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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 693-698

Increased expression of microRNA-301a in nonsmall-cell lung cancer and its clinical significance


1 Department of Emergency Medicine, The Affiliated Hospital of Weifang Medical University, No. 465, Kuiwen, Weifang, Shandong, 261031, People's Republic of China
2 Department of Oncology, The Affiliated Hospital of Weifang Medical University, No. 465, Kuiwen, Weifang, Shandong, 261031, People's Republic of China
3 Department of Cardiothoracic Surgery, The Affiliated Hospital of Weifang Medical University, No. 465, Kuiwen, Weifang, Shandong, 261031, People's Republic of China

Date of Web Publication25-Jul-2016

Correspondence Address:
Shi-zhong Wang
Department of Cardiothoracic Surgery, The Affiliated Hospital of Weifang Medical University, No. 465, Yuhe Road, Kuiwen, Weifang, Shandong, 261031
People's Republic of China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.146130

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 > Abstract 


Aims: Recently, accumulating evidence indicates that dysregulation of microRNAs is associated with the initiation and progression of cancer. Oncogenic miR-301a has been reported upregulation and associated with tumorigenesis and progression in various types of cancer. The aim of this study was to investigate the expression of miR-301a in nonsmall-cell lung cancer. (NSCLC), and to assess its association with malignancy, metastasis and prognosis.
Subjects and Methods: total of 88 NSCLC patients (females = 21 and males = 67), aged 15-81 years were included in the study. miR-301a expression in tumor tissue was estimated by real-time quantitative reverse transcription polymerase chain reaction.
Results: miR-301a was significantly upregulated in NSCLC tissues compared with their paired adjacent nontumor tissues. (P < 0.001). Increased expression of miR-301a was detected in tumors with lymph node metastases. (P =0.003). In addition, high miR-301a expression was significantly associated with poorly differentiation. (P =0.015), lymph node metastasis. (P =0.013) and advanced tumor-node-metastasis. (TNM) stage. (P =0.018). A. comparison of survival curves of low versus high expressers of miR-301a revealed a highly significant difference in NSCLC, which suggests that overexpression of miR-301a is associated with a poorer disease-free survival (DFS) (P =0.002). Moreover, multivariate Cox proportional hazard regression analyses revealed that the miR-301a overexpression was an unfavorable prognostic factor for disease-free survival in addition to TNM stage.
Conclusions: miR-301a may represent a novel prognostic indicator, a biomarker for the early detection of lymph node metastasis and a therapeutic target in NSCLC.

Keywords: miR-301a, nonsmall-cell lung cancer, prognosis


How to cite this article:
Shi Yk, Zang Ql, Li Gx, Huang Y, Wang Sz. Increased expression of microRNA-301a in nonsmall-cell lung cancer and its clinical significance. J Can Res Ther 2016;12:693-8

How to cite this URL:
Shi Yk, Zang Ql, Li Gx, Huang Y, Wang Sz. Increased expression of microRNA-301a in nonsmall-cell lung cancer and its clinical significance. J Can Res Ther [serial online] 2016 [cited 2019 Sep 21];12:693-8. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/693/146130




 > Introduction Top


Nonsmall-cell lung cancer (NSCLC) is one of the most aggressive malignancies with the worst prognosis and is the third leading cause of cancer-related deaths in the world.[1] Despite efforts made in multiple fields, there has been little success in improving the disease-free survival (DFS) rate of patients.[2] New advances in understanding the disease at cellular and molecular level greatly helped researchers in devising novel strategies that limit cancer growth by targeting specific molecules related with tumor progression. Such strategies have shown to be more effective than chemotherapy and radiotherapy and can be complemented to existing therapeutic paradigm in augmenting beneficial clinical outcome.[3] Therefore, revealing the molecular mechanism for the NSCLC development and identifying novel molecular biomarkers is indispensable for developing effective therapy.

