described above. Following PCR, relative miR-23b-3p expression levels in cancerous regions were normalised to their adjacent normal counterparts.
A498 and Caki-2 cells were transiently transfected with either miRNA-23b-3p inhibitor (
Cell cycle analysis was performed 72 h after transfection. The cells were harvested, washed with cold PBS, (UCSF Cell Culture Facility), and resuspended in the nuclear stain 4′,6-diamidino-2-phenylindole (Beckman Coulter, Brea, CA, USA). Stained cells were immediately analysed with a flow cytometer (Cell Lab Quanta SC; Beckman Coulter).
For apoptosis, cells at 72 h after transfection were dual stained with the viability dye 7-amino-actinomycin D(7-AAD) and Annexin V-FITC using an Annexin V-FITC/7-amino-actinomycin D kit (Beckman Coulter) according to the manufacturer’s protocol. Stained cells were immediately analysed by flow cytometry (Cell Lab Quanta SC; Beckman Coulter).
A cytoselect 24-well cell migration and invasion assay kit (Cell Biolabs, Inc., San Diego, CA) was used for migration and invasion assays at 72 hrs after transfection (8 µm, Colorimetric format) according to the manufacturer’s protocol.
Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA; 50 mmol l–1 Tris (pH 8.0), 150 mmol l–1 NaCl, 0.5% deoxycholate, 0.1% SDS and 1.0% NP-40) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein assays were performed using a BCA Protein assay kit (Pierce/Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Total protein (40 µg) was electrophoresed in 12% SDS–PAGE gels, and Western blotting was carried out using standard protocols and proteins detected by chemiluminescence. Antibodies, including PTEN (cat. no. 9552S) and Akt (cat. no. 4691S) were purchased from Cell Signaling (Danvers, MA, USA), PI3-kinase (cat no. ab69870) and IL-32 (cat no. ab62580) were from Abcam (Cambridge, MA, USA), whereas GAPDH (cat. no. sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
The protein expression levels were quantified by optical densitometry using ImageJ Software version 1.36b (
Cells in 24-well plates were transfected with 30 nM pre-miR negative control (NC) or pre-miR-23b-3p (Applied Biosystems) and PTEN construct (Catalog no. HmiT015535, Genecopoeia, Rockville, MD, USA) or control construct, pEZX-MT01 (Genecopoeia) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The control construct lacked miR-23b-3p target sites. All transfection experiments were performed in triplicate. Luciferase activity was assayed at 48 h after transfection, using a dual-luciferase reporter assay system (Promega).
Immunostaining was done on renal cancer tissue slides [made from formalin-fixed, paraffin-embedded (FFPE) renal cell cancer tissue blocks using a microtome (Leica)]. The slides were deparaffinized and antigen retrieval was carried out by microwaving the slides in 10 mmol/L sodium citrate buffer. Slides were incubated overnight with anti-PTEN antibody (Cell signalling). The staining was done using the LABVISION Corporation (Thermo Fisher Scientific) Anti-Rabbit Staining System (LOT-RHD 80924) as per manufacturer’s instructions.
Statistical analysis was performed using StatView version 5.0 for Windows. Student’s t-test was used to compare the different groups. p-values of <0.05 were regarded as statistically significant. Chi-square test was performed to determine the correlation between miR-23b-3p expression levels and PTEN protein expression levels in tissue samples.
We thank Dr Roger Erickson for support and assistance with the preparation of the manuscript.