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Keywords:

  • hypoxia-inducible factor-1α;
  • micro-ribonucleic acid;
  • micro-ribonucleic acid-155;
  • quantitative reverse transcript polymerase chain reaction;
  • renal cell carcinoma

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Objectives

To investigate the clinical significance of micro-ribonucleic acid-155 in clear cell renal cell carcinoma, in particular focusing on the association of expression levels of micro-ribonucleic acid-155 with clinicopathological factors, cancer-specific survival and therapeutic outcomes in clear cell renal cell carcinoma patients.

Methods

Quantitative reverse transcription polymerase chain reaction of micro-ribonucleic acid-155 was carried out on 137 clear cell renal cell carcinoma cases, containing 77 matched pairs of clear cell renal cell carcinoma and normal adjacent kidney tissues from the same patients.

Results

Significant overexpression of micro-ribonucleic acid-155 was found in clear cell renal cell carcinoma compared with normal kidney tissue. Expression of micro-ribonucleic acid-155 was not associated with prognosis in all stage groups. However, in 43 patients with stage III and IV clear cell renal cell carcinoma, low expression levels of micro-ribonucleic acid-155 correlated with a poor prognosis. Regarding cancer-free survival of 26 patients with stage III and IV clear cell renal cell carcinoma who received curative resection and cancer-specific survival of 31 patients who received postoperative therapy with interferon-α after radical nephrectomy, low expression levels of micro-ribonucleic acid-155 correlated with poor clinical outcomes in these two groups.

Conclusions

Low expression of micro-ribonucleic acid-155 represents a valuable marker of poor clinical outcomes in patients with stage III and IV clear cell renal cell carcinoma.


Abbreviations & Acronyms
AJCC

American Joint Committee on Cancer

ccRCC

clear cell renal cell carcinoma

cDNA

complementary deoxyribonucleic acid

CFS

cancer-free survival

CI

confidence intervals

CSS

cancer-specific survival

FFPE

formalin fixed paraffin embedded

HIF-1α

hypoxia-inducible factor-1α

HR

hazard ratio

IFN-α

interferon-α

IHC

immunohistochemistry

miRNA

micro-ribonucleic acid

NS

not significant

PI3K

phosphoinositide 3-kinase

qRT–PCR

quantitative reverse transcription polymerase chain reaction

RCC

renal cell carcinoma

RNA

ribonucleic acid

SE

standard error

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

RCC is the most common neoplasm of the adult kidney and the incidence is increasing worldwide, and the most common subtype is ccRCC.[1] If detected early, ccRCC can be treated surgically, and 5-year survival rates approaching 85% can be achieved for patients with organ-confined disease.[2] In reality, 40–50% of patients develop metastatic disease; 20–30% present with metastases, and 20–30% relapse distantly after curative nephrectomy. Treatment options for these patients are limited, and expected 5-year survival is approximately 10%.[2] Therefore, it is necessary to identify new biomarkers enabling prediction of early metastasis after nephrectomy and to develop new targeted therapies.

MiRNA are short non-coding RNA of 18–25 nucleotides in length that regulate gene expression post-transcriptionally. Aberrant miRNA expression is reported in many cancers, suggesting that they have a novel role as oncogenes or tumor suppressors.[3-5] Recent evidence showed the diverse clinical uses of miRNA for cancer as diagnostic, prognostic and predictive markers.[6] Differential expression of miRNA has been investigated in RCC. MiR-122, miR-155, miR-21, miR-106a, miR-182, miR-106b and miR-210 have been reported to be overexpressed in RCC, whereas miR-141, miR-200c, miR-335 and miR-218 are downregulated.[7-15] MiR-155 has repeatedly been identified through miRNA microarray profiling as upregulated in RCC tissue and its biological role has been studied.[11, 12] A significant correlation was found between miR-155 expression and tumor size.[7, 8, 16] Although miR-155 correlates with prognosis in breast cancer, lung cancer, hepatocellular carcinoma, colorectal cancer and pancreatic tumors,[17-21] there is only one report examining the correlation between miR-155 and prognosis in ccRCC, which was analyzed in just 31 cases and showed no prognostic impact of miR-155.[8] To confirm the correlation between miR-155 and prognosis of ccRCC patients, more validation studies in large sample sets should be carried out.

