Downregulation of microRNA-1274a induces cell apoptosis through regulation of BMPR1B in clear cell renal cell carcinoma
- Hirofumi Yoshino
- Tomokazu Yonezawa
- Masaya Yonemori
- Kazutaka Miyamoto
- Takashi Sakaguchi
- Satoru Sugita
- Youichi Osako
- Shuichi Tatarano
- Masayuki Nakagawa
- Hideki Enokida
- Published online on: November 15, 2017 https://doi.org/10.3892/or.2017.6098
- Pages: 173-181
Renal cell carcinoma (RCC) is a disease in which cells in the tubules of the kidney undergo oncogenic transformation. The most common subtype (approximately 80% of cases) of RCC is clear cell RCC (ccRCC) (1). The 5-year survival rate of advanced-stage RCC patients is extremely poor (5–10%) due to recurrence or distant metastasis (2). At diagnosis, nearly 30% of RCC patients present with metastasis (3). Current treatments for RCC include molecular-targeted therapeutics such as anti-angiogenic multi-tyrosine kinase inhibitors or mTOR inhibitors, which are being widely used for patients with metastatic or recurrent RCC. However, these types of therapies are not expected to have curative effects as they only slightly extend progression-free survival (4). Therefore, to improve the outcome of patients with RCC, it is necessary to fully elucidate the molecular mechanisms underlying the development and progression of RCC and the associated oncogenic pathways.
The discovery of non-coding RNAs (ncRNAs) in the human genome was an important conceptual breakthrough, and the understanding of ncRNAs is important for progress in cancer research. MicroRNAs (miRNAs) are endogenous small ncRNA molecules (19–22 bases in length) that regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner (5). miRNAs are predicted to regulate more than 60% of the protein-coding genes in the human genome (6). Accumulating evidence suggests that miRNAs are aberrantly expressed in many human cancers and play significant roles in human oncogenesis and metastasis (7). Therefore, identifying aberrantly expressed miRNAs is an important first step to elucidate the details of miRNA-mediated oncogenic pathways in cancer cells.
Previously, our miRNA expression signature of ccRCC revealed that microRNA-1274a (miR-1274a) was significantly upregulated in cancer tissues, suggesting that this miRNA functions as an oncogenic miRNA (8). Therefore, the aim of the present study was to investigate the functional significance of miR-1274a and to identify the molecular targets and pathways mediated by miR-1274a in ccRCC cells. Our data demonstrated that downregulation of miR-1274a inhibited cancer cell proliferation and induced apoptosis. Moreover, gene expression data and in silico database analysis showed that bone morphogenetic protein receptor type 1B (BMPR1B) gene was identified as a candidate target of miR-1274a. The discovery of genes and pathways mediated by oncogenic miR-1274a provides important insights into the potential mechanisms of ccRCC oncogenesis and suggests novel therapeutic strategies for the treatment of ccRCC.
Materials and methods
Clinical specimens and cell culture
The ccRCC and adjacent normal tissues were collected from 40 ccRCC patients immediately after undergoing radical nephrectomies at Kagoshima University Hospital between 2003 and 2013. The clinicopathological information of the patients is shown in Table I. The specimens were staged according to the American Joint Committee on Cancer-Union Internationale Contre le Cancer (UICC) tumor-node-metastasis (TNM) classification and histologically graded (9). This study was approved by the Bioethics Committee of Kagoshima University; prior written informed consent and approval were obtained from all patients. We used human ccRCC cell lines, 786O, A498, ACHN and Caki1, obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cell lines were incubated in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and maintained in humidified incubators (5% CO2) at 37°C.
Tissue collection and RNA extraction
Tissues were immersed in RNAlater (Thermo Fisher Scientific) and stored at −20°C until RNA extraction was conducted. Total RNA, including miRNA, was extracted using the mirVana™ miRNA isolation kit (Thermo Fisher Scientific) following the manufacturers protocol.
Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)
Stem-loop RT-PCR (TaqMan MicroRNA Assays; product ID: 2883 for miR-1274a; Applied Biosystems, Foster City, CA, USA) was used to quantify miRNAs according to the manufacturers protocol for PCR conditions. We used human RNU48 (product ID: 001006; Applied Biosystems) as internal control. For investigating the genes, we applied SYBR-Green quantitative PCR-based array approach, and the following primers were used: BMRP1B forward primer, 5′-CTTTTGCGAAGTGCAGGAAAAT-3′ and reverse primer, 5′-TGTTGACTGAGTCTTCTGGACAA-3′; GUSB forward primer, 5′-CGTCCCACCTAGAATCTGCT-3′ and reverse primer, 5′-TTGCTCACAAAGGTCACAGG-3′. qRT-PCR was performed with 500 ng of total RNA using the Power SYBR-Green Master Mix (cat. no. 4367659; Applied Biosystems) on the 7300 Real-Time PCR System (Applied Biosystems). The experimental procedures were conducted according to the protocol recommended by the manufacturer. The specificity of amplification was monitored using the dissociation curve of the amplified product. BMPR1B data values were normalized to GUSB. The ∆∆Ct method was employed to calculate the fold-change of expression level relative to the internal controls.
miRNA inhibitor transfection
ccRCC cell lines were transfected with Lipofectamine RNAiMAX transfection reagent and Opti-MEM (Thermo Fisher Scientific) with 10–60 nM miRNA inhibitor (hsa-miR-1274a; product ID: AM13432, cat. no. AM17000) and negative control #1 anti-miRNA (cat. no. AM17010; Thermo Fisher Scientific).
Cell proliferation and apoptosis assays following anti-miR-1274a transfection
Seventy-two hours after transfection, cell proliferation was determined with an XTT assay using the Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturers instructions. Cell apoptosis assays were carried out using ccRCC cell lines transfected with the transfection reagent, anti-miR-control and anti-miR-1274a, in 6-well tissue culture plates. Cells were harvested 72 h after transfection by trypsinization and washed in cold phosphate-buffered saline (PBS). Double staining with FITC-Annexin V and propidium iodine (PI) was carried out using the FITC Annexin V apoptosis detection kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer's recommendations and analyzed within 1 h by flow cytometry (FACScan; BD Biosciences). Cells were discriminated into viable cells, dead cells, early and late apoptotic cells by CellQuest software (BD Biosciences), and then the percentages of total apoptotic cells from each samples were compared. Experiments were conducted in triplicate.
Putative target gene analysis of miR-1274a
To investigate the expression status of candidate miR-1274a target genes in ccRCC clinical specimens, we examined Oligo Microarray human 44K (Agilent Technologies, Santa Clara, CA, USA) expression profiling of ACHN with anti-miR-control and anti-miR-1274a transfectants. Then, gene expression data were adapted to Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway categories by the GENECODIS program (http://genecodis.cnb.csic.es/). Publicly available expression data from ccRCC specimens in the GEO database (accession number: GSE36895 + GSE22541) were used. We merged these data sets and selected putative genes targeted by miR-1274a according to the TargetScan algorithm (June, 2011 release, http://www.targetscan.org/vert_50/).
Plasmid construction and Dual-Luciferase reporter assays
A partial wild-type sequence of the BMPR1B 3-untranslated region (UTR) or the same region lacking its miR-1274a target site (positions 3237–3243 of the BMPR1B 3-UTR, according to the TargetScan program) was inserted between the XhoI and PmeI restriction sites in the 3-UTR of the hRluc gene in the psi-CHECK-2 vector (C8021; Promega, Madison, WI, USA). ACHN cells were transfected with 50 ng of the vector and 10 nM miR-1274a (product ID: AM13432, cat. no. AM17100; Thermo Fisher Scientific) or control miRNAs (product ID: AM17111; Thermo Fisher Scientific) using Lipofectamine 2000 (Invitrogen). The activities of firefly and Renilla luciferases in cell lysates were determined with a Dual-Luciferase assay system (E1960; Promega). Normalized data were calculated as the ratio of Renilla/firefly luciferase activities.
