Tumor suppressive microRNA‑138 contributes to cell migration and invasion through its targeting of vimentin in renal cell carcinoma
- Authors:
- Published online on: July 3, 2012 https://doi.org/10.3892/ijo.2012.1543
- Pages: 805-817
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Copyright: © Yamasaki et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].
Abstract
Introduction
Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. In this disease, cancer cells form in the tubules of the kidney and approximately 80% of RCC patients are diagnosed with the clear cell RCC subtype (1). Up to 30% of RCC patients present at advanced stages, and approximately 40% of patients who undergo curative surgical resection experience recurrence during subsequent follow-up (2,3). The five-year survival rate of advanced RCC is 5–10% (4). RCC is resistant to radiotherapy and chemotherapy (5,6). Targeted therapies such as sunitinib, sorafenib, everolimus and temsirolimus have been developed and have been used widely in first- and second-line treatments, extending the period of progression-free-survival (7,8). However, these treatments are insufficient for patients who have developed relapse or metastasis. Therefore, increased understanding of the molecular mechanisms of RCC progression and metastasis is needed using the latest approaches to genomic analysis.
RNA can be divided into two categories: protein coding RNA and non-coding RNA (ncRNA). It is important to examine the functions of ncRNAs and their association with human disease, including cancer. 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 (9). A growing body of evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in their initiation, development, and metastasis (10). Some highly expressed miRNAs could function as oncogenes by repressing tumor suppressors, whereas low level miRNAs could function as tumor suppressors by negatively regulating oncogenes (11).
We previously identified many tumor suppressive miRNAs based on our miRNA expression signatures of various types of cancer, such as RCC, bladder cancer, prostate cancer, maxillary sinus squamous cell carcinoma and hypopharyngeal squamous cell carcinoma (12–17). In oncogenic pathways, normal regulatory mechanisms are disrupted by aberrant expression of tumor suppressive or oncogenic miRNAs. Therefore, identification of miRNA-regulated pathways is important for further development in human cancer research. Thus, we have been investigating how tumor suppressive miRNA regulates novel cancer pathways. For example, the miR-1/miR-133a cluster regulates several oncogenic genes, including transgelin-2 (TAGLN2), prothymosin α (PTMA) and purine nucleoside phosphorylase (PNP) (14,15,18).
More recently, we constructed a miRNA expression signature of RCC clinical specimens and successfully identified tumor suppressive miR-1285 targeting transglutaminase 2 (TGM2) (12). Among the signatures, several miRNAs were significantly downregulated in RCC specimens as promising candidate of tumor suppressors. In this study, we focused on miR-138. This miRNA was downregulated in our previous signature, and downregulation of miR-138 has been observed in several malignancies, including anaplastic thyroid carcinoma (19) and lung cancer (20).
The aim of the study was to investigate the functional significance of miR-138 and identify its target genes in RCC cells. To identify miR-138-regulated cancer pathways, we undertook both a genome-wide gene expression analysis (miR-138 transfectants and RCC clinical specimens) and an in silico study. The results showed that vimentin (VIM) was a promising candidate target gene of miR-138. It is well known that VIM is one of the most widely expressed mammalian intermediate filament proteins. Studies have shown that VIM functions in cell adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in cancer cells (21). The existence of a tumor suppressive miR-138-mediated cancer pathway provides new insights into the potential mechanisms of RCC oncogenesis and metastasis.
Materials and methods
Clinical specimens
A total of 33 pairs of clear cell renal cell carcinoma (ccRCC) and adjacent non-cancerous specimens were collected from patients who had undergone radical nephrectomies at Kagoshima University Hospital. The samples were processed and stored in RNAlater (Qiagen, Valencia, CA, USA) at −20°C until RNA extraction. The patient information is summarized in Table I. These samples were staged according to the American Joint Committee on Cancer-Union Internationale Contre le Cancer (UICC) tumour-node-metastasis classification and histologically graded (22). Our study was approved by the Bioethics Committee of Kagoshima University; written prior informed consent and approval were given by the patients.
