MicroRNA-154 Expression is Associated With The Prognosis in Non-Small Cell Lung Cancer.

Background: MicroRNAs are noncoding RNAs that regulate cellular processes during the progression of tumors. Among various microRNAs, MicroRNA-154 (miR-154) has been reported to be involved in many critical processes of human malignancies. This study aimed to evaluate the signicance and prognostic value of miR-154 in human non-small cell lung cancer (NSCLC). Methods: A total of 144 NSCLC tissues samples and matched non-tumor adjacent tissues specimens were obtained from NSCLC patients and the quantitative real-time PCR (qRT-PCR) was performed to investigate expression levels of miR-154. The correlation between miR-154 expression and survival outcomes of NSCLC patients was performed by Kaplan-Meier analysis, univariate and multivariate analysis. Results: MiR-154 expression was signicantly decreased in NSCLC tissues compared with that in matched non-tumor adjacent tissues (P<0.001). In addition, low expression of miR-154 was demonstrated to be associated with tumors size, TNM stages and distant metastasis of NSCLC patients Survival analysis revealed that patients with low expression of miR-154 showed signicantly lower survival rate for OS, DFS and RFS, respectively (all, log rank test, P<0.001) and miR-154 could be an independent prognostic indicator for NSCLC patients. Conclusion: The results suggest that miR-154 has the clinical signicance in the progression of NSCLC and could be a potential prognostic biomarker for NSCLC patients.


Background
As a malignant cancer, non-small cell lung cancer (NSCLC) accounts for 85% of primary lung cancer which is the leading cause of cancer-related deaths worldwide [1,2]. Actually, lung carcinogenesis is a complicated biological process due to mutual dysregulation of different tumor-related genes. Although the treatment including surgery, radiotherapy, and platinum-based combination chemotherapy for NSCLC [3] have improved a lot in recent years, the prognosis of NSCLC remains poor and the ve-year survival rate is less than 15% [4,5]. Therefore, it is still urgent to explore precise and special markers for improving the the survival of NSCLC patients.
MicroRNAs (miRNAs, miR) are a class of small (approximately 18-25 nucleotides), single stranded, conserved and noncoding RNAs, which regulate gene expression by binding to the 3′-untranslated region of speci c target messenger RNAs, thereby result in mRNA degradation or translation inhibition [6][7][8]. It has been widely accepted that miRNAs play an important role in various pathological processes, such as cell proliferation, differentiation, metabolism and apoptosis, indicating their function as suppressor genes or oncogenes [9,10]. Currently, increasing numbers of researches have provided that miRNAs are involved in different human malignancies including pancreas cancer, prostate cancer, colorectal cancer and lung cancer [11,12]. For example, miR-186, miR-205 and miR-21 were all found to promote lung cancer carcinogenesis [13][14][15]. MiR-154 is also one of the cancer-related miRNAs. Recent studies have reported that miR-154 is down-regulated and functions as a candidate tumor suppressor in various cancers, such as hepatocellular carcinoma, colorectal cancer and prostate cancer [16][17][18]. However, the expression level of miR-154 and weather miR-154 has the clinical signi cance in NSCLC has not been reported.
Therefore, the aim of the study was to measure the expression level of miR-154 in NSCLC tissues and investigate the correlation of miR-154 level with clinicopathological features as well as the prognostic value of miR-154 in NSCLC patients.

Patients and samples
A total of 144 NSCLC tissue samples and matched non-tumor adjacent tissues specimens were obtained from patients who had pathologically con rmed as NSCLC in Cangzhou Central Hospital. None of the patients had underwent adjuvant treatments including radiotherapy, chemotherapy or immunotherapy before surgical resection. According to the criteria of American Joint Committee on Cancer (AJCC) tumornode-metastasis (TNM) staging system for lung cancer, 35, 20, 33 and 46 specimens exhibited stage I, II, III and IV cancer, respectively. After resection, all tissues were immediately snap-frozen in liquid nitrogen, and then stored at − 80 ℃ until RNA extraction. The follow-up was performed after surgery from 2 to 86 months (median: 61.5 months) and clinicopathologic characteristics of patients were summarized in Table 1. The protocol of this study was approved by the Ethical Committee of Cangzhou Central Hospital, and all patients signed the informed consent. RNA extraction and quantitative real-time PCR Total RNA was isolated from the frozen tissues using TRIzol reagent (Invitrogen) according to the manufacturer's protocol, and then the rst cDNA was synthesized using a Reverse Transcription Kit (Takara). QRT-PCR was performed with the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) to quantify miR-154. U6 was taken as the endogenous control and the sequences of primers were as follows: miR-154, forward 5′-CGCGAATTCGCATCTAGGACCTCCATCAC-3′ and reverse 5′-ACGGGATCCGAACCATCCCTTCACTTACC-3′; U6 forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′. The relative expression levels of miR-154 were calculated by 2−∆∆Ct method and all experiments were presented in triplicate.

Statistical analysis
All statistical analyses were conducted using SPSS version 18.0 software (SPSS, Chicago, IL) and GraphPad Prism 5.0 (Graphpad Software Inc.). All quanti ed variables were presented as mean ± SD. The Student's t test was used to evaluate the difference between two groups while the differences among three or more groups was evaluated by Chi-square test. Moreover, Kaplan-Meier method with log-rank test was performed to compare survival curves. The analysis of prognostic analysis was carried out using univariate and multivariate Cox proportional hazards model. The difference was signi cant when a P value < 0.05.

