MicroRNA-449a Plays an Indicative Role in Diagnosing Lung Cancer

Background: Lung cancer is one of the most common causes of cancer death among all the malignancies worldwide. Evidences suggest that the incidence and mortality of lung cancer has been on the rise. MicroRNA-449a (miR-449a) as one important member of microRNAs, has been demonstrated acting as a tumor suppressor in lung cancer. In this study, we sought to assess the relationship between miR-449a expression level and diagnostic value of lung cancer. Methods: In this present research, quantitative Real-Time PCR was applied to detect the miR-449a expression in 116 lung cancer patients and 41 healthy volunteers. The diagnostic value of miR-449a in lung cancer patients was determined by receiver operating characteristic (ROC) curve. Results: MiR-449a was signicantly down-regulated in lung cancer patients compared with healthy control (P<0.05). In addition, miR-449a expression was associated with sex (P=0.004), tumor size (P=0.000), TNM stage (P=0.006) and metastasis (P=0.036). However, there was no correlation with age, smoking history and histological type of lung cancer patients (all P (cid:0) 0.05). In the ROC analysis, the results showed that the area under the ROC curve (AUC) was 0.902 with the sensitivity of 94.8% and specicity of 78.0%, and the optimum cutoff value was 2.255. Conclusion: MiR-449a expression was down-regulated in lung cancer patients, and it could be an ecient diagnostic biomarker in lung cancer patients.


Background
Lung cancer is one of the leading causes of cancer related death around the world, and has an increasing incidence and mortality [1]. There is no obvious clinical symptoms at the early stage of lung cancer, with the evidences suggest that almost 75% of lung cancer patients are already at an advanced stage at the time of diagnosis [2,3]. However, few effective test methods of lung cancer are available so far. Several protein markers, such as carcinoembryonic antigen, cytokeratin 19 fragment, cancer-associated antigen (CA) 125, CA 19-9, neuron-speci c enolase, and tissue polypeptide speci c antigen have been used to diagnose lung cancer without a surgical procedure , but the sensitivity is limited [3,4]. Therefore, novel sensitive and speci c diagnostic biomarkers are needed for the detection of lung cancer.
MicroRNAs (miRNAs) are endogenous non-coding RNA molecules of 22 nucleotides in length, which play important roles in regulation of pathological and biological processes [5]. Cellular miRNAs are released into different body uids, such as blood [6]. They are presented in human serum in a highly stable form that is resistant to harsh conditions and RNase digestion [7,8]. It is generally considered that miRNAs target the 3'-untranslated region (3'-UTR) of the targeted mRNAs, causing translation inhibiting or degradation of the mRNA. There are approximately half of miRNA genes are located at genomic regions which frequently ampli ed or deleted in cancer [9][10][11]. Recently studies have found that miRNAs possess regulatory functions in tumorigenesis and closely correlated with tumor differentiation [12][13][14][15]. MicroRNA-449a (miR-449a) is one of these miRNAs, which expressed in wide types of cancers, such as ovarian cancer, gastric carcinoma, bladder cancer and lung cancer [16][17][18][19][20]. Recent evidences suggest that miR-449a performs suppression role in non-small cell lung cancer (NSCLC), which could inhibit tumor cell proliferation and promote tumor cell apoptosis [21]. However, no related studies have reported the diagnostic value of miR-449a in lung cancer.
In this present study, we examined the expression level of miR-449a in lung cancer patients with Quantitative Real-Time PCR (qRT-PCR) method and further evaluated the diagnostic potential of miR-449a for lung cancer patients.

Patients and serum specimens' collection
The study was approved by Ethics Committee of the The First A liated Hospital of Xinxiang Medical University. All the serum specimens and clinical materials in this research were obtained with the informed consents of all the participants.
Serum specimens were obtained from 116 cases with lung cancer and 41 healthy control cases without any malignancy before the study. These serum specimens were collected from patients on the day of diagnosis and before tumor surgery and therapy. All samples taken from lung cancer patients and healthy volunteers were put into blood collection tube of EDTA and stored at -80℃ until RNA extraction.
At the same time, clinicopathological features, which including age, sex, smoking history, histological type, tumor size, TNM stage and metastasis were also collected.
Plasma preparation and RNA extraction A volume of 5 ml of EDTA-anticoagulated blood was obtained from each participant. Serum was separated by centrifugation. Total RNA, including miRNA, was extracted from the serum using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA with OD A260/A280 ratio closed to 2.0 was subsequently used, which suggested that the RNA were pure.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 software. Measurement data were expressed as means ± SD. T-test was used to compare the expression of miR-449a between the lung cancer patients and the healthy group. Chi-square test was used to analyze the differences between miR-449a expression and various clinicopathological characteristics. The diagnostic value of miR-449a in distinguishing lung cancer patients from healthy controls was performed using receiver operating characteristic (ROC) analysis. All tests with P values <0.05 were considered as statistically signi cant.