Cell culture and RNA extraction
We used two human RCC cell lines, A498 and 786-O, obtained from the American Type Culture Collection (Manassas, VA, USA). The cell lines were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified incubator (5% CO2) at 37°C. Total-RNA was extracted, as previously described (12).
Quantitative real-time RT-PCR
TaqMan probes and primers for VIM (P/N: Hs00185584_m1: Applied Biosystems) were assay-on-demand gene expression products. All reactions were performed in duplicate, and a negative control lacking cDNA was included. We followed the manufacturer’s protocol for PCR conditions. Stem-loop RT-PCR (TaqMan MicroRNA Assays; P/N: 002284 for miR-138; Applied Biosystems) was used to quantitate miRNAs according to the earlier published conditions (23). To normalize the data for quantification of VIM mRNA and the miRNAs, we used human GUSB (P/N: Hs99999908_m1; Applied Biosystems) and RNU6B (P/N: 001973; Applied Biosystems), respectively, and we used the ΔΔCt method to calculate the fold-change. As a control RNA, we used Premium total-RNA from normal human kidney (AM 7976; Applied Biosystems).
Mature miRNA and siRNA transfection
As described elsewhere (23), the RCC cell lines were transfected with Lipofectamine™ RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) and Opti-MEM™ (Invitrogen) with 10 nM mature miRNA molecules. Pre-miR™ (Applied Biosystems) and negative-control miRNA (Applied Biosystems) were used in the gain-of-function experiments, whereas VIM siRNA (Cat nos. SASI_ Hs01_00044033 and SASI_HS01_00044036, Sigma-Aldrich, St. Louis, MO, USA) and negative control siRNA (D-001810-10; Thermo Fisher Scientific, Waltham, MA, USA) were used in the loss-of-function experiments. Cells were seeded in 10-cm dishes for protein extraction (8×105 cells per dish), 6-well plates for wound healing assays (20×104 cells per well), in 24-well plates for the mRNA extraction and Matrigel invasion assays (5×104 cells per well) and in 96-well plates for the XTT assays (3,000 cells per well).
Cell morphology
Cells were transfected with miR-138 and si-VIM for 72 h and were then examined by an inverted microscope (CK2-BIP2, Olympus).
Cell proliferation, migration and invasion assays
Cell proliferation was determined using an XTT assay (Roche Applied Science, Tokyo, Japan) that was performed according to the manufacturer’s instructions. Cell migration activity was evaluated with a wound healing assay. Cells were plated in 6-well dishes and the cell monolayer was scraped using a P-20 micropipette tip. The initial gap length (0 h) and the residual gap length 24 h after wounding were calculated from photomicrographs. A cell invasion assay was carried out using modified Boyden Chambers consisting of Transwell-precoated Matrigel membrane filter inserts with 8-mm pores in 24-well tissue cultures plates (BD Bioscience, Bedford, MA, USA). Minimum essential medium containing 10% FBS in the lower chamber served as the chemoattractant as described previously (24). All experiments were performed in triplicate.
Screening of miR-138-regulated genes by microarray
Oligomicroarray Human 60K (Agilent) was used for expression signature in miR-138-transfected A498 cells in comparison with the miR-negative control transfectant, as previously described (23). Briefly, hybridization and washing steps were performed in accordance with the manufacturer’s instructions. The arrays were scanned using a Packard GSI Lumonics ScanArray 4000 (PerkinElmer, Boston, MA, USA). The data obtained were analyzed with DNASIS array software (Hitachi Software Engineering, Tokyo, Japan) that converted the signal intensity. Data from each microarray study were normalized by global normalization.
Expression signature of RCC clinical specimens by microarray
Oligo-microarray Human 60K (Agilent) was used for expression signature in 5 pairs of RCC clinical specimens compared with adjacent non-cancerous tissues. Their age ranged from 42 to 77 years; 3 were G1 and 2 were G2 in their tumor grading; and all were pT1N0M0 tumors.
