# Prognostic Impact of Hypoxia-InduciblemiRNA-210 in Patients with Lung Adenocarcinoma

Authors: Jun Osugi; Yuka Kimura; Yuki Owada; Takuya Inoue; Yuzuru Watanabe; Takumi Yamaura; Mitsuro Fukuhara; Satoshi Muto; Naoyuki Okabe; Yuki Matsumura; Takeo Hasegawa; Athushi Yonechi; Mika Hoshino; Mitsunori Higuchi; Yutaka Shio; Hiroyuki Suzuki; Mitsukazu Gotoh
Journal: Journal of Oncology
 (2015)

---

## Abstract

Objective. The aim of this study was to investigate the prognostic value of MicroRNA-210 (miR-210) expression in patients with non-small-cell lung cancer (NSCLC). Methods. We examined the miR-210 expression of samples of 80 patients, who underwent surgical resection at Fukushima Medical University from 2004 to 2007, by using quantitative RT-PCR. The relationship between miR-210 expression and clinicopathological factors as well as histological subtype was statistically analyzed. Results. miR-210 expression showed an inverse correlation with disease-free and overall survival in patients with NSCLC. Significant correlations were found between miR-210 expression and lymph node metastasis, late disease stages, and poor prognosis in patients with adenocarcinoma. Multivariate Cox analysis indicated that miR-210 expression was an independent prognostic factor for disease-free survival in patients with adenocarcinoma. Conclusions. We showed that miR-210 may be a prognostic biomarker for patients with NSCLC, especially for those with lung adenocarcinoma.


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## Body

## 1. Introduction

Hypoxia is a common feature of pathological conditions such as tissue ischemia and inflammation, as well as of the microenvironment of solid tumors [1]. Many cellular responses to hypoxia are thought to be mediated through changes in targeted gene expression. One major mechanism mediating cellular responses to hypoxia is the pathway of hypoxia inducible factor-1 (HIF-1) [2]. HIF-1 is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family of proteins and binds to hypoxia-response elements (HRE) in the promoters of target genes. HIF-1 consists of an alpha (HIF-1α) and a beta (HIF-1β) subunit and activates the expression of at least 150 genes, which encode proteins that regulate cell metabolism, cell cycle, proliferation, apoptosis, autophagy, erythropoiesis, immune reactions, cytokine production, and angiogenesis as well as many other functions [3]. HIF-1β is a non-oxygen-responsive nuclear protein. In contrast, HIF-1α is highly inducible by hypoxia [4].In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia and of genetic alterations affecting crucial oncogenes and tumor suppressor genes [3]. HIF-1α overexpression has been associated with increased patient mortality in many different human cancers [3]. Similarly, HIF-1α overexpression has been reported at both the protein [5, 6] and the mRNA [7, 8] level in non-small-cell lung cancer (NSCLC) patients with poor prognosis. In preclinical studies, inhibition of HIF-1α activity has marked effects on tumor growth; inhibitors of HIF-1α have therefore attracted much attention as new therapeutic agents for patients with advanced malignancies, and several clinical studies have been performed [3]. Research has shown that HIF-1α antagonists, such as EZN-2968 and PX-478, inhibit tumor cell proliferationin vitro andin vivo [9, 10].miRNAs have emerged as a new class of noncoding genes that are involved in the regulation of cell proliferation, differentiation, and viability [11]. miRNAs are single-stranded small RNA molecules of approximately 22 nucleotides that silence the expression of target genes either through mRNA degradation or suppression of transcription [12–14]. The miRNAs that are regulated by hypoxia were examined in a 2007 study in whichmiR-210 was identified as the most consistently and robustly induced miRNA in hypoxic cells and tissues [15].miR-210 expression is frequently elevated in a variety of cancers [15], including lung cancer [16–20].miR-210 is regulated by both HIF-1α [21–23] and HIF-2α [24], and a recent study demonstrated that HIF-1α directly binds to an HRE on the proximalmiR-210 promoter [23].miR-210 plays numerous crucial roles in the cellular response to hypoxia, such as in apoptosis [15, 25], angiogenesis [26], cell cycle regulation [24, 27], DNA damage repair [22], mitochondrial metabolism [28, 29], and tumor growth [19]. Furthermore,miR-210 is also involved in stem cell biology [30]. Thus,miR-210 is thought to have essential roles in tumorigenesis along with HIF-1α.It has been reported thatmiR-210 overexpression is correlated with poor prognosis in breast [21, 31], pancreatic [32], and head and neck cancer patients [31]. Recently, two systematic reviews and a meta-analysis confirmed thatmiR-210 is useful for prediction of the survival of patients with various tumors, especially breast cancers [33, 34]. However, these two studies did not include the outcome of patients with lung cancer. Therefore, the prognostic impact ofmiR-210 in patients with lung cancer remains unclear. Within this context, we analyzedmiR-210 expression in NSCLC patient samples, and showed that it could be a prognostic biomarker, especially for patients with adenocarcinoma.