In the present study, we analyzed expression levels of miR-155 in 137 ccRCC cases by qRT–PCR, and compared these to the expression levels from 77 corresponding normal kidney tissue samples. Furthermore, the associations between expression levels of miR-155 and clinicopathological factors including prognosis were also investigated.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Tissue samples

In total, 137 primary tumor samples and 77 normal adjacent samples were collected from patients diagnosed with ccRCC. Patients were treated at the Hiroshima University Hospital from 1993 to 2010. Some cases, from a single institution, that did not have available tissue and clinical information about postoperative follow up were excluded.

Clinical stage was determined according to the AJCC Cancer Staging Manual, 7th edition. Patient's ages ranged between 34 and 89 years, with a median of 66 years. Histological diagnosis was established according to the guidelines of the World Health Organization. Cases were selected according to tissue availability and were not stratified for any known preoperative or pathological prognostic factor. The median follow-up time for all cases was 65 months and ranged from 2 to 188 months. We measured tumor sizes in 123 ccRCC tissue samples that could be confirmed macroscopically in pathological samples.

Because written informed consent was not obtained, for strict privacy protection the identifying information for all samples was removed before analysis. This procedure was in accordance with the Ethical Committee for Human Genome Research of Hiroshima University (Hiroshima, Japan). Clinical details of the patients are summarized in Table 1. We analyzed 43 samples to confirm stage III and IV in 137 samples. The patient's clinical details are summarized in Table 2.

Table 1. Clinical characteristics of the 137 ccRCC patients
    Expression of miR-155P-value
LowHigh
  1. The expression levels of miR-155 were divided into two groups, low and high expression of miR-155, based on the median miR-155 expression level (cut-off line = the median miR-155 expression level in this group). Corresponding median value = −0.0831.

Sex (%) Sex (%)   0.0426
Male103 (75)Male103 (75)4657
Female34 (25)Female34 (25)2212
Side (%) Side (%)   NS
Left59 (43)Left59 (43)2435
Right78 (57)Right78 (57)4434
Median age, years (range) Median age, years (%)   NS
 66 (34–89)<6564 (47)3430
  ≥6573 (53)3439
Histological grade (%) Histological grade (%)   NS
G160 (44)G1–2103 (75)5647
G243 (31)G3–434 (25)1222
G331 (23)    
G43 (2)    
Infiltrating type (%) Infiltrating type (%)   NS
INFa113 (82)INFa113 (82)5756
INFb23 (17)INFb/c24 (18)1113
INFc1 (1)    
pT stage (%) pT stage (%)   NS
pT183 (61)pT1–299 (72)5247
pT216 (12)pT3–438 (28)1622
pT336 (26)    
pT42 (1)    
pN stage (%) pN stage (%)   NS
pNx79 (58)pNx/0129 (94)6663
pN050 (36)pN1/28 (6)26
pN1/28 (6)    
Venous invasion (%) Venous invasion (%)   NS
v093 (68)v093 (68)4944
v144 (32)v144 (32)1925
M stage (%) M stage (%)   NS
M0117 (85)M0117 (85)6057
M120 (15)M120 (15)812
Stage grouping (%) Stage grouping   NS
Stage I81 (59)Stage I/II94 (68)5044
Stage II13 (9)Stage III/IV43 (32)1825
Stage III23 (17)    
Stage IV20 (15)    
Observation period (months)      
Median65     
Range2–188     
Table 2. Clinical characteristics of 43 stage III and IV ccRCC patients
    Expression of miR-155P-value
LowHigh
  1. The expression levels of miR-155 were divided into two groups, low and high expression of miR-155, based on the median miR-155 expression level (cut-off line = the median miR-155 expression level in this group). Corresponding median value = −0.0811.

Sex (%) Sex (%)   NS
Male39 (90)Male39 (90)1821
Female4 (10)Female4 (10)31
Side (%) Side (%)   NS
Left14 (33)Left14 (33)68
Right29 (67)Right29 (67)1514
Median age, years (range) Median age, years (%)   NS
 66 (45–89)<6521 (49)1011
  ≥6522 (51)1111
Histological grade (%) Histological grade (%)   NS
G17 (16)G1–222 (59)1111
G215 (35)G3–421 (51)1110
G318 (42)    
G43 (7)    
Infiltrating type (%) Infiltrating type (%)   NS
INFa25 (58)INFa25 (58)1312
INFb17 (40)INFb/c18 (42)810
INFc1 (3)    
pT stage (%) pT stage (%)   NS
pT12 (5)pT1–25 (12)32
pT23 (7)pT3–438 (88)1820
pT336 (84)    
pT42 (5)    
pN stage (%) pN stage (%)   NS
pNx7 (16)pNx/035 (81)1718
pN028 (65)pN1/28 (19)44
pN1/28 (19)    
Venous invasion (%) Venous invasion (%)   NS
v08 (19)v08 (19)53
v135 (81)v135 (81)1619
M stage (%) M stage (%)   NS
M023 (53)M023 (53)1013
M120 (47)M120 (47)119
Observation period (months)      
Median43     
Range2–188     

