Original Article Serum miR-125a-5p, miR-145 and miR-146a as diagnostic biomarkers in non-small cell lung cancer

Original Article

Serum miR-125a-5p, miR-145 and miR-146a as

diagnostic biomarkers in non-small cell lung cancer

Rui-Juan Wang1,3*_{, Yan-Hua Zheng}2*_{, Ping Wang}3_{, Jian-Zhong Zhang}1,4

1_{Chinese PLA General Hospital/Chinese PLA Medical School, Beijing 100853, China;} 2_{Department of Special}

Medical Center, 306 Hospital of PLA, Beijing 100101, China; 3_{Department of Respiratory Medicine, 306 Hospital}

of PLA, Beijing 100101, China; 4_{Department of Pathology, 306 Hospital of PLA, Beijing 100101, China.} *_Equal

contributors.

Received November 5, 2014; Accepted December 23, 2014; Epub January 1, 2015; Published January 15, 2015

Abstract: Background: Lung cancer is becoming the leading cause of cancer-related deaths with high mortality worldwide and in China as well. Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer ac-counting for approximately 85% of all cases. Over 70% of cases are at loco-regionally advanced stages or have distant metastasis at the time of presentation with subsequently poor prognosis. MiRNAs are stable molecules in blood and used as biomarkers for the early diagnosis of various malignancy. The purpose of this study was to evalu-ate whether circulating miR-125a-5p, miR-145 and miR-146a could be used as biomarkers for the diagnosis of NSCLC through measuring their expression and assess their relationship with clinical pathological factors. Methods: Expression levels of serum miR-125a-5p, miR-145 and miR-146a were detected in 70 pairs of NSCLC patients and healthy controls using quantitative real-time PCR analysis. Results: Serum miR-125a-5p, miR-145 and miR-146a were overexpressed in NSCLC patients compared with healthy controls. Their values of the area under the receiver –

operating characteristic curve (AUC-ROC) were 0.71, 0.84 and 0.78. Optimal sensitivity and specificity were 73.53%

and 55.71%, 92.75% and 61.43%, 84.06% and 58.57%, respectively in differentiating NSCLC patients from healthy controls. Conclusions: These preliminary data suggest that serum miR-125a-5p, miR-145 and miR-146a may be useful noninvasive biomarkers for the clinical diagnosis of NSCLC.

Keywords: MicroRNAs, non-small cell lung cancer, serum, diagnosis

Introduction

Lung cancer is becoming the leading cause of cancer-related deaths with high mortality worldwide [1] and in China [2] as well. Non-small-cell lung cancer (NSCLC) is the most com-mon type of lung cancer accounting for approxi-mately 85% of all cases, which is subdivided into three major types: adenocarcinoma (ADC), squamous-cell carcinoma (SCC) and large-cell carcinoma (LCC) [3, 4]. Over 70% of cases are at loco-regionally advanced stages or have distant metastasis at the time of presenta- tion with subsequently poor prognosis [5]. Additionally for the intrinsic resistance and recurrence, the survival rate for NSCLC still remains at low level, including early-stage group. The overall 5-year survival rate is only 17.1% in the United States [6]. It is urgent to explore the pathogenesis and identify more effective biomarkers, especially using

noninva-NSCLC as early as possible and develop effec-tive individualized treatment programs to improve the quality of life and survival.

patients and control subjects are summarized in Table 1.

Total RNA extraction

Total RNA was isolated from 300 μl serum specimens of patients and controls by using miRNA isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. RNA concentration was measured using Nanodrop 2100. Then the RNA samples were preserved at -80°C for use. Repeated freeze-thawing was avoided to ensure the qual-ity of the samples during storage,

Quantitative reverse transcription-PCR (qRT-PCR)

TaqMan MicroRNA Kit (Applied Biosystems)

was used to perform the expression profiling of

serum miRNA of all patients and controls. All reagents, primers and probes were also bought from Applied Biosystems. The cDNA reverse

transcription was first carried out according

to the manufacture’s instruction. The 15 μl reverse transcription reaction system

consist-ed of 0.15 μl of dNTPs mix (100 mM total), 1.0 μl of reverse transcriptase (50 U/μl), 1.5 μl of RT Buffer (10×), 0.19 μl of RNase inhibitor (20

U/μl), 4.16 μl of nuclease free water, 3.0 μl of

gene-specific miRNA primer and 5 μl of RNA extract sample. For synthesis of cDNA, reaction conditions of upper mixtures were: incubated at 16°C for 30 min, at 42°C for 30 min, at 85°C for 5 min, and at 4°C for any length of time. Then cDNA was amplified. The 20 μl qRT-PCR system contained: 1.0 μl of TaqMan Small RNA

