Original Article
Expression of miR-32 in human non-small cell lung
cancer and its correlation with tumor progression
and patient survival
Yu Bai1, Yong-Lian Wang2, Wen-Jian Yao2, Ling Guo2, Hui-Fang Xi3, Song-Yue Li2, Bao-Sheng Zhao2
1Department of Pathology, Xinxiang Medical University, Xinxiang 453000, China; Departments of 2Thoracic
Surgery, 3Pediatric Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, China Received October 27, 2014; Accepted December 22, 2014; Epub January 1, 2015; Published January 15, 2015
Abstract: Introduction: miR-32 has recently been found to be implicated in many critical processes in various types of human cancer. However, its clinical significance in human non-small cell lung cancer (NSCLC) has not yet been elucidated. In the present study, we investigated the expression of miR-32 in NSCLC and analyzed its association with clinical features and prognosis of NSCLC patients. Methods: Quantitative real-time PCR (qRT-PCR) was used to measure expression level of miR-32 in lung cancer cell lines, normal bronchial epithelial cells, 90pairs of tumor samples and adjacent non-tumor tissues. To determine its prognostic value, overall survival was evaluated using the Kaplan-Meier method. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis. Results: The expression of miR-32 was significantly decreased in lung cancer cell lines and NSCLC tis-sues compared with normal bronchial epithelial cells and adjacent non-tumor tistis-sues (P < 0.05). This reduction of miR-32 was associated with tumor stage and lymph node metastasis (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with low 32 expression had shorter overall survival time than those with high miR-32 expression (P < 0.05). Univariate analysis revealed statistically significant correlations between overall survival and miR-32 level, tumor stage and lymph node metastasis (P < 0.05). Furthermore, miR-32 levels, tumor stage and lymph node metastasis were independently associated with overall survival (P < 0.05). Conclusions: Our results pro-vided the first evidence that down-regulation of miR-32 was correlated with NSCLC progression, and miR-32 might be a potential molecular biomarker for predicting the prognosis of patients.
Keywords: Non-small cell lung caner, miR-32, quantitative real-time PCR, prognosis
Introduction
The leading cause of cancer mortality is lung cancer in the worldwide. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for more than 85% of all lung cancer cases [1]. So far, the most effec-tive therapy for NSCLC was still surgical resec-tion [2]. In spite of the progress in understand-ing the molecular mechanisms and treatment for NSCLC in recent years, 5-year overall sur-vival rate of NSCLC patients were still only 13% [3]. The main cause of death in these patients was distant metastasis. Thus, there is a press-ing need for novel specific, sensitive biomark-ers for the early diagnosis and prognosis pre-diction of NSCLC exists.
miRNAs are endogenous non-coding RNAs of
significant role in regulating gene expression by base-pairing to the complementary sites on the target mRNAs, thus inhibiting the translation or triggering the degradation of the target mRNAs [5]. Altered expression of tissue miRNAs has been associated with many diseases, particu-larly cancer, and the use of tissue miRNA expression profiles as diagnostic or prognostic biomarkers in cancer has been demonstrated by several studies [6-9]. Emerging studies have revealed that miRNA is a promising biomarker associated with clinical outcomes in non-small cell lung cancer.
Table 1. miR-32 expression and clinicopathologic features in NSCLC patients
Variable Number miR-32 expression P value
Low High
Age (years) 0.473
< 60 23 14 9
≥ 60 67 35 32
Gender 0.975
Male 66 36 30
Female 24 13 11
Tumor size (cm) 0.317
< 3 61 31 30
≥ 3 29 18 11
Histologic type 0.988
Squamous 57 31 26
Adenoma 33 18 15
Tumor stage 0.004
I-II 58 25 33
III 32 24 8
Lymph nodes metastasis 0.000
No 54 20 34
Yes 36 29 7
proliferation and decreased the migration and invasion capabilities of gastric cancer cells in vitro [11]. However, Petillo et al revealed that over-expression of miR-32 was associated with poor outcome of renal cell carcinomas patients [12]. Jalava et al showed androgen regulated miR-32 was over-expressed in castration-resis-tant prostate cancer, leading to reduced expression of BTG2. Thus, miR-32 was a poten-tial marker for aggressive disease and was a
[image:2.612.92.338.309.574.2]between 2005 and 2009 from the Department of Thoracic Surgery, First Affiliated Hospital of Xinxiang Medical University, The study was approved by the Ethics Committee onXinxiang Medical University, and written informed con-sent was obtained from all patients. All patients recruited in this study were not received radio-therapy and/or chemoradio-therapy before surgery. After collection, all tissue samples were imme-diately stored in liquid nitrogen until use.
