Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. Although several studies have reported high expression of miR-122 in ccRCC, its physiological role remains unclear. To clarify the role of miR-122 in ccRCC, we compared miR-122 expression levels in non-cancerous tissue and ccRCC. Significant upregulation of miR-122 was observed in ccRCC specimens. Moreover, ccRCC patients with high miR-122 expression showed poor progression-free survival compared to those with low miR-122 expression. Overexpression of miR-122 using an miRNA mimic promoted proliferation, migration, and invasion activities of ccRCC cells. miR-122 directly targets occludin, a known component of tight junctions. Occludin knockdown promoted the cell migration activity but not proliferation or invasion activities of ccRCC cells. In human clinical specimens, miR-122 expression inversely correlated with occludin protein expression. These findings show that miR-122 is an oncomiR in ccRCC.
Renal cell carcinoma (RCC) represents the leading cause of death due to urological malignancies (
MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression, mainly at the translational level (
MicroRNA-122 (miR-122), a known tumour suppressor in hepatocellular carcinoma (HCC), functions by targeting oncogenes such as cyclin G1 (
Tight junctions govern the permeability of epithelial and endothelial cells and are the most topical structure of these cell types (
Herein, we showed that highly expressed miR-122 correlated with low progression-free survival compared to low miR-122 expression in ccRCC clinical specimens. We further demonstrated that miR-122 promoted malignant phenotypes partially via targeting occludin in ccRCC cells. Finally we showed that occludin was significantly downregulated in ccRCC specimens compared to normal kidney tissues, and this correlated with the expression levels of miR-122. Our findings identify that miR-122 is as a potent regulator of malignant phenotypes and functions as an oncomiR in ccRCC, leading to a novel therapeutic strategy for treatment of ccRCC.
miRIDIAN miRNA hairpin inhibitor negative control, miRIDIAN miRNA mimic negative control, miRIDIAN hairpin inhibitor or miRIDIAN miRNA mimic for human hsa-miR-122 (MIMAT0000421) were purchased from Thermo Scientific Dharmacon (Waltham, MA, USA). The miRIDIAN hairpin inhibitors and mimics were used at a concentration of 50 nM. Polyclonal anti-occludin and monoclonal anti-β-tubulin antibodies were purchased from Sigma (St. Louis, MO, USA). Polyclonal Anti-ACTIVE p38 (Thr180/Tyr182) and Anti-ACTIVE JNK1/2 (Thr183/Tyr185) antibodies were purchased from Promega (Madison, WI, USA). Monoclonal anti-phospho-Erk1/2 (Thr202/Tyr204), anti-Erk1/2, and anti-p38 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Monoclonal anti-JNK1/2 antibody was purchased from BD Transduction Laboratories (San Jose, CA, USA).
ccRCC specimens were obtained from patients while they underwent primary curative resection at the Osaka University Medical Hospital, Japan. Tumour-associated normal renal tissue was also obtained from a subset of these patients when possible. Prior written and informed consent was obtained from each patient, and the study was approved by the ethics review board of the Osaka University Medical Hospital and the methods were carried out in accordance with the approved guidelines.
Following excision, tissue samples were immediately immersed in RNAlater (Qiagen, Valencia, CA, USA) and stored at −20°C until RNA extraction. miRNAs were purified using the miRNeasy mini kit (Qiagen). Real-time PCR analysis was conducted to validate miR-122 expression in ccRCC using 80 tumour samples and 10 adjacent normal renal samples (for
Three human ccRCC cell lines (Caki-1, Caki-2 and, ACHN), obtained from the American Type Culture Collection (ATCC), were cultured in RPMI-1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 0.1
Protein samples were separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) on a 7.5–15% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane using the Bio-Rad semi-dry transfer system (1 h, 12 V). Immunoreactive proteins reacting with the antibodies described above were visualized by treatment with a detection reagent (ECL Prime western blotting detection reagent; GE Healthcare, Lafayette, CO, USA). Densitometric analysis was performed using NIH ImageJ software.