Assay (20×), 1.33 μl of cDNA solution, 10 μl of TaqMan Universal PCR Master Mix (2×) and 7.67 μl of nuclease free water. Quantitative PCR were performed on Applied Biosystems lung cancer cells by regulating several

down-stream genes involved in EGFR signaling path-way [11], restoration of miR-145 can inhibit cancer cell growth in EGFR mutant lung adeno-carcinoma [12] and miR-146a can target EGFR and its downstream gene with potential

func-tional significance in lung cancer [13]. These

conclusions from above-mentioned studies were drawn in vitro or detecting tissue samples aiming at one single miRNA. Recently circulat-ing miR-145 and miR-146a were found to be overexpressed in breast cancer and might be used for detection of breast cancer [14, 15]. In the present study we performed the miRNAs

expression profiling in NSCLC serum compared

with healthy controls using quantitative RT-PCR and constructed the receiver operating charac-teristic (ROC) curves in order to identify

wheth-er specific miRNAs could discriminate between

NSCLC patients and healthy controls. Material and methods

Patients and samples

Seventy cases were collected from primary NSCLC patients who were diagnosed by histol-ogy or cytolhistol-ogy from March 2013 to May 2014 in our hospital. Histological diagnoses were independently formulated by two pathologists.

Disease stage classifications were determined

according to the world health organization

(WHO) classification and the international asso -ciation for the study of lung cancer staging sys-tem [3, 4]. Clinic pathological characteristics were collected whenever available for all the patients. Peripheral blood samples were col-lected before they received any anticancer treatment, including surgery, chemotherapy, radiotherapy and hormonal treatment, etc. 70

samples from healthy individuals with same age and gender were also collected. Blood samples (3.5 ml) were drawn from subjects using tubes without anticoagu-lant. These samples were processed by centrifugation at 3500 g for 5 minutes at room temperature. Serum was

trans-ferred into RNA-free EP tube every 500 μl

aliquot and stored at -80°C before RNA extraction. This study was approved by the Ethics Committee of 306 Hospital of PLA. All specimens were collected after obtaining the patients’ informed con-sents. The characteristics with respect to age, gender and smoking status of NSCLC Table 1. Clinical characteristics of 70 NSCLC patients

and 70 healthy individuals

Patients Individuals P-value Age (years), median 64.41 ± 10.77 63.74 ± 10.32 0.7189

≥ 60 48 46

< 60 22 24

Gender 1.0000

Male 43 43

Female 27 27

Smoking status 0.1756

Yes 37 29

serum miRNAs and clinic pathological factors of NSCLC patients. P-value < 0.05 was defined as statistical significance.

Results

Patient characteristics

There were 43 males and 27 females among the 70 NSCLC patients, including 48 ADCs, 20 SCCs and 2 LCCs. The mean age and gender distribution were similar between the NSCLC patients and the healthy controls. Smoking rate was higher in NSCLC patients than that in con-trols but without statistical difference (P = 0.1756).

Serum miRNAs expression in NSCLC patients and healthy controls

The serum level of miR-125a-5p, miR-145 and miR-146a were compared between the two 7500 Real Time PCR system (Applied

Biosystems) and the reaction conditions were: 10 min at 95°C, and 40 cycles of 95°C for 15 sec and 60°C for 60 sec. All qRT-PCR assays were undertaken in duplicate, using miR-39 as the internal reference gene to normalize the sample-to-sample variation during the RNA iso-lation procedure. The fold change of gene rela-tive expression was calculated by using the equation of comparative cycle threshold (2-ΔΔCt₎ method.

Statistical analysis

Statistical analysis was performed with SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) and graphs were generated using Graphpad Prism 6.0. Two-tailed unpaired t-test was used to evaluate the differential expres-sion of serum miRNA level between NSCLC patients and normal controls. Receiver

operat-ing characteristic (ROC) curves were generated to assess the diagnostic accuracy of each serum miRNA, and the area under the ROC curve (AUC) were used to assess the

sensi-tivity and specificity. Chi-squared

test was used to assess the correla-tion between the expression level of Figure 1. Serum expression levels of miR-125a-5p, miR-145 and miR-146a in healthy controls and NSCLCs.

Table 2. AUC ROC values of different miRNAs in NSCLC patients

miRNA AUC ROC 95% CI Cut-off Sensitivity Specificity

groups. As shown in Figure 1, the expression of

all three miRNAs was significantly higher in

NSCLC patients than that in healthy controls (P

< 0.0001).

Serum miRNAs as diagnosis signatures for NSCLCs

The ROC curve was plotted to identify a cut-off value that could distinguish NSCLCs from healthy controls. Analysis revealed that the area under the ROC curves (AUC) for serum

miR-125a-5p was 0.71 (95% confidence inter -val 0.62-0.79). At an optimal cut-off -value of

1.41, the sensitivity and specificity was 73.53%

and 55.71%, respectively. Similarly, for miR-145 and miR-146a, results were shown in Table 2 and Figure 2.