## 2. Materials and Methods

### 2.1. Patient and Tissue Samples

In total, 80 snap-frozen NSCLC and 30 matched normal adjacent lung tissue samples were evaluated formiR-210 expression. These consecutive samples were obtained from patients who underwent surgical resection at the Department of Regenerative Surgery, Fukushima Medical University, Fukushima, Japan, from January 2004 to December 2007. The clinical characteristics of the 80 patients included in this study were typical of the characteristics of resected NSCLC reported by the Japan Lung Cancer Society (2004) with respect to age, sex, histology, and pathological stage [35]. None of the patients had received any previous anticancer treatment. Ethical approval for analysis of samples and patient notes was obtained from the local research ethics committee. Tumor types and stages were determined according to the 7th edition of Union for International Cancer Control TNM classification. At the time of surgery, all tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until assay. All samples were analyzed histologically to assess the amount of tumor component (at least 70% tumor cells) and the quality of the material (i.e., absence of necrosis). These 80 cases consisted of 34 female and 46 male patients with a median age of 69 years (range: 51–85), of which 54 were stage I cases, 12 were stage II, and 14 were stage III. The median observation period was 74.5 months (range: 5–117), and the five-year survival rate was 62.4%. Full patient clinicopathological characteristics are provided in Table 1.Table 1
Relationship betweenmiR-210 expression and clinicopathological factors.
Clinicopathological factor
Patient number
miR-210 miRNA expression
P
High group
Low group
Gender
0.258
Male
46
26
20
Female
34
14
20
Age
0.222
≤65
24
9
15
≥66
56
31
25
Tumor size
0.82
<30 mm
48
23
25
≥30 mm
32
17
15
T factor
0.481
T1-T2
61
34
37
T3-T4
9
6
3
Lymph node
0.293
Negative
61
28
33
Positive
19
12
7
P-stage
0.232
Stage I
54
24
30
Stages II-III
26
16
10
ly factor
0.502
Negative
41
19
22
Positive
39
21
17
v factor
0.359
Negative
43
22
27
Positive
27
18
13
Relapse
0.007*
With
37
25
12
Without
43
15
28
Relapse was defined as first evidence of radiographic metastatic disease after surgery.
*
P < 0.05.


### 2.2. RNA Extraction and Quantitative Real-Time PCR

TaqMan-based quantitative real-time polymerase chain reaction (qRT-PCR) was applied to assess miRNA expression levels in tissue samples. RNA was extracted from snap-frozen lung tumor samples or normal lung tissue using the miRVana miRNA Isolation Kit (Ambion, Austin TX, USA). The quantity and quality of the extracted RNA were determined spectrophotometrically by measurement of absorbance at 260 and 280 nm using a DU530 UV-VIS spectrophotometer (Beckman Coulter, Fullerton, CA, USA). Samples with a 260/280 ratio of 1.80 or greater were used for analysis. miRNA-cDNA was synthesized from 5 ng of total RNA using microRNA-specific primers and the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The TaqMan MicroRNA assays formiR-210 (ID: 000512) andRNU6B (ID: 001093) were purchased from Applied Biosystems. Real-time PCR was performed in triplicate using a StepOnePlus Real-Time PCR system (Applied Biosystems). For miRNA assays, each PCR reaction contained 1.33 μL reverse transcription product, 2×TaqMan Universal Master Mix, and 1 μL TaqMan MicroRNA assay. The 20 μL reactions were incubated in a 96-well optical plate at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds, and 60°C for 60 seconds. Changes in miRNA expression between treatment and controls were determined using the 2
-
Δ
Δ
Ct method [36], and results were normalized againstRNU6B expression levels. For lung cancer samples, the controls consisted of the median of 30 normal lung tissues.


### 2.3. Statistical Analysis

Correlations between the status ofmiR-210 expression and clinical characteristics were assessed using Student’s t-test, Pearson and Spearman’s rank test, or the Mann-Whitney U test. Kaplan-Meier survival analysis was performed by applying the long-rank test tomiR-210 expression and was stratified by median values and quartiles. Disease-specific overall survival (referred to as overall survival hereafter) was defined as the time from surgery to last follow-up or time of NSCLC-specific death. Disease-free survival was defined as the time from surgery to the time of first evidence of radiographic metastatic disease. Univariate and multivariate analyses were performed using the Cox proportional hazard model. All statistical analyses were performed using SPSS 17 software (SPSS Inc., Chicago, IL, USA), and P values of <0.05 were considered significant.