For qRT–PCR, 137 ccRCC samples and 77 corresponding normal adjacent samples were used. Samples were FFPE tissues from 137 patients who had undergone surgical excision for ccRCC.

MiRNA extraction and qRT–PCR

MiRNA was extracted using a RecoverAll Total Nucleic Acid Isolation Kit for FFPE tissues (Ambion, Foster City, CA, USA). Reverse transcription was carried out using a TaqMan microRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Hsa-miR-155 was detected using TaqMan MicroRNA assays (Assay ID 000479; Applied Biosystems). Real-time quantification of cDNA was carried out on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Expression data were normalized according to expression of the RNU48 reference DNA (Assay ID 001006; Applied Biosystems).[22]

Immunohistochemistry

IHC was carried out on 4-μm thick sections of FFPE. Immunohistochemical analysis was carried out with a Dako EnVision+ System- HRP Labelled Polymer anti-mouse (Dako Cytomation, Carpinteria, CA, USA). Antigen retrieval for HIF-1α was carried out by microwave heating in citrate buffer (pH 6.0) for 30 min. After peroxidase activity had been blocked with 1% H2O2 / 50% methanol for 10 min, HIF-1α was detected with a mouse monoclonal antibody Mab H1α67 (1:200; Novus Biologicals, Littleton, CO, USA). A total of 64 sections were incubated with primary antibody for 1 h at room temperature, and then with Dako EnVision+ System- HRP Labelled Polymer anti-mouse for 1 h. For color reaction, sections were incubated with 3,3′-diaminobenzidine Substrate-Chromogen Solution (Dako Cytomation) for 3 s. Sections were counterstained with 0.1% hematoxylin. The cut-off point for antibody reactivity necessary to define a result as positive was staining of any cells with nuclear localization in surgically resected specimens.

IHC was carried out on 64 ccRCC samples, including 39 with the lowest expression of miR-155 tissue and 25 with the highest expression of miR-155 tissues of 137 patients who had undergone surgical excision for ccRCC.

Statistical analysis

Statistical differences between miRNA expression levels in ccRCC samples and normal kidney samples were evaluated using the Wilcoxon matched pairs test. Statistical differences of RNU48 reference DNA were evaluated using the non-parametric Mann–Whitney U-test. For analysis of correlation between expression levels of miR-155 and tumor size, we used the non-parametric Spearman's rank correlation coefficient. The correlation between expression levels of miR-155, clinicopathological parameters and expression of HIF-1α in IHC was analyzed with Fisher's exact test. A log–rank test and Kaplan–Meier plots were constructed for the miR-155-high and miR-155-low groups. Univariate and multivariate analysis of factors influencing survival were carried out using the Cox proportional hazards model. And parameters at multivariate analysis were selected by the stepwise method. The HR and 95% CI were estimated from the Cox proportional hazard model. Wilcoxon matched pairs test, Pearson's product-moment correlation coefficient, Fisher's exact test, a log–rank test and Kaplan–Meier plots were calculated using jmp software version 10 (SAS Institute, Cary, NC, USA). The Cox proportional hazards model, stepwise method and concordance, R2 and Wald test were calculated using the R statistical environment (http://www.R-project.org). For all analyses, age was treated as a categorical variable (65 years or more vs less than 65 years). A P-value of less than 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Expression levels of miR-155 between ccRCC and normal kidney tissues

We analyzed 77 matched pairs of ccRCC and normal adjacent kidney tissues from the same patients by qRT–PCR. To show the usefulness of RNU48 as a reference gene, we analyzed the expression of RNU48 between tumors and normal adjacent kidney tissue in 77 matched pairs of ccRCC samples (Fig. S1a). The expression level of RNU48 in some clinicopathological parameters in 137 ccRCC samples was also examined (Fig. S1b). The expression level of RNU48 showed no significant difference between tumors and normal adjacent kidney, and also among all clinicopathological parameters. Therefore, we confirmed that RNU48 is stably expressed in ccRCC and is useful as a reference gene.

