Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer

https://doi.org/10.1016/j.bbrc.2017.06.055Get rights and content

Highlights

  • serum MALAT-1 contained in exosomes is upregulated in NSCLC patients.

  • Exosomal MALAT-1 was positively associated with TNM stage and lymphatic node metastasis.

  • MALAT-1 knockdown inhibited cell proliferation, colony formation and cell migration.

  • MALAT-1 knockdown induced cell cycle arrest and cell apoptosis.

Abstract

Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), MALAT-1 was first identified lncRNA that was related to lung cancer metastasis. However, the relationship between exosomal lncRNAs and the diagnosis and prognosis of NSCLC was poorly understood. The aim of this study is to evaluate the clinical significance of serum exosomal MALAT-1 as a biomarker in the metastasis of NSCLC. In this study, we firstly isolated the exosomes from healthy subjects and NSCLC patients. Then we measured the expression levels of MALAT-1 contained in exosomes, and found that exosomal MALAT-1 was highly expressed in NSCLC patients, more importantly, the levels of exosomal MALAT-1 were positively associated with tumor stage and lymphatic metastasis. In addition, we decreased MALAT-1 expression by short hairpin RNA and conducted a series of assays including MTT, cell cycle, colony formation, wound-healing scratch and Annexin/V PI by flow cytometry in human lung cancer cell lines. These in vitro studies demonstrated that serum exosome-derived long noncoding RNA MALAT-1 promoted the tumor growth and migration, and prevented tumor cells from apoptosis in lung cancer cell lines. Taken together, this study shed a light on utilizing MALAT-1 in exosomes as a non-invasive serum-based tumor biomarker for diagnosis and prognosis of NSCLC.

Introduction

Lung cancer is the main cause of cancer-related deaths in human worldwide, there are about 85% of lung cancer patients being classified histopathologically as non-small cell lung cancer (NSCLC) [1]. At present, local resection of tumor tissue is the main treatment for early stage of NSCLC, but the 5-year overall survival rate is as low as 15% [2], as for late stage patients, lack of sufficient therapeutic methods resulted in cancer exacerbation. Even though multiple oncogenes and tumor-suppressive genes have been reported in NSCLC [3], [4], the precise molecular mechanisms underlying NSCLC pathogenesis are still remaining to be further elucidated. Thus, a sound understanding of the progression of NSCLC is crucial for early diagnosis and treatment.

Long non-coding RNAs(LncRNAs) is a class of RNA molecules with length longer than 200 nucleotides, they were regarded as the “junk” of genome for many years because lack of protein coding ability [5]. Lately, ENCODE project and GENCODE annotation have revealed that these noncoding genome elements play essential roles in normal physiology as well as, when the regulation process of lncRNAs goes to disorder, which may cause human diseases [6], [7]. In recent years, a large amount of lncRNAs have been found in NSCLC, including MALAT-1, GAS5, LINC00673, NEAT1 [8], [9]. These lncRNAs were closely related to tumor metastasis, invasion and proliferation. More importantly, the aberrant expression of lncRNAs also can be detected in serum samples derived from tumor patients [10], therefore, lncRNAs is an ideal diagnostic and prognostic biomarker for cancer patients without invasion procedures.

The lncRNA MALAT-1 (metastasis-associated lung adenocarcinoma transcript 1) also known as nuclear-enriched abundant transcript 2 (NEAT2), is abundant in several solid tumors and its differential expression is associated with cancer metastasis and recurrence [11]. Previous studies have shown that transient over expression of MALAT-1 enhanced cellular proliferation in cell lines and tumor formation in nude mice, while depletion of MALAT-1 in tumor cells reduced tumorigenicity [12]. In NSCLC, MALAT-1 has been evaluated as a candidate blood-based biomarker for diagnosis and prognosis, yielding a sensitivity of 56% and specificity of 96% in distinguishing cancer patients and healthy controls [13]. Meanwhile MALAT-1 in paraffin-embedded tissues can also indicate a poor prognosis in NSCLC and induces migration and tumor growth [11], [13].

Recent observations suggest that nano-sized particles named exosomes may act as transport vesicles for functional cargoes, including proteins, mRNAs, miRNAs and lncRNA, which may result in a phenotypic effect in the recipient cells [14]. Double layer construction of exosomes is an ideal shelter for protecting the “passengers” from lysosome and other enzymes, Wang and his colleagues verified that lncRNA CRNDE-h is stable in exosomes from colorectal cancer patients [15]. Exosomes are abundant in body fluids as well as the blood, therefore circulating exosomes carry regulatory lncRNA molecules and play a crucial role in long distance cell-cell communication, may be participated in cancer metastasis and invasion [16]. Emerging evidences have determined that the specific lncRNAs are selectively packaged into exosomes, which are highly correlated with the clinicopathological characteristics of cancer and thus may function as meaningful biomarkers [17].

In this study, we investigated the expression of serum exosomal MALAT-1 in NSCLC patients and evaluated the correlation between exosomal MALAT-1 expression levels and clinicopathological characteristics. We further examined the functional role of circulating MALAT-1 in NSCLC cell growth, proliferation, migration and apoptosis. Our results suggest that exosomal MALAT-1 expression was significantly increased in NSCLC patients compared with healthy controls, and the increased exosomal MALAT-1 expression was significantly associated with TNM stage and lymphatic metastasis. MALAT-1 was abounding in NSCLC patient-derived exosomes could promote cell growth and migration. The remarkable work provides a potential circulating biomarker for the diagnosis and prognosis of NSCLC.

Section snippets

Serum collection and ethic statement

A total 77 NSCLC patients were obtained from the Center hospital of Xinxiang City, China, from August 2014 to November 2016. All procedures performed in studies were in accordance with the ethical standards. And informed consent was obtained from all individual participants included in the study. Tumor stage was evaluated according to the tumor, node and metastasis (TNM) classification system of the International Union Against Cancer (7th, edn. 2010). The collected blood sample were centrifuged

Exosomes were collected and analyzed from clinical serum samples

To analyze MALAT-1 packaged in exosomes, we first isolated exosomes from serum sample of healthy control and NSCLC patients. Then the morphology and phenotypes of isolated particles were identified according to the characteristics of exosomes described previously [18]. First, the concentration and size range of the particles were measured by using nanoparticle tracking analysis (NTA), the results demonstrated that the concentration of the particles was 1.65 × 109 ± 0.48 × 109 particles per ml,

Discussion

NSCLC is one of the most common causes of lung cancer related death worldwide [2]. Patients diagnosed with NSCLC are usually at late stage, and the prognosis is very poor. It's urgent to find an effective indicator to diagnose NSCLC in earlier stage, thus to get intime treatment. Liquid biopsy was reported to possess higher sensitivity and more convenient for cancer diagnosis compared with traditional imaging and biopsy strategies [19]. As a promising alternative to liquid biopsy, tumor-derived

Conflict of interest

All of the authors declared that there is no conflict of interest.

Acknowledgements

This study was supported by the scientific research foundation of the Central Hospital of Xinxiang City.

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