## 2.1. Patient and Tissue Samples

In total, 80 snap-frozen NSCLC and 30 matched normal adjacent lung tissue samples were evaluated formiR-210 expression. These consecutive samples were obtained from patients who underwent surgical resection at the Department of Regenerative Surgery, Fukushima Medical University, Fukushima, Japan, from January 2004 to December 2007. The clinical characteristics of the 80 patients included in this study were typical of the characteristics of resected NSCLC reported by the Japan Lung Cancer Society (2004) with respect to age, sex, histology, and pathological stage [35]. None of the patients had received any previous anticancer treatment. Ethical approval for analysis of samples and patient notes was obtained from the local research ethics committee. Tumor types and stages were determined according to the 7th edition of Union for International Cancer Control TNM classification. At the time of surgery, all tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until assay. All samples were analyzed histologically to assess the amount of tumor component (at least 70% tumor cells) and the quality of the material (i.e., absence of necrosis). These 80 cases consisted of 34 female and 46 male patients with a median age of 69 years (range: 51–85), of which 54 were stage I cases, 12 were stage II, and 14 were stage III. The median observation period was 74.5 months (range: 5–117), and the five-year survival rate was 62.4%. Full patient clinicopathological characteristics are provided in Table 1.Table 1
Relationship betweenmiR-210 expression and clinicopathological factors.
Clinicopathological factor
Patient number
miR-210 miRNA expression
P
High group
Low group
Gender
0.258
Male
46
26
20
Female
34
14
20
Age
0.222
≤65
24
9
15
≥66
56
31
25
Tumor size
0.82
<30 mm
48
23
25
≥30 mm
32
17
15
T factor
0.481
T1-T2
61
34
37
T3-T4
9
6
3
Lymph node
0.293
Negative
61
28
33
Positive
19
12
7
P-stage
0.232
Stage I
54
24
30
Stages II-III
26
16
10
ly factor
0.502
Negative
41
19
22
Positive
39
21
17
v factor
0.359
Negative
43
22
27
Positive
27
18
13
Relapse
0.007*
With
37
25
12
Without
43
15
28
Relapse was defined as first evidence of radiographic metastatic disease after surgery.
*
P < 0.05.


## 2.2. RNA Extraction and Quantitative Real-Time PCR

TaqMan-based quantitative real-time polymerase chain reaction (qRT-PCR) was applied to assess miRNA expression levels in tissue samples. RNA was extracted from snap-frozen lung tumor samples or normal lung tissue using the miRVana miRNA Isolation Kit (Ambion, Austin TX, USA). The quantity and quality of the extracted RNA were determined spectrophotometrically by measurement of absorbance at 260 and 280 nm using a DU530 UV-VIS spectrophotometer (Beckman Coulter, Fullerton, CA, USA). Samples with a 260/280 ratio of 1.80 or greater were used for analysis. miRNA-cDNA was synthesized from 5 ng of total RNA using microRNA-specific primers and the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The TaqMan MicroRNA assays formiR-210 (ID: 000512) andRNU6B (ID: 001093) were purchased from Applied Biosystems. Real-time PCR was performed in triplicate using a StepOnePlus Real-Time PCR system (Applied Biosystems). For miRNA assays, each PCR reaction contained 1.33 μL reverse transcription product, 2×TaqMan Universal Master Mix, and 1 μL TaqMan MicroRNA assay. The 20 μL reactions were incubated in a 96-well optical plate at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds, and 60°C for 60 seconds. Changes in miRNA expression between treatment and controls were determined using the 2
-
Δ
Δ
Ct method [36], and results were normalized againstRNU6B expression levels. For lung cancer samples, the controls consisted of the median of 30 normal lung tissues.


## 2.3. Statistical Analysis

Correlations between the status ofmiR-210 expression and clinical characteristics were assessed using Student’s t-test, Pearson and Spearman’s rank test, or the Mann-Whitney U test. Kaplan-Meier survival analysis was performed by applying the long-rank test tomiR-210 expression and was stratified by median values and quartiles. Disease-specific overall survival (referred to as overall survival hereafter) was defined as the time from surgery to last follow-up or time of NSCLC-specific death. Disease-free survival was defined as the time from surgery to the time of first evidence of radiographic metastatic disease. Univariate and multivariate analyses were performed using the Cox proportional hazard model. All statistical analyses were performed using SPSS 17 software (SPSS Inc., Chicago, IL, USA), and P values of <0.05 were considered significant.


## 3. Results

### 3.1. Relationship betweenmiR-210 Expression and Clinical Characteristics in Patients with NSCLC

To examine whether miR-210 expression correlates with clinical characteristics in patients with NSCLC, we analyzedmiR-210 expression of NSCLC samples using qRT-PCR. For each sample, the data were normalized usingRNU6B as a reference. The fold-change inmiR-210 expression for each NSCLC sample was calculated by comparison with the median of 30 normal control samples. Patients were divided into two subgroups according to high or lowmiR-210 expression levels. Patients with higher than the median expression level ofmiR-210 were defined as the high group ofmiR-210 expression. No significant association between the status ofmiR-210 expression and clinical characteristics such as sex, age, tumor size, histology, T factor, lymph node status, pathological stage, ly factor, or v factor was observed. However, we found that the status ofmiR-210 expression was significantly correlated with disease relapse (P
=
0.007, Pearson and Spearman’s rank test; Table 1).