A pmirGLO dual-luciferase miRNA target expression vector was used for the 3′-UTR luciferase reporter assay (Promega). The following oligonucleotides were used for the evaluation of efficacy of the miR-122 hairpin inhibitor on occludin 3′-UTR: 5′-CTA GCG GCC GCT AGT TCA ACT GGG CTG AAC ACT CCA G-3′ and 5′-TCG ACT GGA GTG TTC AGC CCA GTT GAA CTA GCG GCC GCT AGA GCT-3′. Luciferase activity was determined using a luminometer (Turner Biosystems 20/20 n luminometer, Promega).
Cell migration was examined by wound healing assay. In brief, Caki-2 cells transfected with the miRIDIAN hairpin inhibitor or the negative control inhibitor, and ACHN cells transfected with the miRIDIAN microRNA mimic or the negative control mimic were seeded in a 24-well plate (Caki-2 cells: 2.0×104 cells/well, ACHN cells: 4.0×104 cells/well) and incubated for 72 h. A wound was created in a monolayer of approximately 90% confluent Caki-2 or ACHN cells using a sterile 1 ml pipette tip. Cell migration pictures were recorded at 0 and 12 h after wound creation using an Olympus IX71 fluorescence microscope.
Cell proliferation was examined by WST-1 assay. Caki-2 cells transfected with the miRIDIAN hairpin inhibitor or the negative control inhibitor, and ACHN cells transfected with the miRIDIAN miRNA mimic or the negative control mimic were seeded in a 96-well plate (0.3×104 cells/well) and incubated for 72 h. After incubation for 2 h with the WST-1 reagent (Dojindo, Osaka, Japan) at 37°C and 5% CO2, the optical density was read at a wavelength of 450/630 nm (Ex/Em).
Caki-2 or ACHN cells were seeded at 1.0×104 cells/well in 6-well plates in RPMI-1640 medium (supplemented with 10% FBS and 0.3% agarose with 0.4% agarose underlay). Dishes were incubated at 37°C and 5% CO2. After 14 days, colonies were stained with crystal violet (Wako) and counted.
The BioCoat tumour invasion system (Corning Inc., Corning, NY, USA) was used to perform the cell invasion assay. Caki-2 cells transfected with the miRIDIAN hairpin inhibitor or the negative control inhibitor were seeded in a 96-well plate (3×104 cells/well). Following incubation for 12 h, the cells were labelled with calcein AM (4
Cells plated on coverslips were washed with phosphate-buffered saline (PBS). The cells were fixed and permeabilized in 4% formaldehyde for 15 min at room temperature and then washed twice with PBS. After blocking with 2% bovine serum albumin in PBS for 1 h, the coverslips were incubated with the anti-occludin antibody (1:500) at 4°C. The coverslips were washed twice with PBS and incubated with the fluorochrome-conjugated secondary antibody for 1 h at room temperature. The cells were then examined under the fluorescence microscope, Bio-Zero (Keyence, Osaka, Japan).
The expression of occludin was determined by immunohistochemical staining of paraffin-embedded tissues of ccRCC samples with the highest and lowest levels of miR-122 expression samples (10 samples of each). Formalin-fixed paraffin-embedded sections (5
ACHN cells transfected with occludin siRNAs or miR-122 mimic for 48 h were reseeded (1.0×105 cells/well) on a 0.4
The results are expressed as the mean ± standard deviation (SD). Differences between the values were statistically analysed using the Student's t-test or one-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. Correlations between the values were analysed using Pearson correlation analysis (GraphPad Prism 6.0, GraphPad software, San Diego, CA, USA). A P-value of <0.05 was considered statistically significant.
In our previous microarray analysis identifying unique miRNA expression signatures (GEO accession number: GSE55138) (
To investigate the role of miR-122 in ccRCC, we first compared the expression levels of miR-122 in three ccRCC cell lines (Caki-1, Caki-2, and ACHN,
We used target prediction programs (miRbase, TargetScan, and miRanda) to identify the potential miR-122 target in ccRCC cells. We then focused on occludin as a potential target, because it is a component of tight junctions and functions as a tumour suppressor in several cancers (
To examine whether the malignant phenotypes upregulated by the miR-122 mimic in ccRCC cells were due to the decrease in occludin expression, we performed occludin knockdown experiments in ACHN cells (
The miR-122 inhibitor upregulated the expression of occludin at the cell-cell adhesive surface in Caki-2 cells (
Although the miR-122 mimic upregulated the cell proliferation, motility, and invasion activities, occludin knockdown only upregulated cell motility. Target prediction programs predict that putative miR-122 binding sequences exist in other genes involved in cell growth [e.g.,
In the present study, we found that high expression of miR-122 was significantly correlated with poor progression-free survival in ccRCC. Overexpression and loss of function experiments revealed that miR-122 functions as an oncomiR by upregulating malignant phenotypes in ccRCC cells. We identified that miR-122 directly targets occludin in ccRCC cells. Moreover, we showed that the miR-122 expression levels and occludin protein levels are negatively correlated in ccRCC clinical specimens.