Western blot analysis
After three days of transfection, protein lysates (40 μg) were separated by NuPAGE on 4–12% bis-tris gels (Invitrogen) and transferred onto polyvinylidene fluoride membranes. Immunoblotting was done with diluted (1:500) polyclonal VIM antibody (HPA001762; Sigma-Aldrich) and GAPDH antibody (MAB374; Chemicon, Temecula, CA, USA). The membrane was washed and then incubated with goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad, Hercules, CA, USA). Specific complexes were visualized with an echochemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK), and the expression levels of these genes were evaluated by ImageJ software (ver. 1.43; http://rsbweb.nih.gov/ij/index.html).
Immunohistochemistry
A tissue microarray of 67 RCC samples and 10 normal kidney samples was obtained from US Biomax Inc. (KD806; Rockville, MD, USA). Detailed information on all tumor specimens can be found at http://www.biomax.us/index.php. Patient characteristics are summarized in Table III. Immunostaining was done on the tissue microarray following the manufacturer’s protocol by UltraVision Detection System (Thermo Scientific). The primary rabbit polyclonal antibodies against VIM (Sigma-Aldrich) were diluted 1:500. The slides were treated with biotinylated goat anti-rabbit. Diaminobenzidine hydrogen peroxidase was the chromogen, and the counterstaining was done with 0.5% hematoxylin. Immunostaining was evaluated according to a scoring method described previously (14). Each case was scored on the basis of the intensity and area of staining. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, 30–60% stained positive; 3+, >60% stained positive. A combined staining score (intensity + extent) of <2 was low expression, a score between 3 and 4 was moderate expression, and a score between 5 and 6 was high expression.
Statistical analysis
The relationships between two variables and numerical values were analyzed using the Mann-Whitney U test, and the relationship between three variables and the numerical values was analyzed using the Bonferroni-adjusted Mann-Whitney U test. Expert Stat View analysis software (ver. 4; SAS institute Inc., Cary, NC, USA) was used in both analyses. In the comparison of three variables, a non-adjusted statistical level of significance of P<0.05 corresponded to the Bonferroni-adjusted level of P<0.0167.
Results
Effect of miR-138 transfection on cell proliferation, migration, and invasion activity of RCC cell lines
In this study, we firstly observed that restoration of miR-138 in RCC cell lines (A498 and 786-O) changed the bleb-like cell morphology characteristic of the epithelial-mesenchymal transition (EMT) (Fig. 1A). A morphological change of cancer cells by miRNA transfection is an important discovery and it suggested that miR-138 functions as a tumor suppressor in RCC cells. To explore that possibility, the following experiments were conducted.
We evaluated the expression levels of miR-138 in two RCC cell lines, A498 and 786-O. RNA was extracted and miRNA expression levels of miR-138 were determined by real-time RT-PCR. The expression levels of miR-138 were significantly lower in both RCC cell lines compared with normal kidney RNA (relative to normal kidney RNA, 0.090±0.008 and 0.102±0.009, respectively) (Fig. 1B).
The XTT assay revealed that cell proliferation was significantly inhibited in miR-138 transfectants in comparison with the transfectant reagent only (mock) and the miR-control transfectants. The percentages of cell proliferation for A498 were 94.6±0.9, 100.0±0.8 and 100.0±1.0, respectively, each P=0.0008. For 786-O, the percentages were 83.5±1.1, 100.0±0.4 and 100.3±0.6, respectively, P<0.0001 (Fig. 1C).
The wound healing assay demonstrated that significant inhibition of cell migration occurred in the miR-138 transfectants in comparison with mock and the miR-control transfectants. The percentages of wound closure for A498 were 6.5±2.3, 100.0±2.7 and 104.8±4.9, respectively, each P<0.0001. For 786-O, the percentages were 30.7±3.8, 100.0±4.4 and 95.9±5.4, respectively, each P<0.0001 (Fig. 1D).