### 3.2.miR-210 Expression Is a Prognostic Factor for Disease-Free Survival and Overall Survival of NSCLC Patients

To confirm the correlation betweenmiR-210 and prognosis, we analyzed the relationship betweenmiR-210 expression and patient survival by performing Kaplan-Meier survival analysis, applying the long-rank test tomiR-210 expression. ThemiR-210-high group showed significantly shorter disease-free survival than themiR-210-low group (log rank chi-square = 11.225, P
=
0.001; Figure 1(a)). Five-year disease-free survival was 37.5% in themiR-210-high group and 70.0% in themiR-210-low group. Furthermore, themiR-210-high group showed significantly shorter overall survival than themiR-210-low group. Five-year overall survival was 47.5% in themiR-210-high group and 77.5% in themiR-210-low group (log rank chi-square = 8.448,P = 0.004; Figure 1(b)).Kaplan-Meier curves of survival of patients with NSCLC (n
=
80). Kaplan-Meier curves of (a) disease-free and (b) overall survival of 80 patients with NSCLC, stratified according tomiR-210 levels. Expression levels were stratified by the median value; follow-up was limited to 60 months.
(a)
(b)


### 3.3.miR-210 Expression Is Significantly Associated with Important Clinicopathological Factors in Patients with Lung Adenocarcinoma

Although we found thatmiR-210 expression is a prognostic factor for disease-free survival and overall survival in NSCLC patient samples; the status ofmiR-210 expression was not significantly correlated with important clinicopathological factors. We therefore analyzed the correlation of each histological type withmiR-210 expression and clinical characteristics. In 62 patient samples with adenocarcinoma,miR-210 expression was significantly correlated with lymph node status, pathological stage, ly factor, v factor, and disease relapse (P
=
0.018, P
=
0.003, P
=
0.0009, P
=
0.044, and P
=
0.002, resp., Mann-Whitney U test), whereas, in 18 patient samples with squamous cell carcinoma, it was not significantly correlated with any clinical characteristic (Table 2).Table 2
Relationship betweenmiR-210 expression and clinical characteristics.
Clinical characteristics
miR-210 expression
Ad cases
P
Sq cases
P
Gender
0.086
1.000
Male
2.32 ± 2.89
2.88 ± 2.00
Female
1.45 ± 1.67
2.93 ± 0.88
Age
0.029*
—
≤65
1.18 ± 1.71
—
≥66
2.17 ± 2.60
2.87 ± 1.89
Tumor size
0.402
1.000
<30 mm
1.84 ± 1.95
2.87 ± 1.62
≥30 mm
1.87 ± 2.98
3.25 ± 2.14
Histology
—
—
Adeno
1.86 ± 2.40
—
Squamous
—
2.87 ± 1.89
T factor
0.417
1.000
T1-T2
1.82 ± 2.48
2.88 ± 1.65
T3-T4
2.34 ± 1.70
3.37 ± 4.40
Lymph node
0.034*
—
Negative
1.44 ± 2.01
2.87 ± 1.89
Positive
2.34 ± 2.97
—
P-stage
0.019*
1.000
Stage I
1.37 ± 2.02
2.88 ± 1.65
Stages II-III
2.62 ± 2.77
3.37 ± 4.40
ly factor
0.025*
0.620
Negative
1.23 ± 1.44
3.30 ± 2.07
Positive
2.24 ± 2.84
2.09 ± 1.23
v factor
0.097
1.000
Negative
1.75 ± 1.97
2.12 ± 1.84
Positive
2.34 ± 2.98
3.01 ± 1.86
Relapse
0.007*
1.000
With
2.72 ± 2.63
2.88 ± 1.96
Without
1.35 ± 1.75
2.87 ± 1.84
Ad: adenocarcinoma.
Sq: squamous cell carcinoma.
Relapse was defined as first evidence of radiographic metastatic disease after surgery.
*
P < 0.05.


### 3.4.miR-210 Expression Is a Prognostic Factor for Disease-Free and Overall Survival in Patients with Lung Adenocarcinoma

BecausemiR-210 expression in adenocarcinoma patient samples was correlated with many important clinicopathological factors, we hypothesized thatmiR-210 expression would be more closely correlated with prognosis in adenocarcinoma patient samples than in NSCLC patient samples. To test this hypothesis, Kaplan-Meier survival analysis was performed by applying the log-rank test tomiR-210 expression of 62 patient samples with adenocarcinoma.miR-210 expression was a strong adverse prognostic factor for disease-free and overall survival when considered as a binary variable divided by median value (log rank chi-square = 12.205, P
<
0.001; Figure 2(a), log rank chi-square = 12.595, P
<
0.001; Figure 2(b), resp.). However, in the 18 patient samples with squamous cell carcinoma, no significant correlation betweenmiR-210 expression and disease-free or overall survival was observed. In the 62 lung adenocarcinoma patient samples, these relationships were also statistically significant when the patients were divided into quartiles on the basis of miR-210 expression levels; miR-210 expression was an adverse prognostic factor for disease-free and overall survival (log rank chi-square = 17.540, P
<
0.001; Figure 2(c), log rank chi-square = 16.651, P
=
0.001; Figure 2(d), resp.).Kaplan-Meier survival curves for patients with adenocarcinoma (n
=
62). Kaplan-Meier curves were constructed of the following: (a) disease-free and (b) overall survival of 62 patients with adenocarcinoma stratified according tomiR-210 levels. Expression levels were stratified by the median value; follow-up was limited to 60 months. (c) Disease-free and (d) overall survival of patients with adenocarcinoma stratified according tomiR-210 levels. Expression levels were stratified by quartiles; follow-up was limited to 60 months.
(a)
(b)
(c)
(d)