Figure 1. miR-32 expression was down-regulated in lung cancer cell lines and NSCLC tissues. A. Relative expression of miR-32 was lower in lung cancer cell lines than in normal bronchial epithelial cell line. B. Relative expression of miR-32 was lower in NSCLC tissues than in matched adjacent non-tumor tissues. miR-32 expression was deter-mined by qRT-PCR and normalized against U6 RNA (an endogenous control). Results are expressed as mean ± SD for three replicate determinations *P < 0.05.
putative drug target in prostate cancer [13]. Wu et al found that over-expression of miR-32 promoted colorectal carcino-ma cells proliferation, migration, and invasion, reduced apoptosis, and result-ed in down-regulation of PTEN at a post-transcriptional level [14], suggesting that miR-32 may act as a tumor sup-pressor or an oncogene in different cancers.
In the present study, we investigated the expression levels of miR-32 in lung caner cell lines and NSCLC tissues. We also analyzed the correlation of miR-32 expression with clinicopathological fea-tures and overall survival of the patients and determined its prognostic role in NSCLC.
Materials and methods
Patients and specimens
Clinical and clinicopathological classification and staging were determined according to the American Joint Committee on Cancer (AJCC) criteria [15]. The clinicopathologic features of the patients with NSCLC are shown in Table 1. All patients with NSCLC were regularly followed up with a mean observation period of 44 months.
Cell culture
The human lung cell lines A549, H157, HEK-293T and normal bronchial epithelial cell line 16HBE were purchased from the American Type Culture Collection (ATCC, USA). All cell lines were routinely maintained in DMEM medi-um (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin sodi-um, and 100 mg/ml streptomycin sulfate at 37°C in a humidified air atmosphere containing 5% CO2.
Quantitative real-time PCR (qRT-PCR) for miRNA
For miRNA analysis, total RNA from cell lines and tissue samples were isolated with the Trizol reagent (Invitrogen). qRT-PCR of miR-32 was performed using Bulge-LoopTM miRNA qRT-PCR
software (IBM). Chi-square test and t test were performed to explore the associations between miR-32 expression and clinical characteristics. Overall survival curves were calculated using the Kaplan-Meier method and the statistical differences between subgroups were com-pared by the log-rank test. The Cox proportional hazards regression model was employed for univariate and multivariate analyses to esti-mate the prognostic factors for survival predic-tion. Data were expressed as mean ± SD. P value of less than 0.05 was considered to sta-tistically significant.
Results
Decreased expression of miR-32 in NSCLC
[image:3.612.91.361.69.310.2]miR-32 expression was examined in three lung cancer cell lines (A549, H157, HEK-293T) and a normal bronchial epithelial cell line 16HBE. after normalization to U6 expression levels, the expression levels of miR-32 was lower in three lung cancer cell lines (A549, H157, HEK-293T) than that in normal bronchial epithelial cell line 16HBE (P < 0.05, Figure 1A). miR-32 expres-sion level was further detected in 90 paired NSCLC and adjacent non-tumor tissues. qRT-PCR results showed that the expression level of
Figure 2. Survival analysis of 90 NSCLC patients by Kaplan-Meier meth-od. Overall survival rate in patients with low miR-32 expression level was significantly worse than that in patients with high miR-32 expression level (log-rank test, P < 0.05).
kits (Ribobio) according to the man-ufacturer’s protocol. Briefly, the miR-32 and U6-specific cDNAs were synthesized from the total RNA using a specific miR-32 stem-loop primer and the reverse primer for U6, followed by real-time PCR with a miR-32-specific forward primer and a universal reverse primer using an ABI-Prism 7900HT system (Applied Biosystems). Each sample was carried out in triplicate. U6 small nuclear RNA was used as an endogenous control. The expres-sion level of miR-32 for each sam-ple was calculated, represented by the ΔCT value. The relative miR-32 expression levels in paired tissues that were obtained from the same patients were analyzed by the com-parative 2−ΔΔCT method.
Statistical analysis
Table 2. Univariate and multivariate analysis of overall survival in NSCLC patients
Variable Univariate analysis Multivariate analysis
Risk ratio 95% CI P Risk ratio 95% CI P
Age (years) 1.227 0.739-1.528 0.425
≥ 60 vs < 60
Gender 1.172 0.526-1.712 0.276
Male vs Female
Tumor size 1.794 0.818-2.673 0.186
≥ 3 cm vs < 3 cm
Histologic type 1.424 0.739-2.216 0.304
Squamous vs Adenoma
Tumor stage 2.117 1.614-4.332 0.011 1.977 1.476-3.995 0.007
III vs I-II
Lymph nodes metastasis 3.107 1.811-5.327 0.008 2.874 1.583-4.917 0.003 Yes vs No
miR-32 3.104 1.627-5.931 <0.001 2.739 1.407-5.083 0.002
Low vs High
miR-32 in NSCLC tissues was significantly lower than that in adjacent non-tumor tissues (P < 0.05, Figure 1B). Accordingly, the mean fold change (0.36)of miR-32 was used as a cutoff value to divide all patients into two groups: the low expression group and the high expression group.