Correlation between miRNAs expression levels and clinic pathological factors

The correlation between these three miRNAs expression levels and various clinic pathologi-cal factors was examined. These factors includ-ed gender, age, smoking status, lymph node

metastasis and TNM stage. NSCLC patients were divided into miRNA high and low groups according to the median fold-change values.

There were no significant associations between

the analyzed miRNAs and the main clinic patho-logical characteristics of the NSCLC patients. Nevertheless, we found that expression of miR-145 in serum was higher in patients in stage IV than that in other stages, even though without

statistical significance.

Discussion

MiRNAs are involved in the whole biological and physiological processed by regulating target mRNA. Severe dysfunctions and aberrant expressions are present by acting in certain cases as oncogenes while in others as tumor suppressor genes through different targets in human malignancy [16]. MiRNAs are released from primary tumor into peripheral circulation, which are stable molecules and resistant to RNase digestion and other harsh conditions in

serum. Since circulating miRNAs were first

for several human cancers, including NSCLC [14, 17-21]. However the technique of detecting miRNAs from tissue is limited owing to its com-plication and need of invasive method to obtain.

In the current study we compared the profile of

three miRNAs in serum from 70 healthy con-trols with 70 NSCLCs and demonstrated that serum expression of miR-125a-5p, miR-145 and miR-146a strongly differentiated the NSCLC patients from healthy controls. A highly

significant increase was found in these three

miRNAs in serum of NSCLC compared with that of healthy individuals. Moreover, receiver oper-ating curve analysis indicated that the AUC of these three miRNAs were all greater than 0.7, indicating that they might be potential biomark-ers in the diagnosis of NSCLC. Especially miR-145 was proved to be the most accurate tumor marker in distinguishing NSCLC patients from healthy controls with AUC ROC reaching 0.84

and the sensitivity and specificity at optimal

cutoff being 92.75% and 61.43%.

We also analyzed the serum expression levels of these miRNAs in relation to the different clin-ical pathologic characteristics in NSCLC

patients. Unfortunately, no significant relation -ships were found between them.

MiR-125a-5p, miR-145 and miR-146a were known as tumor suppressors and down-regu-lated in a variety of malignant tumor tissues. But in some malignant tumors they might be up-regulated and the serum expressions of these miRNAs were not identical, even in the same tumor. For instance, miR125a-5p was down-regulated in gastric cancer and sup-pressed cell proliferation by targeting ERBB2 [22]. In our study we found miR-125a-5p was overexpressed in serum of NSCLC, unlike a prior report which found no difference between NSCLCs and healthy controls [23]. MiR-145 inhibits proliferation of NSCLC cells by targeting c-Myc [24] and plays an inhibitory role in tumor angiogenesis, cell growth and invasion and tumor growth through the post-transcriptional regulation through N-RAS and VEGF-A in breast cancer [25]. Nevertheless conclusions from several studies which focused on the serum expression of miR-145 were not consistent, such as in breast cancer [14, 26, 27]. For miR-146a, its role can also vary in different types of cancer. It worked as tumor suppressor in

pros-tate cancer [28] and gastric cancer [29] expressing lower regulation whereas in ana-plastic thyroid cancer [30] and cervical cancer [31] it was up-regulated as an oncogene. Even converse expression levels of miR-146a in tis-sue were tested in some malignancy like gas-tric cancer [29, 32]. All of these studies indi-cate that the targets and expressions are varied in different tumors, especially the

expression profiling in peripheral blood.

Meanwhile, few literatures were related to their serum expression in NSCLC to date.

In view of these three miRNAs targeting EGFR and its downstream gene to play their roles, we may be able to investigate the relationship between the EGFR mutation status and serum expression level in future studies.

In conclusion, the high stability of serum miR-NAs makes them become noninvasive biomark-ers for NSCLC detection. However, the

sensitiv-ity and specificsensitiv-ity are not satisfied in

distinguishing NSCLCs from healthy controls and their expression is different in various tumors, and therefore their roles and possible targets in NSCLC require further investigation. Acknowledgements

This work was supported by the Capital Medical Development Funds for Special Research Project (SF2011-5005-01) and the Medical Research Funds of 306 Hospital (2013ZD06). Disclosure of conflict of interest

None.

Address correspondence to: Dr. Jian-Zhong Zhang, Department of Pathology, 306 Hospital of PLA, 9 North Anxiang Road, Chaoyang District, Beijing 100101, China. Tel: 10-66356237; Fax: +86-10-64871261; E-mail: zhangjz55@sina.com

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