### 3.5. Multivariate Analysis Indicated ThatmiR-210 Expression in Adenocarcinoma Patient Samples Is an Independent Prognostic Factor for Disease-Free Survival

We performed further studies of diagnostic factors of disease-free survival and overall survival, specifically in adenocarcinoma patients, using univariate and multivariate analysis. These data are summarized in Table3. In a Cox univariate analysis, sex, tumor size, T factor, lymph node metastases, p-stage, ly factor, v factor, andmiR-210 expression were significantly correlated with disease-free survival (P
=
0.024, P
=
0.034, P
=
0.036, P
<
0.0001, P
<
0.0001, P
=
0.001, P
=
0.004, and P
=
0.001, resp.), whereas only age was not correlated with disease-free survival. Furthermore, a Cox multivariate analysis defined sex, ly factor, andmiR-210 expression as independent prognostic factors for disease-free survival (P
=
0.008, P
=
0.021, and P
=
0.020, resp.). Moreover, in a Cox univariate analysis, sex, tumor size, T factor, lymph node metastases, p-stage, ly factor, v factor, andmiR-210 expression were also significantly correlated with overall survival (P
=
0.005, P
=
0.044, P
=
0.018, P
=
0.001, P
<
0.0001, P
=
0.004, P
=
0.032, and P
=
0.001, resp.). Furthermore, Cox multivariate analysis showed that the usefulness ofmiR-210 as a prognostic factor for overall survival was marginal (P
=
0.057).Table 3
Disease-free (a) and overall survival (b) of 62 adenocarcinoma patients according to clinical characteristics andmiR-210 expression, analyzed by Cox proportional regression.
(a)
Disease-free survival
Variables
Univariate analysis
Multivariate analysis
HR (95% CI)
P
HR (95% CI)
P
Gender
Male/female
2.263 (1.113–4.601)
0.024*
3.600 (1.392–9.309)
0.008*
Age
≥66/≤65
0.860 (0.427–1.730)
0.672
Tumor size
≥33/<33
2.130 (1.052–4.735)
0.034*
2.310 (0.819–6.518)
0.114
T factor
T1/T2–T4
2.232 (1.052–4.735)
0.036*
0.469 (0.134–1.645)
0.237
Lymph node
+/−
3.754 (1.857–7.590)
<0.0001*
1.038 (0.300–3.589)
0.953
P-stage
I/II-III
4.969 (2.396–10.304)
<0.0001*
3.270 (0.894–11.964)
0.073
ly factor
+/−
4.078 (1.822–9.128)
0.001*
4.027 (1.229–13.193)
0.021*
v factor
+/−
2.768 (1.376–5.566)
0.004*
0.994 (0.412–2.398)
0.989
miR-210
High/Low
0.284 (0.134–0.604)
0.001*
0.355 (0.148–0.847)
0.02*
(b)
Overall survival
Variables
Univariate analysis
Multivariate analysis
HR (95% CI)
P
HR (95% CI)
P
Gender
Male/female
3.218 (1.434–7.222)
0.005*
2.151 (1.889–14.047)
0.001*
Age
≥66/≤65
1.011 (0.473–2.162)
0.977
Tumor size
≥33/<33
2.173 (1.019–4.630)
0.044*
1.725 (0.618–4.811)
0.298
T factor
T1/T2–T4
2.988 (1.204–7.415)
0.018*
0.801 (0.211–3.039)
0.744
Lymph node
+/−
3.453 (1.614–7.391)
0.001*
1.858 (0.499–6.918)
0.356
P-stage
I/II-III
4.533 (2.027–10.138)
<0.0001*
1.492 (0.380–5.853)
0.567
ly factor
+/−
3.865 (1.554–9.615)
0.004*
3.387 (0.894–12.836)
0.073
v factor
+/−
2.303 (1.075–4.937)
0.032*
0.602 (0.226–1.607)
0.311
miR-210
High/low
0.235 (0.099–0.561)
0.001*
0.361 (0.127–1.031)
0.057
CI: confidence interval.
*
P < 0.05.