Association of miR-32 expression with clinico-pathological features of NSCLC patients
The relationship between miR-32 expression and clinicopathologic features was evaluated. As shown in Table 1, the level of miR-32 in NSCLC was strongly correlated with tumor stage and lymph node metastasis (P < 0.05). However, there were no significant associations between miR-32 expression and other clinical features including age, gender, tumor size and histological type. Taken together, these obser-vations indicated that miR-32 expression was down-regulated in NSCLC and associated with the disease progression.
miR-32 down-regulation associated with poor prognosis of NSCLC patients
To evaluate the prognostic value of miR-32 expression in NSCLC, survival curves were con-structed by Kaplan-Meier method and com-pared by the log-rank test. As shown in Figure 2, NSCLC patients with low miR-32 expression level had poor overall survival than those with high miR-32 expression level (P < 0.05). Cox
proportional hazards regression analysis at the univariate level revealed that there was a sta-tistically significant correlation between overall survival and miR-32 level, tumor stage and lymph node metastasis (P < 0.05). However, the other clinicopathological features, such as age, gender, tumor size and histological type were not associated with overall survival of NSCLC patients (P > 0.05) (Table 2). The param-eters that significantly correlated with overall survival in the univariate analysis were further evaluated by multivariate analysis. Our results of the multivariate analysis showed that miR-32, tumor stage and lymph node metastasis played a significant role of independent prog-nostic markers in overall survival rates (Table 2).
Discussion
So far, more and more deregulated miRNAs were found to be involved in NSCLC carcinogen-esis and patient prognosis. For example, Tejero et al revealed that high miR-141 and miR-200c expression were associated with shorter over-all survival in NSCLC patients with adenocarci-noma through MET and angiogenesis [18]. Li et al found that miR-200c over-expression could reverse the mesenchymal phenotype of cells to epithelial phenotype by targeting ZEB1 in NSCLC, which could also re-sensitize EGFR-TKIs in NSCLC cell lines with primary resis-tance. Moreover, miR-200c over-expression could predict a better efficacy of EGFR-TKIs in advanced NSCLC patients with EGFR-WT [19]. Xu et al demonstrated that miR-9 was up-regu-lated in NSCLC tissues and correup-regu-lated with adverse clinical features and unfavorable sur-vival, indicating that miR-9 might play critical roles in NSCLC progression [20]. Contradictory data concerning the role of miR-32 in the devel-opment and progression of tumor have emerged, with evidence that it can behave both as a promoter and a suppressor in different cancers. However, to our knowledge, the clini-cal significance and function of miR-32 in NSCLC is still unknown.
In the present study, we first explored the expression of miR-32 in lung cancer cell lines, normal bronchial epithelial cells, 90 pairs of tumor samples and adjacent non-tumor tis-sues. We found that the relative expression level of miR-32 in lung cancer cell lines and NSCLC tissues was significantly lower than that in normal bronchial epithelial cells and adja-cent non-tumor tissues. Then, we analyzed its clinicopathologic and prognostic significance. Our date demonstrated that status of miR-32 expression in NSCLC tissues was significantly associated with tumor stage and lymph node metastasis of NSCLC patients; It was observed that patients with low miR-32 expression showed advanced tumor stage and higher inci-dence of lymph node metastasis. But no asso-ciated with other clinicopathological features, suggesting that down-regulation of miR-32 played key roles in lung cancer carcinogenesis and progression.
To explore the potential prognostic value of miR-32, the relationship between expression levels and overall survival in NSCLC patients was analyzed. Kaplan-Meier analysis showed
that patients with low miR-32 expression level had a significantly poorer overall survival rate than those with high miR-32 expression level. Furthermore, multivariate Cox analysis proved that miR-32 was a prognostic indicator inde-pendent of adjusted well-known prognostic fac-tors for NSCLC including tumor stage and lymph node metastasis. Thus, miR-32 could be used as a potential prognostic biomarker to classify subtype of NSCLC patients who have higher risk of death, thus, should receive monitoring more frequently.
Taken together, the current study showed the clinical and prognostic significance of miR-32 in NSCLC. Although this study was limited by the retrospective design and the relatively small number of NSCLC patients, the data proved that miR-32 played important roles in the development and progression of NSCLC and therefore might serve as a potential thera-peutic target. Further studies are needed to evaluate the prognostic value of miR-32 expres-sion in other cohorts and elucidate the mecha-nisms of miR-32 in carcinogenesis and pro-gression of NSCLC.
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Bao-Sheng Zhao, Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, China. E-mail: baoshengzhao68@126.com
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