Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 671-675

Down-expression of circulating micro ribonucleic acid (miRNA)-148/152 family in plasma samples of non-small cell lung cancer patients


Department of Clinical Laboratory, Fuzhou Pulmonary Hospital, Fujian Medical University, Fuzhou, China

Date of Web Publication25-Jul-2016

Correspondence Address:
Ming-Xiang Huang
Department of Clinical Laboratory, Fuzhou Pulmonary Hospital, Fujian Medical University, No. 2 Hubian Cangshan, Fuzhou - 350 008, Fujian
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.150420

Rights and Permissions
 > Abstract 


Aims: Circulating extracellular micro ribonucleic acids (miRNAs) are considered as potential biomarkers for malignancy detection and diagnosis. The aim of this study was to determine whether circulating miRNA-148/152 family (miR-148a, miR-148b, and miR-152) expression in plasma could be used as potential biomarkers for non-small cell lung cancer (NSCLC) patients and healthy individuals.
Subjects and Methods: The levels of miRNA-148/152 family were detected by TaqMan quantitative polymerase chain reaction (qPCR) assay in plasma of 20 NSCLC patients and 10 healthy individuals. The miRNA expression level of each sample was normalized to that of miR-16 and expressed as relative expression (2-ΔΔCt).
Results: The circulating level of all three members of miRNA-148/152 family were significantly lower in plasma samples of NSCLC patients compared with those of healthy controls (P = 0.007, P = 0.003, and P = 0.000, respectively). The expression levels of miR-148a, miR-148b, and miR-152 in the late-stage NSCLC group were all lower than the early-stage NSCLC group (all P < 0.05).
Conclusions: The present study suggests that the expression levels of miR-148/152 family in plasma might be useful biomarkers for NSCLC patients samples in the early diagnosis of NSCLC and monitoring of tumour development.

Keywords: Biomarker, microRNAs, miRNA-148/152 family, non-small-cell lung cancer, plasma


How to cite this article:
Huang MX. Down-expression of circulating micro ribonucleic acid (miRNA)-148/152 family in plasma samples of non-small cell lung cancer patients. J Can Res Ther 2016;12:671-5

How to cite this URL:
Huang MX. Down-expression of circulating micro ribonucleic acid (miRNA)-148/152 family in plasma samples of non-small cell lung cancer patients. J Can Res Ther [serial online] 2016 [cited 2019 Dec 11];12:671-5. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/671/150420




 > Introduction Top


As one of the most aggressive malignancies with the worst prognosis, non-small cell lung cancer (NSCLC) is the most common cancer in China, which increased 465% during the past 30 years and became the leading cancer death cause in the current decade.[1] Moreover, most patients are diagnosed at late-stage, in addition to limitations on timely and standard treatment, both leading to poorer survival.[2] Besides implementing programs for tobacco control and public health campaigns promoting physical activity, a substantial proportion of the cancer burden could be prevented by early detection and treatment. Therefore, searching the blood/serum based biomarker of NSCLC could be not only helpful with the clinical diagnosis but also suggestive of traditional cancer monitoring methods.

Micro ribonucleic acids (miRNAs) is a class of small non-protein coding RNAs, which have recently confirmed as stable molecules in circulatory system, by packed in extracellular vesicular structures, called microsomes and exosomes, they are able to against endogenous RNase activity and evade from degradation.[3] As pivotal regulators involving in almost all aspects of tumorigenesis and development of malignancies, miRNAs is thought to be good indicators of tumor characteristics. Moreover, the tissue/cell-specific expression profiles of miRNAs in cancer patients established a new paradigm for developing non-invasive biomarkers for cancer detection and diagnosis.[4],[5] Recently, it has been found that varies of tumor-associated miRNAs are present in plasma (e. g., miR-21, miR-10a, miR-30d, and miR-145) and serum (e. g., miR-30e, miR-106a, miR-27a/b, miR-92a, let-7b/c) of lung cancer.[6],[7] Thus, circulating miRNAs are emerging as novel blood-based biomarker for lung cancer.

In the present study, we focused on the expression levels of miR-148/152 family (miR-148a, miR-148b, and miR-152) in plasma of NSCLC patients based on previous reports that they are dysregulated in lung cancer.[8],[9],[10],[11] We determined the expression levels of these three miRNAs in plasma of healthy individuals and NSCLC patients by TaqMan quantitative polymerase chain reaction (qPCR) assay, and attempted to analyze the possibility of plasma miR-148/152 family as biomarkers for the diagnosis of NSCLC.