Differences between the two groups were evaluated using the Wilcoxon matched pairs test. Highly significant differences were identified between 77 ccRCC with all stages and normal adjacent kidney tissues in the expression levels of miR-155 (P < 0.001), as well as limited to 63 ccRCC with stage I and II (P < 0.001), and 14 ccRCC with stage III and IV (P = 0.0138; Fig. 1).

figure

Figure 1. Expression of miR-155 between tumor and normal kidney tissue in 77 ccRCC samples in all stages (P < 0.001).

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Relationship between miR-155 expression and tumor size

We analyzed the correlation between expression levels of miR-155 and tumor size in 123 ccRCC tissues in which the tumor sizes had been established. Tumor size ranged between 0.8 and 17 cm, with a median of 4.6 cm. For analysis of the correlation between expression levels of miR-155 and tumor size, we used the non-parametric Spearman's rank correlation coefficient. There were no statistically significant differences, but expression levels of miR-155 tended to be associated with a tumor size (r = 0.2045, P = 0.0943; Fig. 2).

figure

Figure 2. Correlation between expression levels of miR-155 and tumor size in 123 samples. Pearson's product-moment correlation coefficient 0.2045 (P = 0.0943).

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Expression levels of miR-155 and clinical prognosis

To determine the difference in CSS and CFS on the basis of expression levels of miR-155, we divided the sample into two groups (low and high expression levels of miR-155) based on the median miR-155 expression level in the group. Kaplan–Meier plots were constructed in 137 ccRCC with all stages. This analysis showed no association with prognosis (P = 0.7001; Fig. 3a). In contrast, when limited to the stage III and IV groups, the analysis showed significant differences for CSS on the basis of miR-155 expression levels; low expression levels of miR-155 were correlated with poor prognosis in Kaplan–Meier plots and log–rank tests (P = 0.0337; Fig. 3b). Regarding the CFS of 26 patients with stage III and IV ccRCC who had received curative resection, means status of no visible residual tumor clinically after radical nephrectomy with or without metastatectomy. Low expression levels of miR-155 tended to be associated with a high rate of recurrence (P = 0.0614; Fig. 3c). We also analyzed CSS in 31 patients who had received postoperative IFN-α therapy after radical nephrectomy, and found that low expression levels of miR-155 were associated with poor prognosis (P = 0.0464; Fig. 3d).

figure

Figure 3. (a) CSS of 137 patients with ccRCC based on the expression levels of miR-155 (cut-off line = the median miR-155 expression level in this group). Corresponding median value = −0.0831; P = 0.7001. (b) CSS of 43 patients with ccRCC based on the expression levels of miR-155 (cut-off line = the median miR-155 expression level in this group) in stage III and IV. Corresponding median value = −0.0811; P = 0.0337. (c) CFS of 26 patients after undergoing curative resection for ccRCC based on the expression levels of miR-155 (cut-off line = the median miR-155 expression level in this group) in stage III and IV. Corresponding median value = 0.6190; P = 0.0614. (d) CSS of 31 patients with ccRCC based on expression levels of miR-155 (cut-off line = the median miR-155 expression level in this group) in 31 patients who received postoperative therapy with IFN-α after radical nephrectomy. Corresponding median value = 0.59781; P = 0.0464. image, miR-155 high; image, miR-155 low.

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Univariate and multivariate analysis of factors influencing survival

In univariate analyses, M stage, histological grade, infiltrating type, pT stage and pN stage were correlated with poor prognosis. Although high expression levels of miR-155 in univariate analysis were not correlated with poor prognosis, the independent predictors in the multivariate analysis were M stage, pT stage, pN stage and low expression levels of miR-155 (Table 3). In univariate analysis confined to patients with stage III and IV ccRCC, M stage, pN stage and low expression levels of miR-155 were correlated with poor prognosis. Independent predictors in multivariate analysis were M stage, histological grade, pT stage and low expression levels of miR-155. These experiments yielded that low expression of miR-155 was an independent indicator of poor prognosis (Table 4).

