Overexpression of miR-1260b in Non-small Cell Lung Cancer is Associated with Lymph Node Metastasis

: Lymph node (LN) metastasis is often an early event in the progression of malignant tumors and it contributes to the majority of cancer mortalities. MiRNAs play key roles in tumor metastasis. This study aimed to investigate the specific miRNAs as putative indicators of metastasis early diagnosis for non-small cell lung cancer (NSCLC). In this study, five NSCLC cases with LN metastasis and four cases without metastasis (NLN) were enrolled for Agilent Human miRNA array. The interested differentially expressed miRNA was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the LN metastasis (n = 46) and NLN (n = 39) groups. The microarray results revealed that three miRNAs (miR-1260b, miR-423-3p, miR-23a-5p) were differentially expressed in LN metastasis group compared with NLN group. The expression of miR-1260b was tested by qRT-PCR and the mean relative expression fold change (2 -ΔΔCt ) in LN metastasis was significantly higher than that in the NLN group (3.942, 1.743 respectively, P = 1.179E-04). The patients with tumor-node-metastasis (TNM) stage III were identified more frequently in LN metastasis group (P = 1.772E-11) and with a higher expression level of miR-1260b (5.126, P = 1.147E-06). In addition, the LN metastasis cases were associated with a poorly differentiated degree (P = 0.007). The overexpression of miR-1260b in NSCLC with LN metastasis can be regarded as a specific signature for early progression and prognosis of NSCLC.

Lung cancer is currently the leading cause of death worldwide and 80% of patients are diagnosed as nonsmall cell lung cancer (NSCLC) [1,2]. The poor prognosis with a 5-year survival rate of 16% is due not only to the late stage diagnosis but also to the lack of effective treatments [3,4]. Metastasis and invasion are the main characteristics during the progression of malignant tumors, which contributes to the majority of cancer mortalities [5]. Lymph node (LN) metastasis is often an early event in epithelial tumor progression and mainly responsible for tumor recurrence [5].
MicroRNAs (miRNAs) are a large family of posttranscriptional regulators of gene expression with a length of 19-22 nucleotides [6,7]. Numerous reports cited that miRNAs play crucial functions in multiple cellular processes, such as cellular proliferation, apoptosis, differentiation and metabolism [7]. Human microRNA genes are frequently located at cancer-associated genomic regions or in fragile sites [8]. In recent studies, several miRNAs such as miR-145, miR-186 and the let-7 family were considered suppressors for the metastasis and epithelial mesenchymal transition (EMT) in lung cancer [2,9,10] while miR-10b and miR-155 were positively correlated with the NSCLC tumorous process [11,12]. Therefore, there is a need to investigate the specific miRNAs as putative indicators for NSCLC metastasis more comprehensively and provide a sensitive method for the early diagnosis of NSCLC. The focus of this study is

Total RNA isolation
Total RNAs were extracted from NSCLC tissues and para-carcinoma tissues by Trizol (Invitrogen, USA) according to the manufacturer's protocol. RNA quality and quantity were measured by Bio-Rad SmartSpec Plus Spectrophotometer (Bio-Rad, USA). The RNA integrity and purity were analyzed by gel electrophoresis and the ratio of the absorbances at 260 nm and 280 nm (A260/A280≥1.8).

Validation analysis by qRT-PCR
2 μg of total RNA was subjected to reverse transcription (RT) with All-in-One™ miRNA qRT-PCR Detection Kit (No. AOMD-Q050, GeneCopoeia, USA). The prepared RT mixture was incubated at 37 °C for 60 min then inactivated at 85 °C for 5 min. The synthesized cDNA was amplified using the All-in-One TM qPCR Mix (No. AOPR-0200, GeneCopoeia, USA) on the 7500 real-time PCR System (Applied Biosystems, USA). PCR reactions were prepared following the manufacturer's instructions. The quantitative PCR cycling profile was as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and primer extension at 72 °C for 30 s, then a melting curve (90 °C for 10 s, 60 °C for 30 s, and 90 °C 10 s). For normalization, U48 rather than U6 was used as an internal reference gene according to our preliminary experiments that benefited from its stability and sensitivity [16]. The primers used for amplification were supplied by All-in-One TM qPCR Primer (GeneCopoeia, USA). Water was used as a negative and quality control, and each sample was measured in duplicates.

Data analysis
The miRNA array data were analyzed in two steps.

Differentially expressed miRNAs in NSCLC with LN metastasis
MiRNA microarray analysis was performed to evaluate the miRNA expression profiles between LN metastasis and NLN groups in NSCLC. We set the cut-off level as fold change > 2 and P value < 0.05. The original intensities data were normalized using the Quantile method. We found 39 and 18 differentially expressed miRNAs in the LN and NLN groups respectively by comparing NSCLC and their matched para-carcinoma tissues. Six miRNAs were found in both the two groups. After removing the reduplicative miRNAs, we analyzed the 51-pooled miRNAs for the comparison of the LN metastasis and NLN groups, and finally found 3 differentially expressed miRNAs (Fig. 1). Among these altered miRNAs, miR-1260b was overexpressed while miR-23a-5p and miR-423-3p were underexpressed in the LN metastasis group (Table 1). Gene Ontology (GO) analysis was applied to explore the significant biological processes related to cancer development, proliferation, and metastasis. As shown in Table 2, the analyses of cancer related pathways and the predicted targets of miRNAs revealed that miR-1260b showed a deepest connection with cancer development and metastasis.