The Matrigel invasion assay demonstrated that the number of invading cells significantly decreased in the miR-138-transfectants in comparison with mock and the miR-control transfectants. The percentages of cell invasion for A498 were 0.9±0.4, 100.0±8.6 and 83.1±7.4, respectively, each P<0.0001. For 786-O, the values were 10.9±1.1, 100.0±4.4 and 75.3±6.2, respectively, each P<0.0001 (Fig. 1E).
miR-138 regulation of molecular targets assessed by genome-wide gene expression analysis
To confirm that miR-138 regulated molecular targets in RCC cells, we performed genome-wide gene expression analysis using miR-138 transfectants compared with miRNA-control transfectants in A498 cells. A total of 99 genes were downregulated in miR-138 transfectants. Among them, 24 genes had putative target site(s) in their 3′ untranslated region (3′UTR) according to the TargetScan miRNA program (Table II).
Furthermore, we performed gene expression analysis using RCC clinical specimens (5 pairs of RCC and adjacent non-cancerous tissues). Several protein-coding genes were differentially expressed in this signature (data not shown). We selected 99 genes that were downregulated in miR-138 transfectants and demonstrated their expression levels in a heatmap diagram (Fig. 2A). Entries from the microarray data were approved by the Gene Expression Omnibus (GEO), and were assigned GEO accession numbers GSE 36951 (RCC clinical specimens) and GSE 37119 (miR-138 transfectants).
The two expression signatures in this study (miR-138 transfectants and RCC clinical specimens) revealed that VIM was a promising putative target gene in miR-138 in RCC. Thus, we focused on the VIM gene and investigated the functional significance of VIM in RCC cells.
VIM as a direct target of repression by miR-138 in RCC cells
The mRNA and protein expression levels of VIM were markedly downregulated in miR-138 transfectants (A498 and 768-O) in comparison with the mock and miRNA-control transfectants (Fig. 2B and C). The predicted target site of miR-138 in VIM in the 3′UTR is shown in Fig. 2D.
Silencing of VIM in RCC cell lines and the effect on cell proliferation, migration and invasion
First, we assessed the expression level of VIM in cells to be used for functional analysis of VIM. VIM mRNA expression levels in A498 and 786-O were significantly higher than those in normal human kidney RNA (relative to normal kidney RNA, 4.879±0.131 and 9.298±0.255, respectively, each P<0.0001) (Fig. 3A).
To examine the functional role of VIM, we performed loss-of-function studies using two different siRNAs, si-VIM-1 and si-VIM-2 transfected into A498 and 768-O cell lines. The mRNA and protein expression levels of VIM were markedly downregulated in both si-VIM-1 and si-VIM-2 transfectants (A498 and 768-O) in comparison with the siRNA-control transfectants (Fig. 3B and C). This result shows that two siRNA were useful for loss-of-function assays in this study.
Transfection of si-VIM-1 and si-VIM-2 in the RCC cell lines (A498 and 768-O) caused EMT-like changes in cell morphology, as that observed when cells were transfected with miR-138 (Fig. 4A).
The XTT assay revealed that cell proliferation was inhibited in both si-VIM-transfectants in comparison with the si-control transfectants. The percentages of cell proliferation for A498 were 76.1±2.6, 87.5±1.9 and 100.0±3.1, respectively, P<0.0001 and P =0.0038. For 786-O, the values were 43.1±1.0, 89.9±0.7 and 100.0±1.1, respectively, P<0.0001 (Fig. 4B).
The wound healing assay demonstrated that significant inhibition of cell migration occurred in the si-VIM-transfectants in comparison with the si-control transfectants. The percentages of wound closure for A498 were 26.0±10.6, 44.7±8.4 and 100.0±2.6, respectively, each P<0.0001. For 786-O, the values were 60.8±7.3, 73.0±1.7 and 100.0±7.1, respectively, P<0.0001 and P=0.0002 (Fig. 4C).
The Matrigel invasion assay demonstrated that the number of invading cells significantly decreased in the si-VIM-transfectants in comparison with the si-control transfectants. For A498, the percentages of cells invading were 26.5±4.7, 54.8±8.3 and 100.0±12.4, respectively, P<0.0001 and P=0.0019. For 786-O, the values were 43.6±8.3, 61.6±3.3 and 100.0±11.1, respectively, P<0.0001 and P=0.0034 (Fig. 4D).