MicroRNAs (miRNAs) are highly conserved noncoding RNAs during evolution, which have emerged recently as important regulators by involve in several biological events, such as cellular growth, differentiation, apoptosis and tumorigenesis.[4] There is increasing evidence that expression levels of several miRNAs have been found to be correlated with NSCLC, these miRNAs can regulate a variety process of carcinogenesis, function either as oncogenes or tumor suppressors, depending on whether they specifically target tumor suppressor genes or oncogenes.[5],[6],[7],[8] Oncogenic miRNAs are usually overexpressed in tumors, while tumor-suppressive miRNAs down expressed.[9],[10]

Previous studies showed that the miR-301a is aberrantly expressed in multiple malignancies and is involved in the formation, progression and metastasis of cancer. It has been shown to promote nodal or distant relapses in human breast cancer;[11] increase xenograft pancreatic tumor growth;[12] and promotes cell proliferation and invasion in colorectal [13] and gastric [14] cancer cell lines, respectively. Moreover, it has also been showed that in human pulmonary endothelial cells, miR-301a can involve in immediate induction of plasminogen activator inhibitor-1 by modulate placental growth factor.[15] Moreover, inhibition of miR-301a resulted in an increase of the mitotic index and a decrease in colony formation in lung cancer cell lines A549.[16] These results suggest that, miR-301a may acts as an oncogenic miRNA in various tumors, including lung tumorigenesis. However, the expression of miR-301a in NSCLC tissues, and the associations between miR-301a expression levels and NSCLC patients' clinicopathological characteristics and prognosis remain unknown.

In the present study, we clarified the clinical significance of miR-301a expression in NSCLC. Firstly, we examined miR-301a expression in NSCLC tissues and adjacent normal tissues by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). In addition, we assessed the potential relationship between miR-301a levels and clinicopathological features of NSCLC, and we also evaluated its relation with disease-free survival (DFS) of patients.


 > Subjects and Methods Top


Subjects

A total of 88 specimens of cancer tissues and their adjacent benign tissues were collected from January, 2008 to May, 2009 [Table 1]. All surgically excised tumors and matched noncancerous tissues were immediately submerged in liquid nitrogen and stored at −80°C until RNA isolation. Written informed consent for the biological studies was taken from the patients involved in the study. The study was approved by the Ethics Committee. None of the patients received preoperative chemotherapy or radiotherapy. The histological diagnosis was re-examined from the hematoxylin and eosin-stained tissue sections, according to classification guidelines of the World Health Organization.[17] All patients were staged based on the International Association for the Study of Lung Cancer tumor-node-metastasis (TNM) classification, 7th edition.[18]
Table 1: The relationship between miR-301a expression and clinicopathologic parameters

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RNA isolation, reverse transcription and quantitative polymerase chain reaction

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and the RT reactions were performed using a Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA). The gene-specific stem–loop RT primers for miRNAs, which were released by the Sanger Institute, were designed according to Chen et al.[19] qPCR was performed using a standard SYBR Green PCR kit (Toyobo, Osaka, Japan) and a Rotor-Gene RG-3000A (Corbett Life Science, Sydney, New South Wales, Australia) according to the instructions from the respective manufacturer. U6 was used as references for miRNAs. Each sample was analyzed in triplicate. The 2ΔΔCt method was used to quantify the relative levels of gene expression.

Patient follow-up

Follow-up was performed by phone calls or clinic visits at 3-month intervals for 1-year, then at 6-month intervals for 3 years and annually for the remaining duration of the study until disease progression, death or study end. All enrolled patients had completed follow-up. The follow-up examinations consisted of a physical examination and abdominal and pelvic computed tomography scans if necessary. The patients who died from diseases other than NSCLC or from unexpected events were excluded from this study. Survival time was calculated from the date of the resection to the date of death or last follow-up. Follow-up lasted to May 30, 2014, with a median follow-up period of 49.0 months for living patients (range: 1.2-71.4 months). During the follow-up time, 47 deaths from NSCLC were observed.