 > Subjects and Methods Top


Subjects

We analysed plasma samples from 20 patients diagnosed as primary NSCLC and 10 healthy volunteers without any known abnormality of the lung that collected from our hospital between February 2013 and April 2014. All participants provided written informed consent to participate in the study, which was reviewed and approved by the Institutional Review Board of our Hospital. There was no significant difference between the two groups with respect to age, sex, smoking status, or performance status [Table 1]]. The stage of tumor at the time of diagnosis was determined by guidelines of the International Association for the Study of Lung Cancer (IASLC) Tumor-Node-Metastasis (TNM) classification, 7th edition.[12] All samples (5–10 mL) were collected before the start of surgery and were aliquoted into spray-coated K2 EDTA-treated tubes (BD-Becton, Dickinson and Company, Plymouth, UK) and centrifuged to separate the plasma fraction, and then stored at − 80o C until total RNA isolation. The clinical and histopathological parameters of all participants were summarized in [Table 1].
Table 1: Clinical characteristics of patients with lung adenocarcinoma in this study

Click here to view


RNA extraction and real-time reverse transcription quantitative polymerase chain reaction

miRNA was extracted from 1ml thawed plasma using mirVana miRNA Isolation Kit (Ambion, USA, AM1560), according to the manufacturer's instructions. Isolated RNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantification of miRNA was performed by real-time RT-qPCR using an Applied BioSystems 7900HT thermocycler (Applied Biosystems) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. qPCR data was analysed by using the RQ Manager software (Applied Biosystems) with an automatic Ct setting for assigning the baseline and threshold for Ct determination. All tests were performed in triplicates. miR-16 was served as the internal control. All reactions were run in triplicate and Ct data were determined using default threshold settings. Relative miRNA expression levels were calculated using the 2-ΔΔCt method.

Statistical analysis

All statistical analyses were performed using statistical package of social sciences (SPSS) 19.0 (SPSS Inc., Chicago, USA). Differences in miRNA expression between groups were assessed using the Mann-Whitney U test. All P - values were two-sided and P < 0.05 was considered statistically significant.


 > Results Top


Patient's demographic and clinical characteristics

A total of 30 plasma samples (10 patients with stage I NSCLC, 10 patients with stage II–III NSCLC and 10 healthy subjects) were analyzed by RT-qPCR. [Table 1] showed their clinical and pathological data. The median age of the NSCLC patients was 56.55 ± 11.64 years (range, 30 to 76 years) and that of the healthy volunteers was 52.70 ± 15.49 years (range, 26 to 73 years). Ten of all NSCLC patients were diagnosed with squamous cell carcinoma (SCC) and the other ten patients were adenocarcinoma (ADC).

miR-148/152 family expression level comparisons between the early-stage NSCLC and the healthy groups

Our qPCR results showed that all three members of miR-148/152 family could be detected and amplified in all plasma. The mean ± SD value of miR-148a, miR-148b, and miR-152 in the NSCLC group were 0.41 ± 0.27, 0.29 ± 0.30, and 0.58 ± 0.35, respectively, and that of the healthy volunteers were 1.38 ± 0.31, 1.53 ± 0.30, and 1.68 ± 0.25, respectively. As shown in [Figure 1], by using miR-16 as the internal control, our results showed that compared with NSCLC samples, the expression levels of plasma miRNAs were more consistent in healthy samples. The circulating levels of miR-148a, miR-148b, and miR-152 in NSCLC samples were found to be significantly decreased compared with normal controls [P = 0.007, P = 0.003, and P = 0.000; respectively, [Figure 2].
Figure 1: Relative expression levels of micro ribonucleic acid (miRNA)-148/152 family in early-stage non-small cell lung cancer (NSCLC) and healthy individuals. The expression levels of plasma miR-148a (a), miR-148b (b), and miR-152 (c) were more consistent in healthy samples compared with the NSCLC group

Click here to view
Figure 2: Micro ribonucleic acid (miRNA)-148/152 family expression level comparisons between the early-stage non-small cell lung cancer (NSCLC) and the healthy groups. The expression levels of miR-148a (a), miR-148b (b), and miR-152 (c) were significantly higher in the NSCLC groups than the subjects in the healthy volunteer group

Click here to view


miR-148/152 family expression level comparisons between the early- and late-stage NSCLC groups

The expression levels of miR-148/152 family were compared between the early-stage (stage I) and late-stage (stage II–III) NSCLC groups. It is surprisingly to find out that all these three miRNA expression levels change significantly in correlated with the development of the lung cancer [P < 0.05, [Figure 3]. The mean ± SD value of miR-148a, miR-148b, and miR-152 in the late-stage NSCLC group (0.29 ± 0.18, 0.18 ± 0.17, and 0.43 ± 0.31, respectively) were all lower than the early-stage NSCLC group (0.54 ± 0.27, 0.41 ± 0.27, and 0.73 ± 0.26, respectively).
Figure 3: Micro ribonucleic acid (miRNA)-148/152 family expression levels comparisons between the early- and late-stage non-small cell lung cancer (NSCLC) groups. The expression levels of miR-148a (a), miR-148b (b), and miR-152 (c) in the late-stage NSCLC group were all lower than the early-stage NSCLC group