Expression levels of miR-138 and VIM mRNA in RCC clinical specimens
Quantitative stem-loop RT-PCR demonstrated that the expression levels of miR-138 were significantly reduced in 33 RCC samples (Table I) in comparison with adjacent non-cancerous specimens (clinical RCC specimens, 0.346±0.201 versus adjacent normal tissues, 2.983±0.715, P<0.0001) (Fig. 5A).
On the other hand, the mRNA expression level of VIM was significantly higher in RCC than adjacent non-cancerous specimens (clinical RCC specimens, 6.017±0.622, adjacent normal tissues; 1.316±0.224, P<0.0001) (Fig. 5B).
The mRNA expression of ≥T2 specimens (n=7) was significantly higher than that of T1 (n=26) (T1, 5.230±0.607; ≥T2, 8.773±1.349, P=0.0277) (Fig. 5C).
Immunohistochemistry of VIM in tissue microarray
VIM was detected by immunohistochemical staining. Fig. 6A–D shows representative results of immunohistochemical staining of VIM. VIM was strongly expressed in tumor lesions (Fig. 6A–C), whereas no expression was observed in normal tissue (Fig. 6D). The expression score of VIM was significantly higher in 67 RCC specimens in comparison with ten normal kidney specimens (Fig. 6E). The VIM expression of ≥T2 specimens (n=52) was higher than that of T1 (n=15). There was a trend but no significant difference in the expression score of VIM between T1 and ≥T2 specimens (P=0.053, Fig. 6F). While there was a trend between VIM expression and grade, the difference was not significant. Patient characteristics are summarized in Table III.
Discussion
The incidence of RCC has increased over the last few decades, and it currently represents approximately 2% of all cancer-related deaths (25). Although two-thirds of RCC patients have clinically localized disease and will undergo curative surgery, up to 40% of patients develop distant metastasis and their outcomes are poor (2–4). Many studies have indicated that cell adhesion and extra-cellular matrix proteins contribute to the cells’ acquired abilities for invasion, migration and metastasis (26).
EMT is an embryologically conserved genetic program that is an essential step enabling cancer cell invasion and metastasis. For example, epithelial cells lose intercellular tight junctions and polarity (27). Recent data indicate that the microRNA-200 family (miR-200a, -200b, -200c, -141 and -429) is downregulated in aggressive human cancers. Moreover, it plays critical roles in the inhibition of key regulators of EMT and β-catenin/Wnt signaling (28,29). Interestingly, our miRNA expression signature of RCC showed that all miR-200 family members were reduced in clinical specimens and that restoration of the miR-200 family inhibited cancer cell migration and invasion (data not shown).
Our previous study showed that miR-138 was reduced in RCC miRNA expression signature (12), we validated the down-regulation of miR-138 in RCC clinical specimens in this study. Aberrant expression of miR-138 has been observed in several types of cancer such as head and neck squamous cell carcinoma (HNSCC) (30), anaplastic thyroid carcinoma (19) and lung cancer (20). Two miR-138 precursor genes, miR-138-1 and miR-138-2, have identical sequences in the mature miRNA and map to human chromosomes 3p21.33 and 16q13, respectively. Although it is believed that genomic deletion or epigenetic silencing of miRNA in cancer cells, the molecular mechanism of downregulated miRNAs in RCC is not clear. In the human chromosomal region 3p, LOH is frequently observed in many cancers including RCC (31,32). This problem can be solved by genome-based high throughput analysis in each clinical case.
Importantly, we found significant morphologic change in RCC cell lines (A498 and 786-O) by miR-138 transfection. Furthermore, restoration of miR-138 significantly inhibited cancer cell migration and invasion in RCC cells. These data suggested that miR-138 functions as a tumor suppressor that inhibits RCC invasion and metastasis. miRNAs are unique in their ability to regulate many protein-coding genes. A single miRNA is capable of targeting a number of genes to regulate biological processes globally. Bioinformatic predictions suggest that miRNAs regulate more than 30% of protein coding genes (9). The elucidation of new molecular pathways regulated by tumor suppressive miR-138 is important for our understanding of human RCC invasion and metastasis. Based on this view, we performed molecular target searches for miR-138 in cancer cells by combining two genome-wide gene expression studies (miR-138 transfectants and RCC mRNA clinical signature) and in silico analysis.