Although miR-122 mimic significantly downregulated the TEER in ACHN cells, occludin knockdown only slightly suppressed the TEER (
Although miR-122 expression was not correlated with pathological stages and grades, miR-122 expression was significantly high in ccRCC tissues accompanied by lymphatic invasion compared with those without lymphatic invasion (
Originally miR-122 was reported as a tumour suppressor gene in hepatocellular carcinoma (HCC) (
microRNA-122
clear cell renal cell carcinoma
onco-microRNA
This study was supported by a Grant-in-Aid for Scientific Research (25670025) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Project MEET, Osaka University Graduate School of Medicine. The TEER measurement with the Millicell® ERS-2 voltohmmeter was kindly supported by Dr Takefumi Doi and Dr Yoshiaki Okada (Graduate School of Pharmaceutical Sciences, Osaka University). N. Nonomura received commercial research grants from Takeda Pharmaceutical, Novartis Pharma, and Astra Zeneca.
miR-122 expression in ccRCC tissues. (A) miRNA microarray analysis showed upregulated expression of these miRNAs (fold-change >2, p<0.05 normal adjacent kidney vs. ccRCC) in ccRCC specimens. The expression of miR-122 was compared in normal and tumour tissues (B), pathological stages (C), and pathological grades (D) in ccRCC clinical specimens. Number of the samples is given in parentheses. (E) miR-122 expression was compared between ccRCC tissues with (+) and without (−) lymphovascular cancer cell invasion. Number of the samples is given in parentheses. *p<0.05 vs. non-invaded ccRCC tissue. (F) Progression-free survival. ccRCC tissue samples were divided into three groups according to miR-122 expression (low: 41 samples; middle: 41 samples; and high: 40 samples). *p<0.05 low vs. high. ***p<0.001.
miR-122 inhibitor downregulates the migration and invasion activities in Caki-2 cells. (A) Expression of miR-122 in three ccRCC cell lines was examined by quantitative real-time PCR. Duplicated result is shown. (B) Caki-2 cells transfected with the miR-122 inhibitor, or the negative control miRNA inhibitor, for 24 h were reseed in a 96-well plate (0.3×103 cells/well), incubated for the indicated times and examined by WST-1 assay. Values are mean ± SD of three independent experiments. (C–E) Caki-2 cells were transfected with the miR-122 inhibitor, or the negative control miRNA inhibitor, for 72 h. (C) The transfected cell suspension was added to soft agar plates and cultured for 2 weeks, and then the colony number was counted. The results are expressed as mean ± SD of three independent experiments. (D) Cell migration was measured 12 h after a wound was created by scraping. Representative results of cell mobility in the wound-healing assay are shown. The results are expressed as mean ± SD of five independent experiments. **p<0.01 vs. the control inhibitor. (E) The transfected cell suspension was added to the upper chamber of a matrigel-coated CIM-plate. The lower chamber was filled with medium and cultured for 24 h in the xCELLigence system. Values expressed as cell index are mean ± SD of seven experiments. **p<0.01 vs. the control inhibitor.
miR-122 mimic upregulates growth and migration activities in ACHN cells. (A) ACHN cells transfected with the miR-122 mimic, or the negative control miRNA mimic, for 24 h were reseed in a 96-well plate (0.3×103 cells/well), incubated for the indicated times and examined by WST-1 assay. Values are means ± SD of six independent experiments. ***p<0.001 vs. the control mimic. (B–D) ACHN cells were transfected with the miR-122 mimic, or the negative control miRNA mimic, for 72 h. (B) The transfected cell suspension was added to soft agar plates and cultured for 2 weeks, and then the colony number was counted. The results are expressed as mean ± SD of three independent experiments. *p<0.05 vs. the control mimic. (C) Cell migration was measured 12 h after a wound was created by scraping. The results are expressed as mean ± SD of six independent experiments. *p<0.05 vs. control mimic. (D) The transfected cell suspension was added to the upper chamber of matrigel-coated Transwell membrane inserts, and the lower chamber was filled with the medium and then cultured for 12 h. Fluorescence derived from invasive cells was measured. Values are mean ± SD of three independent experiments.