Overall survival analysis of a ccRCC cohort (KIRC) from The Cancer Genome Atlas (TCGA)
The TCGA cohort database for 72 normal kidneys and 534 ccRCC patients (KIRC) was used to determine the relationship between BMPR1B expression in normal and in ccRCCs, and between BMPR1B expression of ccRCC patients and overall survival. Normalized RNA-seq by expectation-maximization (RSEM) value from RNA-seq expression data were used for gene expression quantification (10). Full sequencing information and clinical information were acquired from the cBioPortal (http://www.cbioportal.org/public-portal/) and the TCGA (https://tcga-data.nci.nih.gov/tcga/) (11–13).
A tissue microarray of 67 ccRCC samples and 10 normal renal tissues was obtained from US Biomax, Inc. (KD806; Rockville, MD, USA). Immunostaining was carried out on the tissue microarray according to the manufacturers protocol using the UltraVision Detection System (Thermo Fisher Scientific). Primary rabbit polyclonal antibodies against BMPR1B (GTX102453; GeneTex, Inc., Irvine, CA, USA) were diluted 1:500. Slides were treated with biotinylated goat anti-rabbit. Diaminobenzidine hydrogen peroxidase was the chromogen and counterstaining was conducted with 0.5% hematoxylin. Immunostaining was evaluated according to a previously described scoring method (14).
Relationships between two or three variables and numerical values were analyzed using the Mann-Whitney U test or Bonferroni-adjusted Mann-Whitney U test. Overall survival of ccRCC patients from the TCGA cohort was evaluated by the Kaplan-Meier method. Patients were numerically divided equally into two groups according to BMPR1B expression, and the differences between the two groups were evaluated by log-rank tests. We used Expert StatView software, version 5.0 (SAS Institute, Inc., Cary, NC, USA) for these analyses.
Expression levels of miR-1274a in ccRCC specimens and ccRCC cell lines
We evaluated the expression level of miR-1274a in ccRCC tissues (n=40), adjacent normal kidney tissue (n=40) and ccRCC cell lines (786-O, A498, ACHN and Caki1). The expression levels of miR-1274a were significantly higher in tumor tissues and two ccRCC cell lines (A498 and ACHN) than in normal kidney tissue (P<0.05, Fig. 1A, and P<0.0001, Fig. 1B). miR-1274a expression in Caki1 was higher than that noted in the normal kidneys even though there was no significant difference. Therefore, we used A498, ACHN and Caki1 cells for the next experiments. There were no significant correlations between any of the clinicopathological parameters or overall survival rate and the expression of miR-1274a (Fig. 1C-E). Next, we evaluated the miR-1274a expression level following anti-miR-1274a transfection. The expression of miR-1274a in the A498, ACHN and Caki1 cells was sufficiently downregulated following transfection of 30 and 60 nM anti-miR-1274a (P<0.05; Fig. 1F).
Expression levels of miR-1274a and knockdown efficiency of anti-miR-1274a transfection in ccRCC cells. (A) Expression levels of miR-1274a in normal kidney tissues and ccRCC cell lines, and (B) in normal kidney tissues and ccRCC specimens, were determined by qRT-PCR. Data were normalized to RNU48 expression (*P<0.05). (C and D) Expression levels of miR-1274a in ccRCC patients (C) with pT1 and ≥pT2, and (D) without vein invasion or metastasis and with vein invasion or metastasis. (E) Kaplan-Meier survival curves for overall survival according to miR-1274a expression in 39 ccRCC patients. P-values were calculated using the log-rank test. (F) The expression level of miR-1274a in A498, ACHN and Caki1 cells 48 h after transfection with 10–60 nM anti-miR-1274a (*P<0.05).