## 3.1. Relationship betweenmiR-210 Expression and Clinical Characteristics in Patients with NSCLC

To examine whether miR-210 expression correlates with clinical characteristics in patients with NSCLC, we analyzedmiR-210 expression of NSCLC samples using qRT-PCR. For each sample, the data were normalized usingRNU6B as a reference. The fold-change inmiR-210 expression for each NSCLC sample was calculated by comparison with the median of 30 normal control samples. Patients were divided into two subgroups according to high or lowmiR-210 expression levels. Patients with higher than the median expression level ofmiR-210 were defined as the high group ofmiR-210 expression. No significant association between the status ofmiR-210 expression and clinical characteristics such as sex, age, tumor size, histology, T factor, lymph node status, pathological stage, ly factor, or v factor was observed. However, we found that the status ofmiR-210 expression was significantly correlated with disease relapse (P
=
0.007, Pearson and Spearman’s rank test; Table 1).


## 3.2.miR-210 Expression Is a Prognostic Factor for Disease-Free Survival and Overall Survival of NSCLC Patients

To confirm the correlation betweenmiR-210 and prognosis, we analyzed the relationship betweenmiR-210 expression and patient survival by performing Kaplan-Meier survival analysis, applying the long-rank test tomiR-210 expression. ThemiR-210-high group showed significantly shorter disease-free survival than themiR-210-low group (log rank chi-square = 11.225, P
=
0.001; Figure 1(a)). Five-year disease-free survival was 37.5% in themiR-210-high group and 70.0% in themiR-210-low group. Furthermore, themiR-210-high group showed significantly shorter overall survival than themiR-210-low group. Five-year overall survival was 47.5% in themiR-210-high group and 77.5% in themiR-210-low group (log rank chi-square = 8.448,P = 0.004; Figure 1(b)).Kaplan-Meier curves of survival of patients with NSCLC (n
=
80). Kaplan-Meier curves of (a) disease-free and (b) overall survival of 80 patients with NSCLC, stratified according tomiR-210 levels. Expression levels were stratified by the median value; follow-up was limited to 60 months.
(a)
(b)


## 3.3.miR-210 Expression Is Significantly Associated with Important Clinicopathological Factors in Patients with Lung Adenocarcinoma

Although we found thatmiR-210 expression is a prognostic factor for disease-free survival and overall survival in NSCLC patient samples; the status ofmiR-210 expression was not significantly correlated with important clinicopathological factors. We therefore analyzed the correlation of each histological type withmiR-210 expression and clinical characteristics. In 62 patient samples with adenocarcinoma,miR-210 expression was significantly correlated with lymph node status, pathological stage, ly factor, v factor, and disease relapse (P
=
0.018, P
=
0.003, P
=
0.0009, P
=
0.044, and P
=
0.002, resp., Mann-Whitney U test), whereas, in 18 patient samples with squamous cell carcinoma, it was not significantly correlated with any clinical characteristic (Table 2).Table 2
Relationship betweenmiR-210 expression and clinical characteristics.
Clinical characteristics
miR-210 expression
Ad cases
P
Sq cases
P
Gender
0.086
1.000
Male
2.32 ± 2.89
2.88 ± 2.00
Female
1.45 ± 1.67
2.93 ± 0.88
Age
0.029*
—
≤65
1.18 ± 1.71
—
≥66
2.17 ± 2.60
2.87 ± 1.89
Tumor size
0.402
1.000
<30 mm
1.84 ± 1.95
2.87 ± 1.62
≥30 mm
1.87 ± 2.98
3.25 ± 2.14
Histology
—
—
Adeno
1.86 ± 2.40
—
Squamous
—
2.87 ± 1.89
T factor
0.417
1.000
T1-T2
1.82 ± 2.48
2.88 ± 1.65
T3-T4
2.34 ± 1.70
3.37 ± 4.40
Lymph node
0.034*
—
Negative
1.44 ± 2.01
2.87 ± 1.89
Positive
2.34 ± 2.97
—
P-stage
0.019*
1.000
Stage I
1.37 ± 2.02
2.88 ± 1.65
Stages II-III
2.62 ± 2.77
3.37 ± 4.40
ly factor
0.025*
0.620
Negative
1.23 ± 1.44
3.30 ± 2.07
Positive
2.24 ± 2.84
2.09 ± 1.23
v factor
0.097
1.000
Negative
1.75 ± 1.97
2.12 ± 1.84
Positive
2.34 ± 2.98
3.01 ± 1.86
Relapse
0.007*
1.000
With
2.72 ± 2.63
2.88 ± 1.96
Without
1.35 ± 1.75
2.87 ± 1.84
Ad: adenocarcinoma.
Sq: squamous cell carcinoma.
Relapse was defined as first evidence of radiographic metastatic disease after surgery.
*
P < 0.05.