Statistical analysis

All data were expressed as the mean ± standard deviation (SD). The differences in miR-301a expression levels between groups were analyzed using the Student's t-test, Chi-square test or Wilcoxon test, as appropriate. DFS was calculated using the Kaplan–Meier method, and the log-rank test was performed to compare the differences between survival curves. Variables with a value of P < 0.05 in univariate analysis were used in subsequent multivariate analysis on the basis of Cox proportional hazards model. Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). The difference was deemed statistically significant at P < 0.05.


 > Results Top


Expression of microR-301a was upregulated in nonsmall-cell lung cancer

The first goal of the present study was to investigate whether miR-301a is altered in NSCLC cancer tissues compared with adjacent normal tissues. As shown in [Figure 1]a, miR-301a was significantly up-regulated in 80.68% (71/88) of tumor tissue samples compared with their paired adjacent nontumor tissue samples (P < 0.01). The mean ± SD 2−ΔΔCT value of miR-301a in NSCLC tumor samples was 3.679 ± 0.39, compared with 0.766 ± 0.06 in adjacent nontumor tissue samples. In addition, the expression level of miR-301a in lymph node metastatic tumors (n = 21) were found to be significantly higher than the level in nonmetastatic tumors [n = 67, P = 0.003; [Figure 1]b.
Figure 1: miR-301a level was significantly increased in nonsmall-cell lung cancer tissues. (a) miR-301a expression was significantly higher in tumour tissue compared with adjacent normal tissue (P < 0.001). (b) The expression of miR-301a was higher in lymph node metastatic tumours (n = 67) than in nonmetastatic tumours (n = 21; P= 0.003)

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The association between miR-301a expression and clinicopathologic factors in nonsmall-cell lung cancer

The correlation between miR-301a expression levels and clinic pathological characteristics of patients is summarized in [Table 1]. Using the mean level of miR-301a, the patients were divided into two groups: miR-301 low expressers (n = 40) and miR-301a high expressers (n = 48). High miR-301a expression was significantly correlated with tumor differentiation (P = 0.015), lymphatic metastasis (P = 0.013) and advanced clinical stage (P = 0.018). However, no correlation was observed between miR-301a expression level and other clinicopathological factors, such as age, gender, smoking status tumor size or histology [P > 0.05; [Table 1].

Correlation between miR-301a expression and prognosis of nonsmall-cell lung cancer patients

Disease-free survival curves were plotted according to miR-301a expression level by the Kaplan–Meier method. The DFS rate of the 88 NSCLC patients was 53.4% [Figure 2]a, and the 5-year DFS rates in the low and high miR-301a expression group were 62.5% and 29.16%, respectively [P = 0.002, [Figure 2]b. As shown in [Table 2], univariate Cox proportional hazards regression analysis revealed that tumor size (hazard ratio [HR] 0.267; P = 0.000), lymph node metastasis (HR 0.338; P = 0.022), TNM stage (HR = 0.132; P = 0.000), and miR-301a expression (HR = 0.39; P = 0.002) were predictive factors for prognosis in patients with NSCLC. Multivariate regression analysis confirmed that miR-301a expression (HR = 0.413; P = 0.007) was an independent unfavorable prognostic factor in addition to TNM stage (HR = 0.181; P = 0.000).
Figure 2: Kaplan-Meier survival curves according to miR-301a level. (a) The overall survival rate of 88 nonsmall-cell lung cancer patients. (b) Kaplan-Meier survival curves and log-rank test showing patients with high miR-301a expression had a significantly poorer prognosis than those with low miR-301a expression (P = 0.002)

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Table 2: Univariate and multivariate analysis of different prognostic factors for disease-free survival in 88 patients with NSCLC