Effects of the inhibition of miR-1274a on cell proliferation and apoptosis in ccRCC cell lines
To examine the functional roles of miR-1274a, we performed loss-of-function studies by using anti-miRNA-transfected A498, ACHN and Caki1 cells. XTT assays revealed that there was a significant inhibition of cell proliferation in the 10, 30 and 60 nM anti-miR-1274a-transfectants (P<0.0001; Fig. 2A). We also analyzed the numbers of apoptotic cells and found that miR-1274a inhibition significantly induced apoptosis of the A498, ACHN and Caki1 cells (P<0.05; Fig. 2B).
Effects of anti-miR-1274a on cell growth and apoptosis. (A) Cell growth was determined by XTT assays in the A498, ACHN and Caki1 cells 72 h after transfection with 10–60 nM miR-1274a. **P<0.0001. (B) Apoptosis assays were carried out using flow cytometry. Early apoptotic cells are indicated in the right bottom quadrant and late apoptotic cells are indicated in the right upper quadrant. The normalized ratios of apoptotic cells are shown in the histograms (*P<0.05).
Screening of putative target genes of miR-1274a in ccRCC
To gain further insight into the molecular mechanisms regulated by oncogenic miR-1274a in ccRCC cells, we screened miR-1274a-regulated genes by using in silico and genome-wide gene expression analysis (Fig. 3). As for the target search by oligo microarray, we used ACHN as the knockdown efficiency and cell proliferative inhibition rate by anti-miRNA was the strongest in the ACHN cells. First, we found that 694 genes were upregulated in the oligo microarray by anti-miR-1274a-transfectants of ACHN. These 694 genes were assigned KEGG annotations using singular enrichment analysis of GeneCodis to identify the molecular pathways regulated by miR-1274a. Twenty signaling pathways and 73 genes were identified in this analysis (Table II). Furthermore, we examined gene expression signatures that were downregulated in clinical ccRCC specimens from the GEO database and found that 9 genes were significantly downregulated in 53 ccRCCs compared to 23 normal kidney tissues. Among these, we focused on BMPR1B, as the BMPR1B expression level in ccRCC tissues was most significantly downregulated compared with the level noted in normal tissues (Table III) and TargetScan 5.2 database predicted that there was a binding site for miR-1274a in BMPR1B 3UTR.
Strategy for the analysis of genes regulated by miR-1274a. Flow chart for identifying miR-1274a target genes by in silico analysis using Gene Expression Omunibus (GEO), TargetScan database 5.2, and genome-wide gene expression analysis of anti-miR-1274a transfectants.
Expression of miR-1274a target genes involved in significantly enriched annotations according to gene expression data in ACHN cells.
BMPR1B is regulated by miR-1274a
We performed qRT-PCR to determine whether inhibition of miR-1274a resulted in upregulation of BMPR1B mRNA expression in ccRCC cells. The mRNA levels of BMPR1B were significantly upregulated in the anti-miR-1274a transfectants in comparison with the anti-miR-control transfectants in ACHN and Caki1 cells (P<0.05; Fig. 4A). Next, we performed luciferase reporter assays to determine whether the 3′-UTR of BMPR1B contained an actual binding site of miR-1274a. The TargetScan database predicted that one putative miR-1274a binding site existed in the 3′-UTR of BMPR1B (positions 3237–3243; Fig. 4B). We found that the luminescence intensity in ACHN cells was significantly reduced by transfection of miR-1274a with the wild-type vector carrying the 3′-UTR of BMPR1B, whereas transfection with a deletion vector showed no decrease in luminescence (P<0.05; Fig. 4B). The expression levels of BMPR1B were significantly lower in ccRCCs than in normal kidneys using our clinical samples (P<0.0001; Fig. 4C). These data were consistent with the in silico database analysis (Fig. 3) indicating that downregulated BMPR1B as reported in the GEO data analysis was upregulated when the cells were treated with anti-miR-1274a in the microarray.