Click here to view



 > Discussion Top


Results of the present study indicate that miR-148/152 family (miR-148a, miR-148b, and miR-152) were down-expressed in the plasma of NSCLC patients compared with the healthy volunteers; moreover, all these three miRNAs expression levels were significantly lower in the late-stage NSCLC patients than the early-stage NSCLC patients. Thus, this study is the first to determine the expression levels of miR-148/152 family in plasma from NSCLC patients, and to compare with healthy volunteers. Our results indicate that miR-148/152 family may be valuable biomarkers for future development of diagnosis, monitoring of tumor development, and risk stratification of NSCLC patients.

A considerable effort has been devoted to decode the information carried by circulating miRNAs, and various malignancies were shown to leave specific miRNA fingerprints in the blood of patients.[13],[14],[15],[16],[17],[18],[19] As a class of small non-coding, single-stranded RNAs that play essential roles in the development and progression of carcinomas, miRNAs were found to exist there in a remarkably stable form.[3],[20],[21] miRNAs in tumor tissues can released into the circulation, although plasma from peripheral blood might not be exactly the same from the microenvironment of tumor, these cell-free miRNAs to some extent reflects the general characteristics of patients' tumors, suggesting that they could serve as non-invasive biomarkers for the detection or monitoring of cancer and putative therapeutic targets. miRNAs are suitable for biomarkers because of the characteristic that they are extremely stable than mRNA. Moreover, use peripheral plasma to measure miRNAs expression levels is relatively cheap, time saving, easy to perform and follow-up.

Multiple miRNAs are shown to be dysrugulated and involved in human NSCLC. Previous work has shown that miR-148/152 family (miR-148a, miR-148b, and miR-152) exhibits abnormal expression levels in NSCLC, and all the three members may function as tumor suppressors in tumor development and progression by modulate multiple pathways and mechanisms.[8],[9],[10],[11] In addition, down-expression of miR-152 in NSCLC associated with poor prognosis.[19] However, little is known about the expression of miR-148/152 family in the blood of NSCLC.

In the current study, by analyzed 20 cases of NSCLC patients and 10 healthy individuals using qRT-PCR, we verified that the expression level of miR-148/152 family was significant down-regulated in NSCLC plasma compared with healthy samples, which was in consistent with Wang's [7] report in patients with advanced-stage NSCLC. However, this study is the first to determine expression of miR-148/152 family in NSCLC patients at early-stage. miR-152 down-expression is associated with lymph node metastasis, and TNM stage in NSCLC, and miR-148/152 family were reported to enable to inhibit malignant behaviors including proliferation, metastasis, and epithelial to mesenchymal transition (EMT) of tumor cells.[8],[9],[10],[11] In consistent with previous reports, we demonstrated that high miR-148/152 family expression was significantly decreased along with the developing of NSCLC. According to the International Association for the Study of Lung Cancer (IASLC) Tumor-Node-Metastasis (TNM) classification,[12] patients at TNM stage II ~ III were all suffered from bigger tumor size (≥3 cm) and invasion outside lobe bronchus, which help to interpreted the lower expression of miR-148/152 family appeared in patients with higher tumor stage in our cohort. These observations indicated that miR-148/152 family expression decreases during cancer development, may be potential biomarkers for NSCLC diagnosis and monitors for tumor development.

The current finding has some limitations. First, the reliability of our study is limited by the small sample results, which require confirmation in further large-scale studies. Second, future studies should be conducted over several time intervals to assess miR-148/152 family expression profiles during the preoperative and postoperative periods. Furthermore, studies conducted in various cancer types including hematological and solid malignancies to assess and compare the expression profiles of miR-148/152 family would also be useful in novel biomarkers exploration.

In conclusion, our results showed that the expression levels of miR-148/152 family were significantly down-regulated in plasma of NSCLC patients. In addition, the expression levels of miR-148/152 family detected in later-stage NSCLC patients were lower than that of early-stage NSCLC patients. These findings suggested that miR-148/152 family might be valuable biomarkers for future development of diagnosis, monitoring of tumor development, and risk stratification of NSCLC patients.