Results
The expression level of miR-449a The present analysis used qRT-PCR to estimate the miR-449a expression level in 116 lung cancer patients and 41 healthy volunteers. The result showed that the expression of miR-449a was signi cantly reduced in lung cancer patients compared with the healthy controls (P 0.05; Figure 1).

Relationship between miR-449a and clinicopathological characteristics of lung cancer
In this study, we used statistical analysis to explore the relationship between miR-449a expression level and the clinicopathological data of lung cancer patients. In Table 1, the analysis results showed that miR-449a expression was associated with sex (P=0.004), tumor size (P=0.000), TNM stage (P=0.006) and metastasis (P=0.036). However, there was no signi cant association between miR-449a expression and age, smoking history as well as histological type of lung cancer patients (all P 0.05).
Diagnostic value of miR-449a for lung cancer ROC curves were constructed to evaluate the diagnostic value of miR-449a expression level in lung cancer patients. In Figure 2, the ROC curve showed that the lung cancer patients were distinguished from the healthy volunteers with sensitivity of 94.8%, speci city of 78.0% and associated area under the curve (AUC) value of 0.902. The cutoff value for miR-449a expression level is 2.255. This result indicated that the miR-449a expression has e cient diagnostic value for lung cancer.

Discussion
Lung cancer, as a major death cause, is reported to be with no clinically apparent symptoms until it has reached an advanced stage [23]. Recently evidences demonstrated that the incidence and mortality of lung cancer has been on the rise [9]. The development of lung cancer is a complicated process that involves multiple genes, factors and pathways. Although there are several biomarkers for lung cancer such as SCC antigen, carcinoembryonic antigen and pro-gastrin-releasing peotide, none of them are perfect in terms of sensitivity or speci city. Therefore, we sought to nd an e cient biomarker to achieve the earlier diagnosis for lung cancer patients.
MiRNAs are a class of small noncoding RNAs. There are several researches nd that miRNAs are associated with a wide range of cellular processes, such as cellular proliferation, differentiation, apoptosis, and play a critical role in cancer [24][25][26]. Recently, miR-449a as a member of miRNAs has been found down-regulated in several types of cancers including prostate cancer, gastric cancer, bladder cancer as well as lung cancer [17,19,[27][28][29]. These studies have showed that miR-449a is an important miRNA that has been identi ed as tumor suppressor, and it plays important roles in the occurrence, development, and prognosis of multiple malignancies. However, the role of miR-449a in the diagnosis of lung cancer still needs to be explored.
In our study, we observed that the miR-449a expression level was lower in lung cancer patients than that in healthy volunteers. This nding is consistent with previous reports. For example, Ding et al. demonstrated that the miR-449a expression levels in non-small cell lung cancer are signi cantly lower than those in the surrounding tissue [21]. Ren et al. con rmed that miR-449a acts as a tumor suppressor and is mostly down-regulated in lung cancer patients [29]. In addition, the present study assessed the relationship between miR-449a expression and the clinicalpathological features of lung cancer patients and found that the expression of miR-449a was signi cantly associated with sex, tumor size, TNM stage and metastasis. However, there were no relation with age, smoking history and histological type of the lung cancer patients. Luo et al.
[30] have proved that low expression level of miR-449a appeared to be correlated with various clinical features such as advanced pathological stage, and lymph node metastasis. What's more, they also suggested that miR-449a might be a useful prognostic predictor for NSCLC patients. In the present study, we evaluated the diagnostic value of miR-449a in lung cancer patients, and the results of ROC analysis showed that down-regulation of miR-449a might serve as a novel biomarker to diagnose lung cancer.
Currently, the molecular mechanisms for the expression of miR-449a in tumors still remain unclear. According to the previous reports, single miRNAs with multiple functions are potential candidates for gene therapy. For example, Noonan et al. [28] have showed that HDAC1 was a target of miR-449a and miR-449a regulates cell growth and viability in part by repressing the expression of HDAS-1. Moreover, in the study of Jeon HS et al. [20] showed that the down expression of miR-449a might be one mechanism for over-expression of HDAC1 in lung cancer, and miR-449a could be a potential therapeutic candidate in lung cancer patients.

Conclusions
In summary, the results provided evidences that miR-449a was signi cantly down-regulated in lung cancer patients compared with the healthy controls. The ROC curves indicated that the down-regulation of miR-449a could be used as a novel biomarker to distinguish patients with lung cancer from the healthy ones. However, there are still some limitations, for example, the sample size is small and we did not give subdivision of lung cancer. Therefore, more detailed research will be needed in the further studies.