Table 3. Univariate and multivariate analysis of factors influencing survival in 137 patients with ccRCC
 Univariate analysisMultivariate analysis
Hazard ratio95% CIP-valueHazard ratio95% CIRobust SEP-value
  1. Concordance = 0.946 (SE = 0.059), R2 = 0.493, Wald test = 52.93 on 6 d.f. (P < 0.0001).

Sex       
Female1 0.0539Non-selected
Male4.120.98–17.42 
Age (years)       
<651 0.411Non-selected
≥651.010.98–1.05 
Side       
Right1 0.99301 0.6120.1500
Left1.010.46–2.17 2.440.73–8.10  
M stage       
cM01 <0.00011 0.485<0.0001
cM118.318.18–41.02 8.63.33–22.25  
Histological grade       
G1/21 <0.00011 0.6080.0710
G3/45.302.46–11.42 3.000.91–9.99  
INF       
INF a1 <0.0001Non-selected
INF b/c6.513.02–14.08 
pT       
pT1/21 <0.00011 0.9590.0055
pT3/419.736.77–57.52 14.372.19–94.17  
pN stage       
pNx/01 <0.00011 0.4610.0380
pN1/210.684.33–26.38 2.601.05–6.42  
Expression of miR-155       
High (>median)1 0.98501 0.4210.0001
Low (≤median)0.990.47–2.11 5.492.40–12.52  
Table 4. Univariate and multivariate analysis of factors influencing survival in 43 patients with stage III and IV ccRCC
 Univariate analysisMultivariate analysis
Hazard ratio95% CIP-valueHazard ratio95% CIRobust SEP-value
  1. Concordance = 0.818 (SE = 0.063), R2 = 0.561, Wald test = 27.42 on 6 d.f. (P = 0.0001).

Sex       
Female1 0.6570Non-selected
Male1.380.33–5.89 
Age (years)       
<651 0.4260Non-selected
≥651.010.98–1.05 
Side       
Right1 0.06731 0.5840.1700
Left2.110.46–2.17 0.450.14–1.41  
M stage       
cM01 0.00201 0.452<0.0001
cM13.691.61–8.48 11.564.77–28.01  
Histological grade       
G1/21 0.06111 0.5310.0110
G3/42.130.97–4.71 3.881.37–10.97  
INF       
INFa1 0.0947Non-selected
INFb/c1.900.86–4.01 
pT       
pT1/21 0.92101 0.685<0.0001
pT3/41.060.32–3.57 22.555.88–86.41  
pN stage       
pNx/01 0.04331 0.5740.1300
pN1/22.541.03–6.27 2.390.78–7.37  
Expression of miR-155       
High (>median)1 0.03841 0.3890.0001
Low (≤median)2.311.05–5.11 4.672.18–10.00  

Relationship between HIF-1α expression and expression of miR-155

We used IHC to investigate the association between HIF-1α expression and expression of miR-155 in 64 ccRCC samples that included 39 lowest expression of miR-155 tissue and 25 highest expression of miR-155 tissues in the 137 samples we analyzed.

In the 39 low expression levels of miR-155 ccRCC, the HIF-1α positive and HIF-1α negative tumor frequency were 17 out of 39 (44%) and 22 out of 39 (56%). In contrast, in the 25 high expression of miR-155 ccRCC, the HIF-1α positive and HIF-1α negative tumor frequency were 17 out of 25 (66%) and eight out of 25 (32%; Fig. 4). For analysis of the correlation between expression of miR-155 and HIF-1α, we used the Fisher's exact test. This difference was not statically significant, but high expression levels of miR-155 tended to be associated with a high HIF-1α expression (P = 0.0744; Table 5).

figure

Figure 4. IHC staining for HIF-1α in ccRCC. HIF-1α immunoreactivity in nuclei of tumor cells.