Results
The down-regulated expression of miR-154 in NSCLC We measured the expression levels of miR-154 in 144 samples of NSCLC tissues and matched adjacent specimens by qRT-PCR. As shown in Fig. 1A, miR-154 expression level in NSCLC tissues was signi cantly lower than those in the corresponding adjacent normal tissues (0.290 ± 0.152 vs 0.725 ± 0.280, P < 0.001).

Association between miR-154 expression and clinical features
To analyze whether miR-154 was associated with the development and progression of NSCLC, we divided the samples into high and low miR-154 expression groups according to the mean value of miR-154 levels, and then investigated its relationship with the clinicopathological features. As summarized in Table 1, the signi cant associations between miR-154 expression and tumor size (P = 0.001), TNM (P = 0.003) and distant metastasis (P = 0.005) were identi ed. Concretely, low level of miR-154 expression was signi cantly correlated with larger tumor size ( ≧ 3 cm), TNM stages in III, IV, and present distant metastasis (all, P < 0.001, Fig. 1B). All these results demonstrated that miR-154 was related to NSCLC progression and it might act as a tumor suppressor. However, no signi cant relationship had been found between miR-154 expression and other clinical factors including age, gender, lymph node metastasis, differentiation or the or history of smoking.
The correlation between miR-154 level and survival of NSCLC patients To further evaluate the correlation between miR-154 expression and clinical outcomes, we determined the prognostic value of miR-154 expression on overall survival (OS), disease-free survival (DFS) and recurrence-free survival (RFS) in NSCLC patients. As determined by Kaplan-Meier method with log rank test, the patients with low expression of miR-154 presented a shorter OS (P < 0.001; Fig. 2A), shorter DFS (P < 0.001; Fig. 2B) and shorter RFS (P < 0.001; Fig. 2C) respectively than those with high miR-154 expression, indicating reduced expression of miR-154 predicted a poor prognosis in patients with NSCLC.
Moreover, as respect to the in uence of miR-154 levels and clinicopathological characteristics on patient survival, we performed univariate Cox regression analysis. As shown in Table 2

Discussion
As the most common malignant disease in the world, lung cancer is the leading cause of mortality in China [19] According to the past researches, several prognostic factors and biomarkers for NSCLC have been identi ed [20][21][22][23], which have improved the outcome of the NSCLC patients. However, the 5-year survival rate for the NSCLC is still unsatisfactory. Therefore, it is still urgent to identify novel and reliable prognostic markers to improve the prognosis of NSCLC patients.
Previous researches reveal that, till now, approximately 1,900 human miRNAs have been identi ed [7], which have great importance in the regulation of gene expression by regulating cellular functions such as proliferation, apoptosis and differentiation [24,25]. Additionally, the roles of miRNAs in the progression and tumorigenesis of lung cancer have been gradually recognized. miR-10b in lung cancer is closely associated with lymph node metastasis [26]. Moreover the abnormal expression of several miRNAs are found to be involved in lung cancer [27][28][29]. For example, Foss et al. reported that the expression of miR-1254 and miR-574-5p was signi cantly increased in the early-stage of NSCLC samples and they may serve as valuable biomarkers for NSCLC early detection [27]. Jia et al. have demonstrated that the miR-146a expression was signi cantly decreased in NSCLC patients [28]. In addition, the study of Nasser et al. found that miR-1 was down-regulated in human primary lung cancer tissues and cell lines and it has potential therapeutic application against lung cancers [29].
MiR-154 is located on human chromosome 14q32, which is frequently lost in human cancers [30,31]. According to recent studies, miR-154 has been reported to be down-regulated in various types of cancer tissues, and closely associated with the outcome of prognosis for several tumors. For instance, Pang et al. showed that miR-154 functioned as a tumor suppressor in hepatocellular carcinoma by targeting ZEB2 and it may serve as a potential target for HCC [16]. Yang et al. indicated that low expression of miR-154 was associated with tumor progression of colorectal cancer and it may be an independent prognostic marker for CRC patient [17]. Moreover, Zhu et al. reported that miR-154 expression signi cantly decreased in primary prostate cancer samples and played a prominent role in prostate cancer proliferation by suppressing CCND2 [18]. However, the clinical signi cance of miR-154 in lung cancer are poor characterized.
In the present study, we explored the expression of miR-154 and its clinical value in predicting survival outcome in patients with NSCLC. The results con rmed that the miR-154 expression was signi cantly down-regulated in NSCLC tissues compared to the adjacent normal specimens. Subsequently, the association between miR-154 expression levels and clinicopathological characteristics in NSCLC revealed that down-regulation of miR-154 was closely correlated with tumor size, TNM stage and distant metastasis. In contrast, there was no correlation between miR-154 expression and age, gender, lymph node metastasis, differentiation or smoking history. All these results suggest that miR-154 plays an important role in the progression of NSCLC. Moreover, survival analysis revealed that patients with low miR-154 expression have worse survival outcome. This result is consistent with report examining the prognostic role of miR-154 [17]. According to multivariate analyses, miR-154 was an independent prognostic factor for OS, DFS and RFS.
This present study had several limitations. First, the sample size was relatively small. Secondly, the underlying mechanisms of miR-154 in NSCLC have not been well characterized. Therefore, identi cation of miR-154 function and its downstream genes in lung cancer would be an important goal in future studies.

Conclusions
In conclusion, in the present study, we explored that the expression of miR-154 was down-regulated in NSCLC, and the patients with low miR-154 expression had shorter survival time in the OS, DFS and RFS analysis than those with high miR-154 expression. All these ndings demonstrated that miR-154 could be regarded as an independent prognostic factor for patients with NSCLC. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.