 
 > References Top

1.
Zhao P, Dai M, Chen W, Li N. Cancer trends in China. Jpn J Clin Oncol 2010;40:281-5.  Back to cited text no. 1
    
2.
Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.  Back to cited text no. 2
    
3.
Zen K, Zhang CY. Circulating microRNAs: A novel class of biomarkers to diagnose and monitor human cancers. Med Res Rev 2012;32:326-48.  Back to cited text no. 3
    
4.
Lujambio A, Lowe SW. The microcosmos of cancer. Nature 2012;482:347-55.  Back to cited text no. 4
    
5.
Di Leva G, Garofalo M, Croce CM. MicroRNAs in cancer. Annu Rev Pathol 2014;9:287-314.  Back to cited text no. 5
    
6.
Markou A, Sourvinou I, Vorkas PA, Yousef GM, Lianidou E. Clinical evaluation of microRNA expression profiling in non small cell lung cancer. Lung Cancer 2013;81:388-96.  Back to cited text no. 6
    
7.
Wang Y, Gu J, Roth JA, Hildebrandt MA, Lippman SM, Ye Y, et al. Pathway-based serum microRNA profiling and survival in patients with advanced stage non-small cell lung cancer. Cancer Res 2013;73:4801-9.  Back to cited text no. 7
    
8.
Li J, Song Y, Wang Y, Luo J, Yu W. MicroRNA-148a suppresses epithelial-to-mesenchymal transition by targeting ROCK1 in non-small cell lung cancer cells. Mol Cell Biochem 2013;380:277-82.  Back to cited text no. 8
    
9.
Cheng Z, Ma R, Tan W, Zhang L. MiR-152 suppresses the proliferation and invasion of NSCLC cells by inhibiting FGF2. Exp Mol Med 2014;46:e112.  Back to cited text no. 9
    
10.
Su Y, Wang Y, Zhou H, Lei L, Xu L. MicroRNA-152 targets ADAM17 to suppress NSCLC progression. FEBS Lett 2014;588:1983-8.  Back to cited text no. 10
    
11.
Liu GL, Liu X, Lv XB, Wang XP, Fang XS, Sang Y. miR-148b functions as a tumor suppressor in non-small cell lung cancer by targeting carcinoembryonic antigen (CEA). Int J Clin Exp Med 2014;7:1990-9.  Back to cited text no. 11
    
12.
Goldstraw P, Crowley J, Chansky K, Giroux DJ, Groome PA, Rami-Porta R, et al. International Association for the Study of Lung Cancer International Staging Committee; Participating Institutions. The IASLC Lung Cancer Staging Project: Proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol 2007;2:706-14.  Back to cited text no. 12
    
13.
Schrauder MG, Strick R, Schulz-Wendtland R, Strissel PL, Kahmann L, Loehberg CR, et al. Circulating micro-RNAs as potential blood-based markers for early stage breast cancer detection. PLoS One 2012;7:e29770.  Back to cited text no. 13
    
14.
Borel F, Konstantinova P, Jansen PL. Diagnostic and therapeutic potential of miRNA signatures in patients with hepatocellular carcinoma. J Hepatol 2012;56:1371-83.  Back to cited text no. 14
    
15.
van Schooneveld E, Wouters MC, Van der Auwera I, Peeters DJ, Wildiers H, Van Dam PA, et al. Expression profiling of cancerous and normal breast tissues identifies microRNAs that are differentially expressed in serum from patients with (metastatic) breast cancer and healthy volunteers. Breast Cancer Res 2012;14:R34.  Back to cited text no. 15
    
16.
Leidinger P, Keller A, Borries A, Reichrath J, Rass K, Jager SU, et al. High-throughput miRNA profiling of human melanoma blood samples. BMC Cancer 2010;10:262.  Back to cited text no. 16
    
17.
Zhou G, Shi X, Zhang J, Wu S, Zhao J. MicroRNAs in osteosarcoma: From biological players to clinical contributors, a review. J Int Med Res 2013;41:1-12.  Back to cited text no. 17
    
18.
Fu SW, Chen L, Man YG. miRNA biomarkers in breast cancer detection and management. J Cancer 2011;2:116-22.  Back to cited text no. 18
    
19.
Sanfiorenzo C, Ilie MI, Belaid A, Barlesi F, Mouroux J, Marquette CH, et al. Two panels of plasma microRNAs as non-invasive biomarkers for prediction of recurrence in resectable NSCLC. PLoS One 2013;8:e54596.  Back to cited text no. 19
    
20.
Zandberga E, Kozirovskis V, Abols A, Andrejeva D, Purkalne G, Line A. Cell-free microRNAs as diagnostic, prognostic, and predictive biomarkers for lung cancer. Genes Chromosomes Cancer 2013;52:356-69.  Back to cited text no. 20
    
21.
Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513-8.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  >Abstract>Introduction>Subjects and Methods>Results>Discussion>Article Figures>Article Tables
  In this article
>References

 Article Access Statistics
    Viewed2148    
    Printed25    
    Emailed0    
    PDF Downloaded119    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]