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Table 5. Association between expression of HIF-1α and expression of miR-155 (P = 0.0744)
 Expression of miR-155P-value
Low, (n = 39)High, (n = 25)
n (%)n (%)
HIF-1α   
Positive17 (44%)17 (68%)0.0744
Negative22 (56%)8 (32%)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

RCC remains to be one of the leading causes of death, so finding new molecular targets for its diagnosis, prognosis and treatment has the potential to improve the clinical strategies and outcomes of this disease. One of the most frequently studied miRNA in cancer biology, miR-155, has also been repeatedly identified through miRNA microarray profiling as upregulated in ccRCC tissue. It has been reported that miR-155 levels are almost 30-fold higher in ccRCC compared with normal tissues.[16] Consistent with this result, our qRT–PCR analysis showed that expression levels of miR-155 were 6.2-fold higher in 77 matched pairs of ccRCC than in normal adjacent kidney tissues (P < 0.001). The correlation between high expression levels of miR-155 and tumor size in ccRCC has been reported,[7, 8] and we also confirmed a significant association between expression of miR-155 and tumor size (r = 0.2045, P = 0.0238). The available experimental evidence indicates that miR-155 is overexpressed in a variety of malignant tumors. MiR-155 behaves as an oncogenic miRNA in breast cancer, lung cancer, hepatocellular carcinoma, colorectal cancer and pancreatic tumors.[17-21] It has been reported that miR-155 downregulates many tumor suppressor genes that repress PI3K or apoptosis-related signaling.[17, 23, 24] MiR-155 can be attributed to ccRCC tumor progression.

A significant correlation was found between low expression levels of miR-155 and prognosis in 43 patients with stage III and IV ccRCC using the log–rank test, and univariate and multivariate analysis. Especially in multivariate analysis, low expression levels of miR-155 showed a strong correlation with poor prognosis (P = 0.0010), and the CFS of 26 patients who had received curative resection tended to be associated with low expression levels of miR-155. We also found a significant correlation between low expression levels of miR-155 and prognosis in 31 patients who had received postoperative therapy with IFN-α after radical nephrectomy. These results are inconsistent with the results of all 137 cases. In general, the central part of a tumor will be in an ischemic and hypoxic state. Tumor cells undergo a variety of biological responses when placed in hypoxic conditions, including activation of signaling pathways that regulate proliferation, angiogenesis and cell death by the transcription factor HIF-1α.[25] The overexpression of HIF-1α is one of the important characteristic genetic abnormalities in ccRCC.[26] It has also been reported that hypoxia-induced miR-155 plays a role as a component of a network of negative feedback loops that controls HIF-1α translation.[27] It is well known that HIF-1α overexpression is a marker of unfavorable prognosis in human cancers and increases with tumor size.[26, 28-30] Based on the results of the present study, miR-155 is expressed with increasing tumor size, much as HIF-1α. Consistent with these results and past reports, expression of HIF-1α also tended to be more frequently found in ccRCC cases with high expression levels of miR-155 in IHC (P = 0.0720). In 137 ccRCC of all stages, there were no associations with prognosis in Kaplan–Meier plots, because this group includes many small tumor cases where it is suspected that there is a low expression of both HIF-1α and miR-155. However, when limited to the stage III and IV ccRCC groups, low expression levels of miR-155 showed a strong correlation with poor prognosis in Kaplan–Meier plots. This group includes many large tumor cases where it is suspected that both HIF-1α and miR-155 are highly expressed. It is suspected that expression of miR-155, in part, suppressed by certain factors might lead to breaking the state of the negative feedback loop and accumulation of HIF-1α, and worsening of the prognosis. Further study should be carried out to clarify which factors downregulate expression of miR-155 in advanced ccRCC showing poor prognosis.

In summary, the present study showed that miR-155 was significantly upregulated in ccRCC compared with normal tissue, and high expression levels of miR-155 were correlated with increased tumor size. We also identified that the expression of miR-155 was significantly suppressed in patients with stage III and IV ccRCC and was associated with poor prognosis. Thus, miR-155 might be a valuable biomarker for predicting the survival of patients with stage III and IV RCC, and play an important role in ccRCC progression.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Mr Shinichi Norimura for his excellent technical assistance and advice. This work was carried out with the kind cooperation of the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University. We thank the Analysis Center of Life Science, Hiroshima University, for the use of their facilities. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
iju3182-sup-0001-si.tif969K

Fig. S1 (A) Expression of RNU48 between tumor and normal kidney tissue in 77 matched pairs of clear renal cell carcinoma samples (P = 0.9896). (B) Expression level of RNU48 in different sexes (P = 0.6206), sides (P = 0.9654), histological grades (P = 0.9849), infiltrating type (P = 0.5084), pT stages (P = 0.8572), pN stages (P = 0.7671), venous invasion (P = 0.9540), M stages (P = 0.6206) and stages (P = 0.8576) in 137 clear renal cell carcinoma samples. Whiskers depict the 5 and 95 percentiles.

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