BMPR1B is regulated by miR-1274a. (A) BMPR1B mRNA expression was evaluated by qRT-PCR in A498, ACHN and Caki1 cells 72 h after transfection with anti-miR-1274a. GUSB was used as an internal control. (B) Luciferase reporter assays in ACHN cells using vectors encoding a putative miR-1274a target site at position 3237–3243 of BMPR1B 3-UTR. Renilla luciferase values were normalized to firefly luciferase values (*P<0.05). (C) Expression levels of BMPR1B in normal kidney and ccRCC specimens were determined by qRT-PCR. Data were normalized to GUSB expression.
BMPR1B expression as a prognostic marker of ccRCC
The expression levels of BMPR1B were significantly lower in ccRCC tissue samples than that noted in the normal kidneys using the TCGA database (P<0.0001; Fig. 5A). We then evaluated the correlation of BMPR1B expression levels with overall survival of the ccRCC patients. The cohort was numerically divided equally into two groups based on BMPR1B expression. Unexpectedly, we found that the low BMPR1B expression group (n=264) had better median overall survival in comparison with the high BMPR1B expression group (n=265) (P=0.0167; Fig. 5B). This findings were supported by the results of the immunohistochemical staining of BMPR1B in tissue specimens (Fig. 6). The expression scores of BMPR1B were significantly lower in 67 ccRCCs in comparison with 10 normal renal tissues (P=0.0015; Fig. 6A). On the other hand, the scores were significantly higher in ccRCCs with high-grade tumors (more than G2, n=13) in comparison with scores in ccRCCs with low-grade tumors (G1, n=53) (P=0.0136; Fig. 6B).
Analysis of TCGA kidney clear cell carcinoma (KIRC) datasets. (A) Expression levels of BMPR1B in normal kidney tissues and ccRCC tissue specimens were determined by the KIRC dataset. (B) Kaplan-Meier survival curves for overall survival rate according to BMPR1B expression in 529 ccRCC patients. P-values were calculated using the log-rank test.
Immunohistochemical staining of BMPR1B in tissue specimens. (A) Expression levels of BMPR1B were significantly lower in 67 ccRCCs in comparison with 10 normal renal tissues, (B) and in ccRCCs with G1 (n=53) in comparison with ccRCCs with G2 or higher (n=13). (C) Representative images of immunohistochemical staining of BMPR1B in tissue specimens (magnification, ×40 and ×200).
There is considerable evidence that normal miRNA regulatory mechanisms are disrupted in cancer cells (7). However, the regulatory mechanisms by which upregulated miRNAs exert their effects has not been fully understood compared to downregulated miRNAs. Upregulated miRNAs have been mainly viewed as markers (15), and functional studies have not been performed in detail, even though many studies have successfully inhibited miRNA expression using miRNA inhibitors, morpholinos, sponges, or CRISPR/Cas9 (8,16–18). One of the possible reasons is that miRNA gain-of-function studies in downregulated miRNAs can be easily performed by transfection of precursor miRNAs with sufficient efficiency. In the present study, we suppressed upregulated miR-1274a with miRNA antisense inhibitor. Even though we obtained nearly 85% knockdown efficiency at most, high concentration of the miRNA antisense inhibitor was needed. Therefore, other approaches such as CRISPR/Cas9 to gain adequate knockdown efficiency without off-target effects will be promising in subsequent research.