In this study, we focused on VIM as a putative candidate of miR-138 in RCC cells. We chose VIM for the following reasons. First, downregulation of VIM was recognized in the expression signature of miR-138 transfectants. Second, overexpression of VIM was observed in RCC clinical specimens. Third, VIM has a putative miR-138 target site in its 3′ untranslated region. Our data demonstrated that restoration of miR-138 significantly inhibited both mRNA and protein expression levels of VIM in RCC cells, suggesting VIM was regulated by tumor suppressive miR-138. It is well known that VIM is an essential constituent of cytoskeletal proteins of mesenchymal cells and VIM is a marker of EMT (21). During EMT, cytoskeletal proteins are changed from keratin-rich networks to VIM-rich networks connected to focal adhesions. Morphological changes of RCC cells and accelerated cell migration and invasion is caused by the reduction of miR-138 and the upregulation of VIM pathways. Interestingly, it has been shown that miR-138 regulated cell migration and invasion by targeting RhoC, ROCK, ZEB2, EZH2 and VIM in HNSCC cells (33,34). Importantly, restoration of miR-138 in an HNSCC cell line changed the EMT-like cell morphology and suppressed cell migration and invasion (34). This result is in accord with the data obtained in our study of RCC. Furthermore, overexpression of miR-138 reduced cell viability and colony formation in HCC cell lines targeting CCND3(35). The report also showed that protein expression of CCND3 was negatively correlated with miR-138 expression in HCC tissues. Our data of miR-138 transfectants in RCC cell lines demonstrated that CCND3 is a putative target of miR-138 in RCC, suggesting that miR-138 regulation of the CCND3 pathway is important for RCC oncogenesis.
The results of this study and previous data indicate that VIM is a functional target of tumor suppressive miR-138, and this pathway contributes to cancer cell migration, invasion, and metastasis. In this study, we also demonstrated overexpression of VIM in clinical specimens of RCC. Previous studies indicated that VIM was a sensitive and specific marker for conventional RCCs (36,37). The combination of VIM and CD9 staining was found to distinguish clear cell RCC and chromophobe RCC (37). Our tissue microarray data showed a positive correlation between VIM expression and tumor grade in RCC specimens. In this analysis, we were not able to obtain a correlation of VIM expression and metastasis in RCC patients. Silencing of VIM in RCC cell lines changed cell morphology and significantly inhibited cell migration and invasion in this study. Thus, we propose that overexpression of VIM participates in metastasis of RCC. Studies of a large number of samples with balanced pathological backgrounds are needed to elucidate the precise correlation between VIM and/or miR-138 expression and clinicopathological parameters.
In summary, the reduction of miR-138 and the increased expression of VIM are frequent events in RCC clinical specimens. Restoration of miR-138 in RCC cells changed the EMT-like morphology and suppressed cell migration and invasion. The tumor suppressive miR-138-mediated cancer pathway provides new insights into the potential mechanisms of RCC oncogenesis and metastasis.
Acknowledgements
This research was supported by the Ministry of Education, Science, Sports and Culture Grant-in-Aid for Scientific Research (C), 20591861 and 21592187.