miR-122 targets occludin in ccRCC cells. (A) Predicted miR-122-binding site within the 3′-UTR of the human occludin gene. (B) Caki-2 cells were co-transfected with the luciferase reporter construct containing the predicted miR-122-binding site from the occludin 3′-UTR and miR-122 inhibitor, or the negative control miRNA inhibitor. Values are mean ± SD of three independent experiments. **p<0.01 vs. each control. (C) ACHN cells were co-transfected with the luciferase reporter construct containing the predicted miR-122-binding site within the occludin 3′-UTR and miR-122 mimic, or the negative control miRNA mimic. Values are mean ± SD of four independent experiments. *p<0.05 vs. each control. (D) Caki-2 cells were transfected with the control and miR-122 inhibitors for 72 h, and then protein samples were collected and subjected to western blot analysis with anti-occludin antibody. The membrane was re-probed with the anti-β-tubulin antibody. Relative occludin expression is shown as mean ± SD of six independent experiments. **p<0.01 vs. the control inhibitor. (E) ACHN cells were transfected with the control and miR-122 mimics for 72 h, then protein samples were collected and subjected to western blot analysis with an anti-occludin antibody. The membrane was re-probed with the anti-β-tubulin antibody. Relative occludin expression is shown as mean ± SD of four independent experiments. *p<0.05 vs. the control mimic.
miR-122 expression correlates with occludin protein expression in ccRCC specimens. (A) ACHN cells were transfected with the occludin siRNAs for 72 h, and then protein samples were collected and subjected to western blot analysis with anti-occludin antibody. The membrane was re-probed with the anti-β-tubulin antibody. Representative results of three independent experiments are shown. (B) Cell migration was measured 12 h after a wound was created by scraping. Representative results of cell mobility in the scratch wound-healing assay are shown. The results are expressed as means ± SD of four independent experiments. *p<0.05 vs. the control siRNA. ACHN cells were transfected with the miR-122 mimic (C) and two kinds of occludin siRNAs (D) for 48 h, and TEER analysis was performed. Values are mean ± SD of four independent experiments for (C) and three independent experiments for (D). (E) Caki-2 cells were transfected with the miR-122 inhibitor for 72 h and then immunofluorescence staining was performed with the anti-occludin antibody and 4′,6-diamidino-2-phenylindole (DAPI). Representative images of three independent experiments are shown. White bars indicate 10
The effect of miR-122 on MAPKs activation in ccRCC cells. Caki-2 cells were transfected with the miR-122 inhibitor (A, left), and ACHN cells were transfected with miR-122 mimic (A, right) or the occludin siRNAs (B) for 72 h. Protein samples were collected and subjected to western blot analysis with anti-phospho Erk1/2 (Thr202/Tyr204), anti-Erk1/2, anti-phospho p38 (Thr180/Tyr182), anti-p38, anti-phospho JNK1/2 (Thr183/Tyr185), and anti-JNK1/2 antibodies. Representative results of three independent experiments are shown.
Clinical and pathological data related to the clinical samples.
Characteristics | Validation cohort for miR-122 expression | For the PFS analysis |
---|---|---|
Age (year) | ||
Mean | 62.6 | 63.8 |
Range | 35–86 | 34–82 |
Gender | ||
Male | 58 | 38 |
Female | 31 | 83 |
Unknown | 1 | 1 |
Pathologic stage | ||
Normal | 10 | 0 |
pT1a | 11 | 56 |
pT1b | 69 | 65 |
Unknown | 0 | 1 |
Pathologic grade | ||
G1 | 23 | 47 |
G2 | 52 | 72 |
G3 | 5 | 2 |
Unknown | 0 | 1 |
Lymphatic invasion | ||
− | 74 | 127 |
+ | 6 | 10 |