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The prognostic value of miR-301a expression in patient subgroups classified by lymph node metastasis status and clinical stage was also evaluated. Low miR-301a expression was found to significantly correlate with poor survival in patients with lymph node metastasis [P = 0.021; [Figure 3]b and at TNM stage III [P = 0.016; [Figure 3]d. However, no significant differences between low and high miR-301a expression were identified between patients without lymph node metastasis [P = 0.088; [Figure 3]a and at clinical stage I-II [P = 0.117; [Figure 3]c. These results indicate that the miR-301a may present an improved prognostic biomarker for advanced-stage NSCLC patients.
Figure 3: miR-301a shows improved prognostic value in advanced-stage nonsmall-cell lung cancer (NSCLC) patients. Kaplan–Meier analysis and log-rank test showing the disease-free survival of 88 NSCLC patients with low and high miR-301a levels categorized according to (a and b) lymph node metastasis status and (c and d) tumour-node-metastasis stage

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 > Discussion Top


Multiple miRNAs are dysregulated and involved in various human malignancies.[20] Previous work has shown that miR-301a exhibits abnormal expression levels in different cancers such as gastric,[21] pancreatic,[12],[22] hepatocellular,[23] and colorectal [13] cancers, and miR-301a may function as an oncogene in carcinogenesis by modulate multiple pathways and mechanisms.[12],[13],[14] In addition, abnormal expression of miR-301a in gastric cancer associated with progression and poor prognosis.[21] In particular, miR-301a has been reported overexpressed in NSCLC cells relative to human bronchial epithelial cells.[24] However, little is known about the expression of miR-301a in NSCLC.

In the current study, by analyzed 88 cases of NSCLC using qRT-PCR, we firstly verified that the expression level of miR-301a was significant upregulated in NSCLC tissue samples compared with their paired adjacent nontumor tissue samples, and the level of miR-301a expression in lymph node metastatic tumor tissues were found to be significantly higher than level for nonmetastatic tumor tissues. These observations indicated that miR-320a expression increases during cancer development may be a potential biomarker for NSCLC diagnosis.

miR-301a overexpression is associated with tumor size, necrotic volume, invasion depth, lymph node metastasis, and TNM stage in various cancers.[21],[25] In consistent with previous reports, we demonstrated that high miR-301a expression was significantly associated with poorly differentiation, present of lymph node metastasis and advanced TNM stage in patients with NSCLC. A comparison of survival curves of low versus high expression of miR-301a revealed a significant difference in NSCLC. Moreover, high miR-301a expression was associated with a poor prognosis in patients with lymph node metastasis or at TNM stage III. These data suggest that miR-301a is a novel prognostic indicator in patients with NSCLC, moreover, it may have increased prognostic value for advanced-stage cancer.

Studies have begun to reveal the underlying molecular mechanisms that link miR-301a overexpression to cancer, and several functional targets have been identified. Upregulated microRNA-301a in breast cancer promotes tumor metastasis by targeting PTEN and activating Wnt/β-catenin signaling;[26] increase pancreatic tumor growth by downregulate NKRF, activate nuclear factor-κB target genes (MMP2, COX-2, MYC, VEGFC and FAM33A);[12] and acts as oncogene in colorectal [13] and gastric [14] cancer by targeting Smad4 or RUNX3, respectively. In human lung tumorigensis, miR-301a was shown to feedback regulates its host gene, ska2 in A549 cells by targeting MEOX2 to affect ERK/CREB pathways, and the inhibition of miR-301a resulted in an increase of the mitotic index and a decrease in colony formation [16]. A typical miRNA may have several targets and a single mRNA may be regulated by a number of miRNA,[27] the same miRNA may target different mRNAs in different cancer types and different stages of tumor progression, therefore, the underlying mechanisms of miR-301a-mediated tumorigensis and development in NSCLC need to be well elucidated in further research. Determining the molecular characteristics of miR-301a is an important aspect of future investigations.


 > Conclusions Top


Our results showed that the expression level of miR-301a was significantly upregulated in NSCLC tissues. Increased expression of miR-301a was detected in tumors with lymph node metastasis. In addition, high miR-301a expression was associated with poorly differentiation, present of lymph node metastasis and advanced TNM stage. Furthermore, the upregulation expression of miR-301a was associated with poor prognosis. These findings suggested that miR-301a might be a novel prognostic indicator in NSCLC, a biomarker for the early detection of metastasis and a potential target for gene therapy.

 
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