References
|
Karumanchi SA, Merchan J and Sukhatme VP: Renal cancer: molecular mechanisms and newer therapeutic options. Curr Opin Nephrol Hypertens. 11:37–42. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Janzen NK, Kim HL, Figlin RA and Belldegrun AS: Surveillance after radical or partial nephrectomy for localized renal cell carcinoma and management of recurrent disease. Urol Clin North Am. 30:843–852. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Koul H, Huh JS, Rove KO, Crompton L, Koul S, Meacham RB and Kim FJ: Molecular aspects of renal cell carcinoma: a review. Am J Cancer Res. 1:240–254. 2011.PubMed/NCBI | |
|
Hadoux J, Vignot S and De La Motte Rouge T: Renal cell carcinoma: focus on safety and efficacy of temsirolimus. Clin Med Insights Oncol. 4:143–154. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Huang Y, Dai Y, Yang J, Chen T, Yin Y, Tang M, Hu C and Zhang L: Microarray analisis of microRNA expression in renal cell carcinoma. Eur J Surg Oncol. 35:1119–1123. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Motzer RJ, Bander NH and Nanus DM: Renal-cell carcinoma. N Engl J Med. 335:865–875. 1996. View Article : Google Scholar | |
|
Motzer RJ, Aqarwal N, Beard C, Bhayani S, Bolger GB, Carducci MA, Chang SS, Choueiri TK, Hancock SL, Hudes GR, Jonasch E, Josephson D, Kuzel TM, Lavine EG, Lin DW, Margolin KA, Michaelson MD, Olencki T, Pili R, Ratliff TW, Redman BG, Robertson CN, Ryan CJ, Sheinfeld J, Spiess PE, Wang J and Wilder RB: Kidney cancer. J Natl Compr Canc Netw. 9:960–977. 2011.PubMed/NCBI | |
|
Agarwala SS and Case S: Everolimus (RAD001) in the treatment of advanced renal cell carcinoma: a review. Oncologist. 15:236–245. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Fillipowicz W, Bhattacharyya SN and Sonenberg N: Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 9:102–114. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Nelson KM and Weiss GJ: MicroRNAs and cancer: past, present, and potential future. Mol Cancer Ther. 7:3655–3660. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Esquela-Kerscher A and Slack FJ: OncomiRs-microRNAs with a role in cancer. Nat Rev Cancer. 6:259–269. 2006. View Article : Google Scholar | |
|
Hidaka H, Seki N, Yoshino H, Yamasaki T, Yamada Y, Nohata N, Fuse M, Nakagawa M and Enokida H: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma. Oncotarget. 3:44–57. 2012.PubMed/NCBI | |
|
Chiyomaru T, Enokida H, Tatarano S, Kawahara K, Uchida Y, Nishiyama K, Fujimura L, Kikkawa N, Seki N and Nakagawa M: miR-145 and miR-133a function as tumour suppressors and directly regulate FSCN1 expression in bladder cancer. Br J Cancer. 102:883–891. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Yoshino H, Chiyomaru T, Enokida H, Kawakami K, Tatarano S, Nishiyama K, Nohata N, Seki N and Nakagawa M: The tumoursuppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer. Br J Cancer. 104:808–818. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Kojima S, Chiyomaru T, Kawakami K, Yoshino H, Enokida H, Nohata N, Fuse M, Ichikawa T, Naya Y, Nakagawa M and Seki N: Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase (PNP) in prostate cancer. Br J Cancer. 106:405–413. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Nohata N, Hanazawa T, Kikkawa N, Sakurai D, Fujimura L, Chiyomaru T, Kawakami K, Yoshino H, Enokida H, Nakagawa M, Katayama A, Harabuchi Y, Okamoto T and Seki N: Tumor suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma. Br J Cancer. 105:833–841. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Kikkawa N, Hanazawa T, Fujimura L, Nohata N, Suzuki H, Chazono H, Sakurai D, Horiguchi S, Okamoto Y and Seki N: miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC). Br J Cancer. 103:877–884. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Yamasaki T, Yoshino H, Enokida H, Hidaka H, Chiyomaru T, Nohata N, Kinoshita T, Fuse M, Seki N and Nakagawa M: Novel molecular targets regulated by tumor suppressors microRNA-1 and microRNA-133a in bladder cancer. Int J Oncol. 40:1821–1830. 2012.PubMed/NCBI | |
|