## 3.4.miR-210 Expression Is a Prognostic Factor for Disease-Free and Overall Survival in Patients with Lung Adenocarcinoma

BecausemiR-210 expression in adenocarcinoma patient samples was correlated with many important clinicopathological factors, we hypothesized thatmiR-210 expression would be more closely correlated with prognosis in adenocarcinoma patient samples than in NSCLC patient samples. To test this hypothesis, Kaplan-Meier survival analysis was performed by applying the log-rank test tomiR-210 expression of 62 patient samples with adenocarcinoma.miR-210 expression was a strong adverse prognostic factor for disease-free and overall survival when considered as a binary variable divided by median value (log rank chi-square = 12.205, P
<
0.001; Figure 2(a), log rank chi-square = 12.595, P
<
0.001; Figure 2(b), resp.). However, in the 18 patient samples with squamous cell carcinoma, no significant correlation betweenmiR-210 expression and disease-free or overall survival was observed. In the 62 lung adenocarcinoma patient samples, these relationships were also statistically significant when the patients were divided into quartiles on the basis of miR-210 expression levels; miR-210 expression was an adverse prognostic factor for disease-free and overall survival (log rank chi-square = 17.540, P
<
0.001; Figure 2(c), log rank chi-square = 16.651, P
=
0.001; Figure 2(d), resp.).Kaplan-Meier survival curves for patients with adenocarcinoma (n
=
62). Kaplan-Meier curves were constructed of the following: (a) disease-free and (b) overall survival of 62 patients with adenocarcinoma stratified according tomiR-210 levels. Expression levels were stratified by the median value; follow-up was limited to 60 months. (c) Disease-free and (d) overall survival of patients with adenocarcinoma stratified according tomiR-210 levels. Expression levels were stratified by quartiles; follow-up was limited to 60 months.
(a)
(b)
(c)
(d)


## 3.5. Multivariate Analysis Indicated ThatmiR-210 Expression in Adenocarcinoma Patient Samples Is an Independent Prognostic Factor for Disease-Free Survival

We performed further studies of diagnostic factors of disease-free survival and overall survival, specifically in adenocarcinoma patients, using univariate and multivariate analysis. These data are summarized in Table3. In a Cox univariate analysis, sex, tumor size, T factor, lymph node metastases, p-stage, ly factor, v factor, andmiR-210 expression were significantly correlated with disease-free survival (P
=
0.024, P
=
0.034, P
=
0.036, P
<
0.0001, P
<
0.0001, P
=
0.001, P
=
0.004, and P
=
0.001, resp.), whereas only age was not correlated with disease-free survival. Furthermore, a Cox multivariate analysis defined sex, ly factor, andmiR-210 expression as independent prognostic factors for disease-free survival (P
=
0.008, P
=
0.021, and P
=
0.020, resp.). Moreover, in a Cox univariate analysis, sex, tumor size, T factor, lymph node metastases, p-stage, ly factor, v factor, andmiR-210 expression were also significantly correlated with overall survival (P
=
0.005, P
=
0.044, P
=
0.018, P
=
0.001, P
<
0.0001, P
=
0.004, P
=
0.032, and P
=
0.001, resp.). Furthermore, Cox multivariate analysis showed that the usefulness ofmiR-210 as a prognostic factor for overall survival was marginal (P
=
0.057).Table 3
Disease-free (a) and overall survival (b) of 62 adenocarcinoma patients according to clinical characteristics andmiR-210 expression, analyzed by Cox proportional regression.
(a)
Disease-free survival
Variables
Univariate analysis
Multivariate analysis
HR (95% CI)
P
HR (95% CI)
P
Gender
Male/female
2.263 (1.113–4.601)
0.024*
3.600 (1.392–9.309)
0.008*
Age
≥66/≤65
0.860 (0.427–1.730)
0.672
Tumor size
≥33/<33
2.130 (1.052–4.735)
0.034*
2.310 (0.819–6.518)
0.114
T factor
T1/T2–T4
2.232 (1.052–4.735)
0.036*
0.469 (0.134–1.645)
0.237
Lymph node
+/−
3.754 (1.857–7.590)
<0.0001*
1.038 (0.300–3.589)
0.953
P-stage
I/II-III
4.969 (2.396–10.304)
<0.0001*
3.270 (0.894–11.964)
0.073
ly factor
+/−
4.078 (1.822–9.128)
0.001*
4.027 (1.229–13.193)
0.021*
v factor
+/−
2.768 (1.376–5.566)
0.004*
0.994 (0.412–2.398)
0.989
miR-210
High/Low
0.284 (0.134–0.604)
0.001*
0.355 (0.148–0.847)
0.02*
(b)
Overall survival
Variables
Univariate analysis
Multivariate analysis
HR (95% CI)
P
HR (95% CI)
P
Gender
Male/female
3.218 (1.434–7.222)
0.005*
2.151 (1.889–14.047)
0.001*
Age
≥66/≤65
1.011 (0.473–2.162)
0.977
Tumor size
≥33/<33
2.173 (1.019–4.630)
0.044*
1.725 (0.618–4.811)
0.298
T factor
T1/T2–T4
2.988 (1.204–7.415)
0.018*
0.801 (0.211–3.039)
0.744
Lymph node
+/−
3.453 (1.614–7.391)
0.001*
1.858 (0.499–6.918)
0.356
P-stage
I/II-III
4.533 (2.027–10.138)
<0.0001*
1.492 (0.380–5.853)
0.567
ly factor
+/−
3.865 (1.554–9.615)
0.004*
3.387 (0.894–12.836)
0.073
v factor
+/−
2.303 (1.075–4.937)
0.032*
0.602 (0.226–1.607)
0.311
miR-210
High/low
0.235 (0.099–0.561)
0.001*
0.361 (0.127–1.031)
0.057
CI: confidence interval.
*
P < 0.05.