Our previous studies of miRNA expression signatures in ccRCC identified several upregulated miRNAs (miR-885-5p, miR-1274a, miR-210-3p, miR-224 and miR-1290) (8). In this study, we focused on miR-1274a, as increased expression of miR-1274a has been reported in other types of human cancers (19,20). Wang et al (19) reported that miR-1274a was overexpressed in gastric cancer tissues or cancer cell lines, and that miR-1274a promoted proliferation, migration and epithelial-mesenchymal transition (EMT) by suppressing FOXO4, which is a transcriptional factor and leads to cell cycle arrest or apoptosis. In melanoma, high expression of miR-1274a was also reported (20). On the other hand, contrasting roles of miR-1274a have also been reported in cancers to date. Zhou et al (21) reported that miR-1274a was upregulated by sorafenib, which is a multi-kinase inhibitor clinically used for cancer treatment. They also found that miR-1274a could significantly suppress one of the protease proteins ADAM9 and accelerate antitumor immunity in hepatocellular carcinoma (HCC). Yan et al (22) also reported similar results in HCC where miR-1274a was upregulated by paclitaxel. In fields other than cancers, Ezzie et al (23) reported that miR-1274a was one of the highly expressed miRNAs in lungs from subjects with chronic obstructive pulmonary disease (COPD) compared with smokers without COPD. Thus, diverse types of roles for miR-1274a have been reported not only in cancers but also in chronic disease.
In the present study, we investigated the pathways and targets that were regulated by oncogenic miR-1274a in ccRCC cells. In the process of the identification of miR-1274a-regulated molecular pathways and target genes, we used a combination of expression data and in silico database analysis (24). Using this strategy, we have identified molecular targets and pathways in several types of cancer that are regulated by tumor-suppressive miRNAs. In this study, ‘Cytokine-cytokine receptor interaction’ and ‘TGF-β signaling pathway’ were significantly selected as candidate pathways regulated by miR-1274a in ccRCC cells. In these pathways, BMPR1B was selected as a candidate target of miR-1274a. BMPR1B is a member of the BMP receptor family of transmembrane serine/threonine kinases, and the ligands of this receptor are BMPs (25,26). BMPs, members of the TGF-β superfamily, bind their receptor including BMPR1B, which lead to activation of the SMAD-dependent pathway subsequently through phosphorylation of SMAD1/5/8 (25,26). Then phosphorylated SMADs form a complex with the common signaling transducer SMAD4, resulting in the induction of apoptosis (26–30). Therefore, our finding that suppression of miR-1274a caused apoptosis through BMPR1B upregulation is plausible. However, A498 ccRCC cells did not exhibit increased BMPR1B expression, even though ACHN and Caki1 cells had rescued expression, and miR-1274a suppression in all 3 cell lines induced apoptosis in this study. Even though low expression of BMPR1B was observed in ccRCC compared to normal kidneys, BMPR1B expression was significantly higher in high-grade ccRCCs in comparison with low-grade ccRCCs as detected by immunohistochemistry. In addition, the low expression patient group had prolonged median overall survival in comparison with the high expression patient group according to the TCGA analyses. BMPR1B upregulation might be an early event in ccRCC carcinogenesis, and its functional changes might have occurred as the tumor acquires malignant potential at a late stage. Further study is necessary to elucidate the further functional role of BMPR1B. Concerning miR-1274a, we did not find any significant correlation between expression of miR-1274a and any of the clinicopathological parameters including prognosis. This may be due to the small sample numbers and short follow-up periods. A larger scale and longer follow-up period of study should be considered in future research.
In conclusion, upregulation of miR-1274a was observed in ccRCC clinical specimens and cell lines. Suppression of miR-1274a significantly inhibited cancer cell proliferation through apoptosis, suggesting that miR-1274a functions as an oncogenic miRNA in ccRCC cells. The identification of tumor-suppressive BMPR1B regulated by miR-1274a may lead to a better understanding of ccRCC oncogenesis and the development of new therapeutic strategies to treat this disease.
We thank Mutsumi Miyazaki for the excellent laboratory assistance and the critical editing of this manuscript. The present study was supported by the KAKENHI (B) 16H05464, the KAKENHI (C) 16K11015 and 17K11148, the KAKENHI (HOUGA) 16K15691, and the KAKENHI (WAKATE)17K16799.
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