Mitomo S, Maesawa C, Ogasawara S, Iwaya T, Shibazaki M, Yashima-Abo A, Kotani K, Oikawa H, Sakurai E, Izutsu N, Kato K, Komatsu H, Ikeda K, Wakabayashi G and Masuda T: Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines. Cancer Sci. 99:280–286. 2008. View Article : Google Scholar | |
|
Seike M, Goto A, Okano T, Bowman ED, Schetter AJ, Horikawa I, Mathe EA, Jen J, Yang P, Sugimura H, Gemma A, Kudoh S, Croce CM and Harris CC: MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers. Proc Natl Acad Sci USA. 106:12085–12090. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Satelli A and Li S: Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci. 68:3033–3046. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Sobin LH and Wittekind C: TNM classification of malignant tumours In: International Union Against Cancer (UICC). 6th edition. John Wiley & Sons; Hoboken, NJ: pp. 255–257. 2009 | |
|
Nohata N, Hanazawa T, Kikkawa N, Mutallip M, Fujimaru L, Yoshino H, Kawakami K, Chiyomaru T, Enokida H, Nakagawa M, Okamoto T and Seki N: Caveolin-1 mediates tumor cell migration and invasion and its regulation by miR-133a in head and neck squamous cell carcinoma. Int J Oncol. 38:209–217. 2011.PubMed/NCBI | |
|
Nohata N, Sone Y, Hanazawa T, Fuse M, Kikkawa N, Yoshino H, Chiyomaru T, Kawakami K, Enokida H, Nakagawa M, Shozu M, Okamoto T and Seki N: miR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma. Oncotarget. 2:29–44. 2011.PubMed/NCBI | |
|
Siegel R, Naishadham D and Jemal A: Cancer statistics, 2012. CA Cancer J Clin. 62:10–29. 2012. View Article : Google Scholar | |
|
Friedl P and Alexander S: Cancer invasion and the microenvironment: plasticity and reciprocity. Cell. 147:992–1009. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Soini Y: Tight junctions in lung cancer and lung metastasis: a review. Int J Clin Exp Pathol. 5:126–136. 2012.PubMed/NCBI | |
|
Brabletz S and Brabletz T: The ZEB/miR-200 feedback loop - a motor of cellular plasticity in development and cancer? EMBO Rep. 11:670–677. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Mongroo PS and Rustgi AK: The role of the miR-200 family in epithelial-mesenchymal transition. Cancer Biol Ther. 10:219–222. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Li X, Jiang L, Wang A, Yu J, Shi F and Zhou X: MicroRNA-138 suppresses invasion and promotes apoptosis in head and neck squamous cell lines. Cancer Lett. 286:217–222. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Hogg RP, Honorio S, Martinez A, Agathanggelou A, Dallol A, Fullwood P, Weichselbaum R, Kuo MJ, Maher ER and Latif F: Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma. Eur J Cancer. 38:1585–1592. 2002. View Article : Google Scholar | |
|
Sukosd F, Kuroda N, Beothe T, Kaur AP and Kovacs G: Deletion of chromosome 3p14.2-p25 involving the VHL and FHIT genes in conbentional renal cell carcinoma. Cancer Res. 63:455–457. 2003.PubMed/NCBI | |
|
Jiang L, Liu X, Kolokythas A, Yu J, Wang A, Heidbreder CE, Shi F and Zhou X: Downregulation of the Rho GTPase signaling pathway is involved in the microRNA-138-mediated inhibition of cell migration and invasion in tongue squamous cell carcinoma. Int J Cancer. 127:505–512. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Liu X, Wang C, Chen Z, Jin Y, Wang Y, Kolokythas A, Dai Y and Zhou X: MicroRNA-138 suppresses epithelial-mesenchymal transition in squamous cell carcinoma cell lines. Biochem J. 440:23–31. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Wang W, Zhao LJ, Tan YX, Ren H and Qi ZT: Mir-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma. Carcinogenesis. 33:1113–1120. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Young AN, Amin MB, Moreno CS, Lim SD, Cohen C, Petros JA, Marshall FF and Neish AS: Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers. Am J Pathol. 158:1639–1651. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Williams AA, Higgins JP, Zhao H, Ljunberg B and Brooks JD: CD9 and vimentin distinguish clear cell from chromophobe renal cell carcinoma. BMC Clin Pathol. 9:92009. View Article : Google Scholar : PubMed/NCBI |