Clinical characteristics
A total of 85 NSCLC patients were recruited in a validation study with their clinical characteristics presented in Table 3. Tumor types can be classified as the two most common NSCLC type (adenocarcinoma and squamous carcinoma) and other types (like sarcomatoid carcinoma, large cell carcinoma, adenosquamous carcinoma, etc.) [17]. The analyses showed that there was no significant difference between LN metastasis and NLN groups with respect to age, gender, and tumor type (Table  3). Nevertheless, the LN metastasis cases were accompanied by a higher TNM stage and poorly differentiated degree (Table 3; P = 1.772E-11, 0.007, respectively).

Validation of the miRNA-1260b by qRT-PCR
To validate the results from the miRNA array, miR-1260b was assayed by qRT-PCR in the validation cohort. The selection of this miRNA was according to the overexpression in the miRNA array and the potential function analyzed by GO. The result obtained by qRT-PCR showed that the mean relative expression fold change (2 -ΔΔCt ) in the LN metastasis group (3.942) was significantly higher than that in the NLN group (1.743), Aging and Disease • Volume 6, Number 6, December 2016 482 which was in agreement with the data from microarray ( Fig. 2, P = 1.179E-04). The value of fold-change between the LN metastasis and NLN groups was 2.262. These results suggest that overexpression of miR-1260b is associated with a higher risk of LN metastasis. Furthermore, we also discovered the association of miR-1260b expression with different clinical stages (P = 1.147E-06). Patients with TNM stage Ⅲ had a higher relative expression fold change than patients in TNM stageⅠandⅡ (Fig. 3, Ⅰ:1.145; Ⅱ: 2.159; Ⅲ: 5.126; Ⅰ vs. Ⅲ : P = 1.347E-06; Ⅱvs. Ⅲ: 1.662E-05, respectively). No significant difference was observed between patients in TNM stagesⅠandⅡ (P = 0.159).

DISCUSSION
Non-small cell lung cancer is regarded as a heterogeneous disease with multiple histopathological and clinical features. Patients with metastasis that frequently occurs in NSCLC often have poor prognosis and high mortality. Previous high-throughput array analyses have demonstrated that the expression of miRNAs present substantial variances in tumors [18,19]. Overexpression of miRNAs may contribute to its oncogenesis by downregulating tumor suppressors, whereas underexpression of miRNAs may negatively regulate oncogenes or factors related to tumorigenesis and progression [20]. Thus, we explore comprehensive miRNAs profile in NSCLC with LN metastasis.
In this study, we discovered three abnormally expressed miRNAs (miR-1260b, miR-23a-5p and miR-423-3p). Through further qRT-PCR validation, overexpressed miR-1260b was demonstrated in LN metastasis tissue of NSCLC patients compared with LN free NSCLC patients. A variety of studies have identified certain miRNAs that suppress the metastasis in lung cancer [9,10,21]. Let-7 is one of the earliest identified miRNAs with significantly reduced expression in a large number of malignant tumors and is known to attenuate the development of lung cancer [22,23]. As for miR-10b and miR-155, the upregulated expression level was significantly positively correlated with TNM stage, lymph node involvement, and poor survival [11,12]. The higher expression of miR-31 was observed in lung adenocarcinoma tissues from patients with LN metastases rather than those without LN metastases [24]. In line with our verification, miR-1260b was highly expressed in prostate cancer (PC) tissues and renal cell carcinoma (RCC) tissues [25,26]. On the other hand, miR-1260b was underexpressed in periodontitis tissues compared with healthy tissues [27]. Interestingly, miRNAs were not only tissue specific but also body fluid specific. MiR-1260b was first discovered to be specifically expressed in vaginal secretions [28]. The discrepancy of these studies might be derived from different tumor types and heterogeneity. Two miRNAs were newly detected in NSCLC tissue i.e., miR-423-3p and miR-23a-5p, which were underexpressed in the LN group. MiR-423-3p can promote tumor progression through similar signaling pathways as in laryngeal hepatocellular carcinomas [29,30]. It could also be a signature to differentiate hereditary and non-hereditary breast cancers [31]. The miR-23a-5p was only identified as a participant in the pathogenesis of traumatic injury [32]. No study has explored the association of miR-1260b expression and metastasis. One study revealed that the higher expression of miR-1260b was observed in stage II, III, and IV of PC patients rather than stage I patients [25]. In addition, in vitro experiments showed that miR-1260b promoted renal cell carcinoma cells proliferation and invasion [26]. Consistent with the results, the expression of miR-1260b was significantly higher in stage III patients than stage I and II in our study. Clinically, TNM stage III patients almost always present with metastasis. Therefore, we can infer that the miR-1260b may play an important role in lung cancer metastasis. Our study is the first to reveal the role of miR-1260b in NSCLC metastasis. The overexpression of miR-1260b can be regarded as a specific signature for early progression and prognosis of NSCLC lymph node metastasis. However, formal verification for these findings is required in a larger cohort of tissue samples. Furthermore, the information of smoking history and the 5-year survival rate ought to be followed up. The predicted target genes and pathways responsible for cancer metastasis and development also need to be elucidated in future studies. We plan to dissect the mechanism for metastasis and find more miRNAs that might intervene with the metastatic process. We also hope that some of the miRNA strategies will be further developed and used, alone or in combination with chemotherapy, to improve the specificity of diagnosis and treatment for NSCLC patients.