## 4. Discussion

By univariate and multivariate analyses our study provides clear evidence that upregulation ofmiR-210 is a prognostic factor in patients with lung adenocarcinoma and is correlated with important clinicopathological factors including nodal involvement, pathological stage, lymphatic vessel invasion, and cancer relapse.Recently, a meta-analysis of human lung cancer microRNA expression profiling studies that compared cancer tissues with normal tissues showed that the top two most consistently reported upregulated microRNAs weremiR-210 andmiR-21 [37]. In addition, systematic reviews and meta-analyses of two studies confirmed that upregulation ofmiR-210 is predictive of poor survival of patients with various tumors, especially breast cancers [33, 34]. However, these two systematic reviews did not include the outcome of patients with lung cancer. Thus, the prognostic impact ofmiR-210 in patients with lung cancer remained unclear.Recently, Eilertsen et al. reported a large-scale study of the prognostic role ofmiR-210 in NSCLC [38]. In that study, upregulation ofmiR-210 expression was a positive prognostic factor for disease-free survival in 335 NSCLC patients. This result is not consistent with our findings. One reason for the differences between these results might be the different methods used, since our study assessedmiR-210 expression using qRT-PCR, whereas the previous study usedin situ hybridization. However, other previous studies strongly suggest that high expression ofmiR-210 could be a biomarker of bad prognosis in lung cancer. Puisségur et al. reported thatmiR-210 was significantly elevated in patients with advanced disease such as stages II-III disease compared with stage I A disease (n
=
20) [39]. Li et al. reported that miR-210 was significantly elevated in patients with stages III-IV disease compared with stages I-II disease (n
=
60) [40]. Furthermore, in the majority of the previous studies,miR-210 upregulation was significantly correlated with poor prognosis in patients with various cancers such as breast cancer [21], pancreatic cancer [32], head and neck cancer [31], colorectal cancer [41], and glioblastoma [42], though not renal cancer [43]. Our data are consistent with these previous important findings. Further study is still needed to clarify the clinical impact ofmiR-210 as a prognostic factor in patients with NSCLC.In vitro functional studies regardingmiR-210 function in cancer progression provide even further contradictory results. For example, Zhang et al. found thatmiR-210 inhibits MNT, an antagonist of c-MYC, and promotes cell proliferation in transformed cells such as colon and cervical cancer cells [24]. However, Giannakakis et al. found thatmiR-210 acts as a tumor suppressor by inhibiting cell proliferation via E2F3 regulation in ovarian cancer cell lines [27]. It is unclear how to reconcile these paradoxical findings thatmiR-210 acts primarily as a positive or a negative regulator of proliferation.To understand these conflicting findings, we hypothesized thatmiR-210 may play various roles depending on the cancer type or histological subtype in which it is expressed. In the present study, we first analyzed the correlation ofmiR-210 expression in NSCLC patient samples with each histological subtype of NSCLC and with the clinical characteristics of patients with each subtype. We then focused onmiR-210 expression in samples with histology specific for adenocarcinoma. In patients with adenocarcinoma, we clearly showed thatmiR-210 expression was strongly associated with important clinical parameters such as age, lymph node metastasis, pathological stage, ly factor, v factor, and relapse, while, in patients with squamous cell carcinoma,miR-210 was not associated with any clinical characteristic. In our study, squamous cell carcinoma showed high levels of baselinemiR-210 expression compared with adenocarcinoma. The uniformly high expression levels ofmiR-210 in most squamous cell carcinomas meant that a prognostic impact ofmiR-210 on squamous cell carcinoma could not be determined. However,miR-210 could be a biomarker of adenocarcinoma because adenocarcinomas showed varying levels ofmiR-210 expression.


## 5. Conclusions

In conclusion, this study demonstrated for the first time thatmiR-210 was correlated with poor prognosis in patients with NSCLC, especially in lung adenocarcinoma. This evidence could contribute to biomarker studies in patients with lung adenocarcinoma. Because this was an exploratory study and the sample size was very small, our results warrant further investigation and require independent validation. In particular,miR-210 levels in the plasma may have special prognostic significance since miRNA has been shown to circulate in various body fluids in remarkably stable forms, resulting in the identification of novel noninvasive biomarkers for the diagnosis and prognosis of various cancers and other diseases [44, 45]. Based on these evidences, the prognostic signature ofmiR-210 in the plasma of NSCLC patients should be examined in future studies.



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*Source: 316745-2015-02-05.xml*